www.aging-us.com AGING 2021, Vol. 13, No. 9 Research Paper Shikimic acid protects skin cells from UV-induced senescence through activation of the NAD+-dependent deacetylase SIRT1 Alfredo Martínez-Gutiérrez1,2,3, Irene Fernández-Duran1, Anna Marazuela-Duque1,2, Nicolás G. Simonet1,2, Ibraheem Yousef4, Immaculada Martínez-Rovira4,5, Josefina Martínez-Hoyos3, Alejandro Vaquero1,2 1 Chromatin Biology Laboratory, Josep Carreras Leukaemia Research Institute (IJC), Barcelona 08916, Badalona, Spain 2 Chromatin Biology Laboratory, Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), Barcelona 08908, Spain 3 Mesostetic Pharma Group, Barcelona 08840, Viladecans, Spain 4 MIRAS Beamline, ALBA-CELLS Synchrotron, Barcelona 08290, Cerdanyola del Vallès, Spain 5 Ionizing Radiation Research Group, Physics Department, Universitat Autònoma de Barcelona (UAB), Barcelona 08193, Cerdanyola del Vallès, Spain Correspondence to: Alejandro Vaquero, Josefina Martínez-Hoyos; email: avaquero@carrerasresearch.org, jmartinez@mesoestetic.com Keywords: SIRT1, senescence, human dermal fibroblasts, UV irradiation, shikimic acid Received: September 18, 2020 Accepted: March 31, 2021 Published: April 26, 2021 Copyright: © 2021 Gutiérrez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT UV radiation is one of the main contributors to skin photoaging by promoting the accumulation of cellular senescence, which in turn induces a proinflammatory and tissue-degrading state that favors skin aging. The members of the sirtuin family of NAD+-dependent enzymes play an anti-senescence role and their activation suggests a promising approach for preventing UV-induced senescence in the treatment of skin aging. A two- step screening designed to identify compounds able to protect cells from UV-induced senescence through sirtuin activation identified shikimic acid (SA), a metabolic intermediate in many organisms, as a bona-fide candidate. The protective effects of SA against senescence were dependent on specific activation of SIRT1 as the effect was abrogated by the SIRT1 inhibitor EX-527. Upon UV irradiation SA induced S-phase accumulation and a decrease in p16INK4A expression but did not protect against DNA damage or increased polyploidies. In contrast, SA reverted misfolded protein accumulation upon senescence, an effect that was abrogated by EX-527. Consistently, SA induced an increase in the levels of the chaperone BiP, resulting in a downregulation of unfolded protein response (UPR) signaling and UPR-dependent autophagy, avoiding their abnormal hyperactivation during senescence. SA did not directly activate SIRT1 in vitro, suggesting that SIRT1 is a downstream effector of SA signaling specifically in the response to cellular senescence. Our study not only uncovers a shikimic acid/SIRT1 signaling pathway that prevents cellular senescence, but also reinforces the role of sirtuins as key regulators of cell proteostasis. INTRODUCTION impairment of skin biological functions [1–3]. It can be classified as intrinsic or extrinsic, depending on the Skin aging is defined as the degenerative process by origin of the type of stress that causes aging. Intrinsic which structural and physiological alterations cause the skin aging, also known as chronological aging, depends www.aging-us.com 12308 AGING on the passage of time and is influenced by the genetic dependent deacetylases conserved throughout evolution, background and hormonal levels. In contrast, extrinsic from bacteria to humans. These proteins regulate the skin aging, or photoaging, is caused by external stress response of the cell through their involvement in stressors such as UV light, pollution, or tobacco smoke many cellular processes, including chromatin [1]. UV light is the main contributor to photoaging [4– regulation, DNA repair, metabolism and inflammation. 6], and may be divided into UVB, which reaches the There are seven sirtuins in humans, named SIRT1- epidermis and the upper dermis, and UVA, which SIRT7, which are located in different cellular involves longer UV wavelength radiation able to compartments and perform specific and redundant penetrate into the dermis. functions [16]. SIRT1, SIRT2, SIRT3, SIRT6, and to a lesser extent SIRT7, are known to play a role in The development of skin aging is associated with several senescence [17]. changes at the molecular level, including the accumulation of DNA damage, genome instability, Given the increasing evidence linking sirtuins, and SIRT1 epigenetic dysregulation, extracellular matrix degradation, in particular, to skin senescence and aging, the modulation mitochondrial dysfunction, inflammation, loss of of sirtuin activity in this functional context is a promising proteostasis, ER-stress and autophagy dysfunction. Many approach for delaying the development of UV-induced of these alterations are directly associated with cellular skin aging. In this sense, a number of SIRT1 specific senescence, one of the hallmarks of skin aging. Cellular activators have been shown to prevent senescence, inhibit senescence is a state in which cells enter permanent cell inflammation and promote wound healing in dermal cycle arrest and acquire a variety of phenotypic changes fibroblasts [18–20]. Moreover, some of them are currently related to gene expression, cellular functions and used in the treatment of psoriasis or are being evaluated morphology [7]. This process is normally induced after clinically for this purpose [21]. However, although several exposure to mild, chronic damage, such as the exposure of sirtuin activators have been described, the few that are of the skin to UV rays from sunlight. Other known inducers relevance in dermatology have limited efficacy and skin of cellular senescence include telomere shortening, penetration [22, 23]. Therefore, the identification of novel oncogene activation, genomic damage and mitochondrial sirtuin activators in skin is potentially very significant for dysfunction [8, 9]. Upon DNA damage, several pathways, the prevention of skin aging. including p53, p38-MAPK, NF-κβ and mTOR pathways, are activated to establish and maintain cellular senescence In the work presented here, we have performed a [10–12]. These pathways are then involved in cell cycle screening to identify novel molecules that induce sirtuin arrest and induction of a pro-inflammatory state (known activation and protect against UV-induced cellular as the senescence-associated secretory phenotype, SASP), senescence. We selected the most promising among other processes, which is one of the major features compounds from the screening and characterized in of cell senescence. The SASP creates an inflammatory greater detail their effect on senescence, sirtuin environment that favors the degradation of extracellular activation and targets in order to describe novel matrix proteins and promotes senescence in the compounds that are effective at prevent skin surrounding cells, which contribute to tissue damage, photoaging. aging and tumorigenesis. Senescent cells are also characterized by other features, which are routinely used RESULTS as biomarkers to identify senescence in vitro and in vivo. These features include large and flattened cell In our efforts to identify novel factors that could prevent morphology, induction of senescence-associated β- skin cell senescence, we developed a screening process galactosidase (SA-β-Gal) activity, chromatin to identify new modulators of the protective role of rearrangement, increased glycolysis, expression of sirtuins in UV-induced senescence. For this purpose, we immunogenic-related proteins, activation of pro-survival established a culture of primary human dermal pathways, DNA damage, loss of proteostasis and fibroblasts (HDFs). For the screening, we selected 30 mitochondrial damage [13]. Of these, increased levels of compounds that had previously been shown to regulate SA-β-Gal activity is currently the most widely accepted skin senescence and/or aging or had been linked to biomarker to identify senescence cells. However, as none sirtuin function (Table 1, left). Our first step in the of the aforementioned senescence features is specific to screening was to establish the optimal concentrations of senescence, the detection of more than one marker is these compounds in the case of HDFs. For this purpose, required to confirm the senescent state [14]. we calculated for each compound the lethal dose 50 (LD50) and the maximal non-cytotoxic dose conditions Of the factors associated with this process, sirtuins play (WST-1 cell viability test), which we used to perform essential roles by protecting against cellular senescence the screening (Table 1, right). Once established, and aging [15]. Sirtuins are a family of NAD+- we took these parameters into account www.aging-us.com 12309 AGING Table 1. List of compounds chosen to be tested. Max. non-cyt. Compound Target Biological process Species References LD50 (mM) doses (mM) Resveratrol Sir2, SIRT1 SIRT1 activation Y, H [24–26] 0.173 0.05 Quercetin Sir2, SIRT1 SIRT1 activation Y, H [24–26] 0.604 0.25 Dexpanthenol - SH, WH H [27] 262.48 100 Allantoin AMPK GM M [28] 100 50 BAX/Bcl-2 Idebenone SEN H [29] 0.021 0.01 ratio Ferulic acid FOXO3a AG M [30] 16.5 7.5 Lipoic acid SIRT1-AMPK LM M [31] 34.69 1 Gallic acid AMPK-SIRT1 AP, GM, LM H, M [32] 0.179 0.1 Taurine - SEN M [33] 396.58 150 Salicylic acid AMPK LM H, M [34] 20.88 1 Shikimic acid Nrf2-NF-κβ OS M [35] 58.44 25 Ectoine - PA H [36] 359.12 300 3,4-dihydroxybenzaldehyde JMJD2a L D [37] 1.08 0.1 PI3K/Akt/Nrf2 Andrographolide INF H [38] 0.124 0.01 PI3K/Akt/AP-1 Kinetin - PA H [39] 1.835 0.5 Zeatin - AG H [40] 2.462 1 Carnosine SEN H [41] 85.46 25 Damascenone PA M [42] 0.121 0.05 Betaine NF-κβ INF R [43] 507.81 50 Pyridoxine - OS M [44] 16.92 5 Verbascoside - PA H [45] 0.122 0.05 Hamamelitannin - PA M [46] 0.247 0.1 Phloretin SOD-Sir2 RL Y [47] 0.166 0.05 Vanillin ATM-p53-JNK UVDD H [48] 5.912 1 Sesamol - PA M [49] 4.358 0.5 Salicyladehyde thiosemicarbazone - OS H [50] 4.175 0.5 Syringic acid NF-κβ-TLR4 INF, LM M [51] 3.65 1 Sclareol - INF M [52] 0.04 0.02 Gentiopicrin p38-ERK-JNK INF R [53] 13.825 2.5 Irisflorentin ERK1/2-AP-1 INF M [54] 0.049 0.01 Left columns show the molecular targets, biological processes and species. Right columns show the calculated LD50 and the maximum non-cytotoxic dose (mM) in our cell viability assay for each compound. Aging (AG), autophagy (AP), glucose metabolism (GM), inflammation (INF), lifespan (L), lipid metabolism (LM), oxidative stress (OS), photoaging (PA), senescence (SEN), skin hydration (SH), UV-induced DNA damage (UVDD), wound healing (WH). Species: chicken (C), C. elegans (CE), D. melanogaster (D), hamster (HA), human (H), mouse (M), rat (R), yeast (Y). to decide the specific working range of concentrations purpose, we treated the cells for 24 h and monitored the for each compound. effect of the compounds on two of the best characterized sirtuin targets, the histone marks Shikimic acid is a senescence inhibitor that promotes H4K16ac (the target of SIRT1 and SIRT2) and H3K9ac sirtuin activity (the target of SIRT1, SIRT2 and SIRT6) (Figure 1B). In these studies we included the well described SIRT1 We performed the screening by merging two sequential activator resveratrol [18], and quercetin, which has been approaches. The first one was a cellular assay in which shown to activate or inhibit SIRT1 in different we tested the ability of these compounds to promote a functional contexts [26, 55]. Seven compounds decrease in sirtuins targets that would indicate an (Resveratrol, Quercetin, Dexpanthenol, Shikimic increased sirtuin activity. The second one was a acid, 3,4-dihydroxy-benzaldehyde, Andrographolide, senescence assay in which we determined the effect of Sesamol) induced a similar effect on both targets the compounds on cellular senescence induced by UV (Figure 1B–1D and Supplementary Figure 1). Three irradiation. compounds reduced the levels of H3K9ac only (Ectoine, Kinetin, Zeatin), and one compound In the first approach, we tested the ability of the 30 (Phloretin) had an effect solely on H4K16ac compounds to activate sirtuin activity, for which (Supplementary Figure 1). www.aging-us.com 12310 AGING In the second approach, we established a method for showed that, of the 30 compounds, only shikimic acid inducing senescence in these cells with 2 cycles of (SA), one of the positive candidates identified by the UVB irradiation 72h apart (Figure 2A). The use of first approach, prevented the development of UVA to induce senescence was discarded due to its senescence, reflected by both a significant decrease in considerably longer time requirement, being unsuitable SA-β-Gal accumulation and an increase in HAS2 to screen 30 compounds within a reasonable period. expression (Figure 2C–2E, Supplementary Figures 2, Cells were treated with these compounds throughout 3). Interestingly, SIRT1 reported activators the duration of the assay and cellular senescence was Resveratrol and Quercetin either only promoted HAS2 monitored with two markers: accumulation of SA-β- upregulation (Quercetin) or increased SA-β-Gal galactosidase (SA-β-Gal) activity, and the decrease in staining (Resveratrol) (Figure 2B and Supplementary expression of HAS2 (hyaluronic acid synthase 2), a Figure 2). This observation was in part explained by previously reported marker of skin senescence [56]. the fact that the identified range of concentrations Our senescence analysis considered positive candidates able to activate Sirtuin activity in the first stage to be only those compounds capable of inducing (Figure 1), induced cell death in the second stage of changes in both markers (Figure 2B). This first stage the screening. Moreover, the other four Figure 1. (A) Schematic representation of the compound testing for sirtuin activity evaluation in HDF. Cells were treated with or without the compounds for 24h and harvested for analysis. (B) Sirtuin activity assay results including H4K16 and H3K9 acetylation for cells treated with each compound. The results are shown by arrows that indicate increase/decrease (↑, ↓) and hyphens that indicate no effect (-). (C) Western Blot analysis and (D) quantification of H4K16ac and H3K9ac levels in cells treated with Resveratrol or Shikimic acid at the indicated concentrations. H4K16 and H3K9 levels were normalized to H4 and H3 levels respectively. Student T-test, *p<0.05, **p<0.01, ***p<0.001. www.aging-us.com 12311 AGING compounds (Dexpanthenol, 3,4-dihydroxy-benzaldehyde, SA prevents UV-induced senescence through SIRT1 Andrographolide, Sesamol) that induced the deacetylation of sirtuin targets in the previous screening (Figure 1B), Next, we set out to understand the role of SA in this did not prevent cellular senescence. Although six other context. The decreased levels of both H4K16ac and compounds induced HAS2 expression (Ferulic and Lipoic H3K9ac pointed to the involvement of SIRT1, since it acids, Taurine, Carnosine and Verbascoside), they did not is the only one of these sirtuins that efficiently targets change the level of SA-β-Gal staining (Figure 2B). both marks. To confirm this involvement, we Overall, the combined result of the two approaches pharmacologically inhibited SIRT1, SIRT2 and identified SA as being the only bona fide candidate for SIRT6 with the compounds EX-527, AGK2 and preventing cellular senescence through sirtuin activation. OSS128167, respectively, and tested the ability of SA Figure 2. (A) Schematic representation of the protocol for senescence induction using UVB irradiation. Cells were treated -/+ the compounds from day 1 to 7 after seeding, irradiated with UVB on days 2 and 5 and harvested for analysis on day 7. (B) Senescence assay results including β-Gal staining and HAS2 gene expression analysis for cells treated with each compound. The results are shown by arrows that indicate increase/decrease (↑, ↓) and hyphens that indicate no effect (-). (C) Phase-contrast (left column) and bright field (right column) microscope images of SA-β-Gal staining showing non-irradiated cells, UVB-irradiated cells non-treated and treated with Gallic acid or Shikimic acid at the indicated concentrations. (D) Percentage of SA-β-Gal positive cells and (E) Relative HAS2 mRNA levels in non-irradiated cells, UVB-irradiated cells and UVB-irradiated cells treated with Gallic acid or Shikimic acid at the indicated concentrations. Student T-test, *p<0.05, **p<0.01, ***p<0.001. www.aging-us.com 12312 AGING to prevent senescence (Figure 3A, 3B and these observations, siRNA-mediated downregulation of Supplementary Figure 4). The working conditions were SIRT1 reverted the anti-senescent effect of SA 25mM chosen either based on previous reports (EX-527, and 50mM measured by SA-β-Gal levels. In contrast, OSS128167) [57, 58] or determined experimentally to downregulation of SIRT2 did not have any effect while avoid excessive cell death (AGK2, data not shown). In SIRT6 downregulation induced by itself a SA- the case of EX-527, our working concentration was independent increase in the levels of senescence 1μM, as it has been shown to inhibit SIRT1 specifically (Figure 3E). (IC50 100nM) in contrast to the other two described targets SIRT2 (IC50 19μM) and SIRT3 (IC50 40μM) In agreement with a SA-dependent modulation of [57]. As expected, treatment of EX-527 significantly SIRT1 activity, SA 25mM induced increased inhibited the protective effect of SA on senescence at deacetylation of H3K9ac and H4K16ac under UV the level of SA-β-Gal and HAS2 expression (Figure 3C, conditions in these cells while EX-527 reverted this 3D), while AGK2 and OSS128167 produced no clear effect (Figure 3F). We discounted the possibility that effect (Supplementary Figure 4A, 4B). Confirming the effect of SA was due to an increase in SIRT1 levels, Figure 3. (A) Schematic representation of the protocol for senescence induction using UVB irradiation and study of EX527 involvement in the effect of Shikimic acid. Cells were treated with or without shikimic acid -/+ EX-527 from day 1 until day 7 after seeding, irradiated with UVB on days 2 and 5 and harvested for analysis on day 7. (B) Phase-contrast (left column) and bright field (right column) microscope images of β- Gal staining showing non-irradiated cells non-treated and treated with shikimic acid (10, 25 and 50 mM), UVB-irradiated cells non-treated and treated with shikimic acid (10, 25 and 50 mM), shikimic acid (10, 25 and 50 mM) + EX-527 1 μM and EX-527 1 μM. (C) Percentage of SA-β- Gal positive cells and (D) relative HAS2 mRNA levels in non-irradiated cells non-treated and treated with Shikimic acid (10, 25 and 50 mM), UVB-irradiated cells non-treated and treated with Shikimic acid (10, 25 and 50 mM), Shikimic acid (10, 25 and 50 mM) + EX-527 1 μM and EX- 527 1 μM. (E) Percentage of SA-β-Gal as in (C) of SA 25 or 50mM upon siRNA mediated downregulation of SIRT1, SIRT2 or SIRT6. siScr: scramble siRNA. Student T-test, *p<0.05, **p<0.01, ***p<0.001. (F) Levels of H3K9ac/H3 and H4K16ac/H4 under UV-induced senescence in HFD incubated with -/+25mM SA -/+1μM EX-527. (G) IL-6 mRNA levels of the assays as in (D). (H) The activity of FLAG-SIRT1 purified from HEK293 cells was tested in in vitro deacetylation assays -/+ NAD+ -/+ Shikimic acid (1 μM-10mM) in presence of hyperacetylated core histones. SIRT1 activity was then evaluated by analyzing acetylation of H3K9, H4K16 and p53K382 by Western blot. Student T-test, *p<0.05, **p<0.01, ***p<0.001. www.aging-us.com 12313 AGING since we did not observe any significant correlation probably overcoming the G1/S checkpoint, but is with gene expression and protein levels unrelated to the control of genome integrity. (Supplementary Figure 5A–5C). Interestingly, SA induced a reduction in the levels of expression of the SA regulates cell proteostasis through a SIRT1- inflammatory cytokine IL-6 (Figure 3G), previously dependent mechanism shown to be regulated by SIRT1 [59]. Moreover, this decrease was further enhanced by EX-527, indicating Given the direct role of SA in metabolism, and that the anti-inflammatory role of SA is independent particularly in aminoacid biosynthesis in many of SIRT1. This suggested that SA may not be a direct organisms, we considered the possibility that SA might regulator of SIRT1. Consistently, SA did not alter regulate the recycling of unfolded or damaged proteins. SIRT1 deacetylation activity in vitro towards In this sense, the unfolded protein response (UPR) and three different SIRT1 targets, H3K9ac, autophagy have been associated to cellular senescence. H4K16ac and p53K382ac (Figure 3H and Thus, although both signaling pathways play positive Supplementary Figure 5D). roles in the maintenance of a healthy proteostasis and therefore prevent the development of cell senescence, Overall, these results suggested that SIRT1 is an hyperactivation of these pathways under certain indirect downstream target of shikimic acid in this conditions, such as chronic stress, can favor several functional context. senescence-associated phenotypes [61, 62]. FTIRM allowed us to evaluate biochemical changes in the most The protective effect of SA in HDF cellular important biomolecules. Principal Component Analysis senescence is not related to genome stability or cell (PCA) in the Fingerprint region (mostly accounting for death proteins, DNA and carbohydrates) showed differences among the groups along PC3 (Figure 4G). ‘UVB’ and Our next step was to understand the effect of SA in ‘UVB SA’ groups overlapped and were separated from these HDF cells. We discounted the possibility that the control group (‘No UVB’). Interestingly, the distribution effect was due to increased cell death as the SRB/trypan of ‘UVB SA+EX-527’ group was closer to control. An blue assay revealed no differences in cytotoxicity analysis of the loading plot (Supplementary Figure 6D) (Figure 4A), and annexin V assays showed very mild shows that most of the differences were due to changes and mostly insignificant levels of early apoptosis in in the Amide I, mostly related to conformational UVB-irradiated cells upon exposure to SA (Figure 4B, modifications in the proteins. The Amide I 4C). SA did not alter the cell cycle in the absence of deconvolution allowed us to estimate changes in the UVB irradiation (Supplementary Figure 6A). While secondary protein structure, which revealed a global UVB induced an accumulation of cells in G2/M and a increase of the β-sheet/α-helix ratio in the proteins of reduction in the abundance of cells in G1, SA induced a these cells upon UVB (Figure 4G, 4H and progressive increase in the number of cells in S-phase Supplementary Figure 6D). This ratio increase has been that was associated with a mild decrease in G1 and in previously linked to increased levels of misfolded G2/M (Figure 4D and Supplementary Figure 6B). This proteins [63–66], suggesting that SA prevents the observation suggests that SA could participate in cell accumulation of misfolded proteins accumulated upon cycle checkpoint regulation, which was further UV. Strikingly, while SA treatment inhibited these supported by the downregulation of the cell cycle levels well below the ratio observed in control cells in regulator p16 observed upon SA treatment (Figure 4E). the absence of UVB irradiation, treatment with EX-527 In contrast, we did not observe any clear effect on the completely reverted the effect of SA (Figure 4G, 4H and levels of the cell cycle regulator, p21 (Supplementary Supplementary Figure 6D). This suggested that the Figure 6C). These results suggest that SA exerts its SIRT1-dependent effect of SA on senescence may be protective function by specifically impeding the de- associated with an altered pattern of misfolded and/or repression of the INK4/ARF locus. Indeed, activated aggregated proteins, which pointed to an effect on UPR SIRT1 has been suggested to be able to regulate p16 and/or autophagy. Interestingly, while SA treatment expression through epigenetic and genetic mechanisms induced upregulation of chaperone BiP (also known as [60], although how SIRT1 regulates p16 expression GPR78), a major regulator of cell proteostasis, that during cellular senescence remains to be fully effect was partially reverted by EX-527 (Figure 5A, 5B). elucidated. Consistently, incubation with SA did not In agreement with a key role of BiP in SA signaling, alter the accumulation of DNA damage or the frequency siRNA-induced downregulation of BiP partially reverted of polyploidies induced by UVB irradiation, nor did it the anti-senescence effect of SA (Figure 5C and have any effect in the absence of the stress (Figure 4D– Supplementary Figure 7A). The excess of available BiP 4F). Overall, this indicates that the effect of SA in protein has been linked with UPR inhibition [67], so we senescence favors the entry of cells in S-phase, hypothesized that under these conditions SA blocks UPR www.aging-us.com 12314 AGING signaling, and subsequently UPR-induced autophagy. DISCUSSION Accordingly, SA treatment induced a decrease in spliced-XBP1, a marker of UPR activity (Figure 5D). Our work reveals a role for SA in preventing cellular Consistently with a direct connection between the effect senescence in HDFs and identifies SIRT1 as a key of SA on BiP and UPR, we detected a SA-dependent downstream effector of this function (Figure 5D). Our decrease in the levels of LC3-II, a well-established evidences suggest that the signaling axis involving marker of autophagy. In agreement with a link with the SA/SIRT1 contributes to maintenance of proteostasis. SA/SIRT1 signaling axis, this effect was mostly reverted However, at this point we cannot discard that the by EX-527 treatment (SA 10mM). Interestingly, this SA/SIRT1 signaling could prevent senescence through EX-527 effect was in turn reversed by increased levels other mechanisms, or that SA may regulate senescence of SA (25 and 50 mM) (Figure 5C and Supplementary through other SIRT1-independent mechanisms. In fact, Figure 7B). Taken together, these results suggest that the our study suggests that SIRT1 is not targeted directly SIRT1-dependent protective effect of SA on senescence and it’s instead a downstream effector of SA based on is associated with preventing the cellular accumulation several evidences: i) SA does not alter SIRT1 activity in of misfolded proteins. vitro; ii) SA does not have a clear effect in UV-induced Figure 4. (A) SRB and Trypan blue cell viability assays performed in cells treated with or without Shikimic acid (5, 10, 25 and 50 mM) for 24h. (B) Apoptosis detection using the Annexin V-FITC/PI double staining followed by flow cytometry in non-irradiated and non-treated cells, irradiated and non-treated cells and irradiated cells treated with Shikimic acid (10, 25 and 50 mM). (C) Representation and statistical analysis of the percentage of apoptotic cells in each of the conditions analyzed. (D) Cell cycle analysis using PI staining followed by flow cytometry. (E) Relative p16INK4a mRNA levels monitored by qPCR in the indicated conditions. Student T-test, *p<0.05. (F) Immunofluorescence of γH2AX in non-irradiated and non-treated cells, irradiated and non-treated cells and irradiated cells treated with Shikimic acid (10, 25 and 50 mM). (G) Principal component analysis (PCA) on Savitzky–Golay second derivatized spectra in the fingerprint region (1800–950 cm−1) for non-irradiated cells (No UVB), irradiated non-treated cells (UVB), irradiated cells treated with Shikimic acid 25 mM (UVB SA) and irradiated cells treated with Shikimic acid 25 mM plus EX-527 1 μM (UVB SA+EX-527). Student T-test, *p<0.05, **p<0.01, ***p<0.001. (H) Beta-sheet to alpha-helix ratio obtained by curve-fitting analysis of the amide I band (1700-1600 cm-1). www.aging-us.com 12315 AGING cell death, DNA damage or genome stability while tissues, given that SA protects against oxidative stress- SIRT1 has important roles in these pathways; and iii) induced damage in hepatocytes and neuroblastoma cells SIRT1 inhibitor EX-527 cannot revert the effect of SA [35, 68]. Despite all these evidences, the mechanism of on the expression levels of pro-inflammatory IL-6. This SIRT1 activation by SA signaling remains unaddressed. suggests that SA signaling activates SIRT1 activity One interesting possibility is that the observed specifically in this context of senescence, but has no activating effect of shikimic acid may involve SIRT1 further effect on other SIRT-associated functions. This phosphorylation and subsequent activation. Thus, is also suggested by the different behavior of the other SIRT1 activators JNK2 and AMPK, two key stress- screening candidates, such as resveratrol and quercetin, dependent kinases, have been functionally linked with which promoted sirtuin activity but had different effect protective effect upon ER stress and autophagy, which on senescence markers. Nevertheless, the lack of effect may involve SIRT1 activation [69, 70]. Although not of SA on genome stability may not be the case in other present in animals, in many organisms such as plants, Figure 5. (A) Western-blot of BiP proteins levels in UVB or non UVB irradiated cells treated with SA at the indicated concentrations in presence or absence of EX-527 1 μM. (B) Quantification of n=3 experiments as (A). The levels of BiP are normalized by the loading control (histone H3) and represented related to the conditions in UVB irradiated cells in absence of SA and EX-527. Student T-test, *p<0.05. (C) Percentage of SA-β-Gal in assays performed as in Figure 3C. (D) Levels of spliced and unspliced XBP1 mRNA monitored by qPCR analysis in cells treated in the indicated conditions in presence or absence of SA and/or EX-527. The levels were normalized by internal controls and represented in each case related to the conditions in UVB irradiated cells in absence of SA and EX-527. Student T-test, *p<0.05, **p<0.01, ***p<0.001. (E) LC3-II protein levels in experiments like in (A). The levels of LC3-II are normalized by the loading control (tubulin) and represented related to the conditions in UVB irradiated cells in absence of SA and EX-527. Student T-test, *p<0.05. (F) Proposed Model for the effect of SA on senescence through SIRT1. Other possible unexplored connections between SA and senescence are indicated by blue broken lines. www.aging-us.com 12316 AGING bacteria and fungi, SA is a key metabolic intermediate senescence at different levels [62]. We hypothesize that at the crossroads of several essential metabolic under senescence, SA induces BiP upregulation to pathways, such as glycolysis and the biosynthesis of promote the recovery of a healthy proteostasis, which in aromatic aminoacids and metabolites like coenzyme Q turn promotes a block of UPR receptors by available and vitamin K [71]. Moreover, SA is commonly used in BiP [67], and a concomitant decrease in the signaling of many applications, given that it has an antibacterial and UPR and associated autophagy. In particular, our results antifungal activity and is widely used in biomedicine as indicate that the addition of SA on HDFs undergoing a precursor in the chemical synthesis of several UV-induced senescence impairs the formation of anticancer and antiviral compounds [72]. In the case of spliced XBP1, a UPR-dependent transcription factor skin, SA is used to treat bacterial infections and acne previously shown to promote autophagy under ER and is known to induce skin exfoliation. The important stress [76]. metabolic role of SA in non-animal organisms suggests that the effect of SA in animals may take place through Our findings together with the low toxicity of SA other conserved pathways associated to SA signaling in suggest that it has considerable potential in the these non-animal organisms. In this sense, it would be treatment against skin senescence and aging induced by relevant to determine whether SA has an anti-senescent UV irradiation. Future studies should confirm whether role also in these non-animal organisms. Moreover, we this is the case and define the precise contribution of cannot discard that the observed anti-senescent effect in SIRT1 in this context. human cells is tissue-specific and that it may not have the same protective effect beyond skin. Further studies MATERIALS AND METHODS will be required to define how conserved is the role of SA in this context and the mammalian pathways Cell culture involved in this protective role. Human dermal fibroblasts (HDFs) (Promocell, The role of this compound as a metabolic intermediate Germany) from 40-year old Caucasian women were in other organisms and its high turnover in the cell used as the in vitro model for compound viability and probably explain the very low toxicity seen in our efficacy screening. Cells were cultured in Dulbecco's experiments, and the concentration levels required to modified eagle medium (DMEM) low glucose protect senescence and induce SIRT1 activity. This is a (Biowest, France) supplemented with 10% fetal bovine similar case to that of nicotinamide, a well-established serum (FBS) (Capricorn Scientific, Germany) and 10 sirtuin inhibitor and key metabolic intermediate that is U/mL penicillin/streptomycin (Biowest, France) in a used in cellular concentrations of 5-10 mM in a variety humidified incubator at 37° C and 5% CO2. For cell of human cell types, from neural to stem cells [73]. passage, cells at 80% confluence were trypsinized and plated at 60% confluence. Cells between passage 13 and The lack of specific senescence markers is a current 18 were used for the assays. For the SIRT1 in vitro challenge in the field and there is a clear requirement assay, HEK293 cells (ATCC, USA), used to transfect for novel simple combinations in each specific context and immunoprecipitate SIRT1 protein, were grown in that may allow easy and efficient senescence detection. DMEM high glucose (Thermo Fisher Scientific, USA), Our studies imply that the SA-β-Gal/HAS2 combination supplemented with 10% FBS in a humidified incubator is a bona fide marker of skin cell senescence, in contrast at 37° C and 5% CO2. The following experiments were to other factors, such as p21 or p16. In the case of skin, all performed in triplicate. several reports already indicated that p21 or p16 levels might not be reliable markers of senescence, as the Cell viability assays, SiRNAs, compound treatments pathways activated during senescence are influenced by and senescence the stress stimulus and cell type [74, 75]. This probably reflects a specific feature of UV-induced senescence in HDFs were plated in 100x20 mm2 cell culture plates skin cells. and after 24h, fresh cell culture medium containing the compounds was added. After 24h, cells were detached Another interesting finding of our work is the effect of with cell scrapers, harvested, and processed for qPCR or SA in cell proteostasis, UPR and autophagy. Previous western blot. results have portrayed the complex relationship between UPR, autophagy and skin senescence, and it is generally The protocol for senescence induction using UVB accepted that abnormal hyperactivation of UPR and irradiation is described in the Supplementary Methods. autophagy promotes the development of cellular SA-β-Gal staining, WST-1, sulforhodamine B (SRB) senescence [61, 62]. UPR sustained signaling has been and trypan blue assays were performed as previously shown to activate autophagy and to promote cell described [77–80]. For the siRNA experiments, the www.aging-us.com 12317 AGING protocol for senescence induction using UVB purified SIRT1-FLAG from HEK293F cells were irradiation was the same as in the Supplementary incubated with 1200ng of hyperacetylated histones Methods. Specific siRNA (Horizon discovery, UK) for (purified by acid extraction) or 500ng of purified SIRT1, SIRT2, SIRT6, BiP or scramble siRNA were FLAG-p53 (anti-FLAG resin, Sigma-Aldrich) in 30μl of incubated for 6 hours before each irradiation step. deacetylation buffer (50 mM-Tris pH7.8, 4mM MgCl2, 0.2mM DTT) for 15min at 37° C in the presence or Gene expression quantification by qPCR absence of 1 mM NAD+ and/or SA at 0-10mM (dissolved in Tris 50mM pH 7.8). The reaction was RNA was extracted from cell pellets using the Total RNA stopped by adding Laemmli sample buffer. Purification Kit (Norgen, Canada), according to the Deacetylation was monitored by quantification of manufacturer’s protocol. Extracted RNA was H3K9ac/H3, H4K16ac/H4 and p53K382ac/p53 levels. quantified using a NanoDrop® ND-1000 UV-Vis Spectrophotometer (Thermo Fisher Scientific, USA) and Fourier transform infrared microspectroscopy 300 ng of RNA were retrotranscribed to cDNA using the (FTIRM) analysis PrimeScriptTM RT reagent kit (Takara Bio Inc., Japan). cDNA was diluted 1/10 in ultrapure water and qPCR mix FTIRM measurements were performed at the MIRAS was prepared by adding cDNA (12 ng), SYBR green beamline of ALBA Synchrotron using the internal supermix (Bio Rad, USA), forward and reverse primers (Globar®) source. The Hyperion 3000 microscope (0.2μM). The primers and their respective sequences are (Bruker) coupled to a Vertex 70 spectrometer and listed in Supplementary Table 1. qPCR protocol included equipped with liquid Nitrogen cooled Mercury one step at 95° C for 30 s, 40 cycles at 95° C for 10s, and Cadmium Telluride (MCT) detector was used for the 60° C for 1min. The results were analyzed with Bio-Rad measurements. The spectra of individual cells (more CFX Manager software. BACT was used as a than 100 per sample) were collected using a single mask housekeeping gene to normalize gene expression. aperture of 20×20 μm2. The infrared spectra were acquired in transmission mode in the 4000–900 cm−1 Protein level quantification by western blot mid-infrared range at a spectral resolution of 4 cm−1 with 512 co-added scans per spectrum. Principal Protein levels were analyzed as previously described Component Analysis (PCA) was performed on vector [81]. Cells were directly lysed in 1x Laemmli Buffer normalized second derivative spectra (Savitzky-Golay (Bio-Rad, USA), incubated at 95° C and centrifuged. algorithm, 11 smoothing points) using the Orange Supernatant was subjected to SDS-polyacrylamide gel toolbox (Bioinformatics Lab) [63, 84]. The structure electrophoresis, and successive steps were performed as content of the amide I peak (1710-1598 cm−1) was described [81]. The list of antibodies used is included in determined by band deconvolution using Gaussian band Supplementary Table 2. fitting profiles (Origin 7.0 software). The position and number of peaks, which were assigned to specific types Flow cytometry of secondary structures [85], were determined using the second derivative of the spectra. Fluorescence-activated cell sorting (FACS) analyses of the cell cycle and apoptosis were carried out using, Data availability statement respectively, a Propidium Iodide Flow Cytometry Kit (Abcam, UK), and an Annexin V-FITC Apoptosis The data supporting the findings of this study are Staining/Detection Kit (Abcam, UK), following the included in the article and the supplementary manufacturer’s instructions. information. Additional data or material related to this paper is available upon request to the authors. γH2AX immunofluorescence assay AUTHOR CONTRIBUTIONS The staining and detection of γH2AX foci by immunofluorescence was performed as previously A.M. designed and performed experiments, and described [82]. Slides were analyzed by confocal participated in the writing of the manuscript. A.V. and microscopy using a Confocal Leica SP5 microscope J.M. designed and supervised the experiments and A.V. (Leica Microsystems) with a 63x objective. wrote the manuscript. IFD quantified the β-gal staining experiments, studied BIP protein levels and designed In vitro assay for SIRT1 activity experiments. NGS participated in the in vitro assay of SIRT1 and performed XBP1 qPCR measurements. The in vitro SIRT1 deacetylation assay was performed A.M. participated in the treatments and the as previously described [83]. Briefly, 500ng of the measurement of the H4K16ac/H3K9ac levels in the first www.aging-us.com 12318 AGING stage of the screening. IMR and IY performed the 6. Farage MA, Miller KW, Elsner P, Maibach HI. Intrinsic FTIRM studies. All authors participated in the and extrinsic factors in skin ageing: a review. Int J discussion. Cosmet Sci. 2008; 30:87–95. https://doi.org/10.1111/j.1468-2494.2007.00415.x ACKNOWLEDGMENTS PMID:18377617 7. Kuilman T, Michaloglou C, Mooi WJ, Peeper DS. The We thank the members of the Vaquero laboratory and essence of senescence. Genes Dev. 2010; 24:2463–79. Mesostetic Pharma group for stimulating discussions https://doi.org/10.1101/gad.1971610 PMID:21078816 and support. We acknowledge the granted beamtime at the MIRAS BL of ALBA Synchrotron. 8. Muñoz-Espín D, Serrano M. Cellular senescence: from physiology to pathology. Nat Rev Mol Cell Biol. 2014; CONFLICTS OF INTEREST 15:482–96. https://doi.org/10.1038/nrm3823 A.M and J.M are employees of mesoestetic Pharma PMID:24954210 Group, s.l. Mesoestetic Pharma Group, s.l has applied 9. Gorgoulis V, Adams PD, Alimonti A, Bennett DC, for a patent, PCT 2019/070704, for the use of shikimic Bischof O, Bishop C, Campisi J, Collado M, Evangelou K, acid in the treatment of skin aging. Ferbeyre G, Gil J, Hara E, Krizhanovsky V, et al. Cellular Senescence: Defining a Path Forward. Cell. 2019; FUNDING 179:813–27. https://doi.org/10.1016/j.cell.2019.10.005 This work was supported by the Industrial Doctorate PMID:31675495 Program (2015DI76) of the Catalan Government Agency AGAUR (2014-SGR400, 2017-SGR148 to 10. Chien Y, Scuoppo C, Wang X, Fang X, Balgley B, Bolden A.V.), the Spanish Ministry of Economy and JE, Premsrirut P, Luo W, Chicas A, Lee CS, Kogan SC, Competitiveness - MINECO (SAF2014-55964R, Lowe SW. Control of the senescence-associated SAF2017-88975R to A.V.), cofunded by FEDER secretory phenotype by NF-κB promotes senescence funds/European Regional Development Fund (ERDF) - and enhances chemosensitivity. Genes Dev. 2011; A Way to Build Europe, and the Spanish Ministry of 25:2125–36. Science, Innovation and Universities (RYC2018- https://doi.org/10.1101/gad.17276711 024043-I to I.M.R.). PMID:21979375 11. Qian Y, Chen X. Senescence regulation by the p53 REFERENCES protein family. Methods Mol Biol. 2013; 965:37–61. https://doi.org/10.1007/978-1-62703-239-1_3 1. Sjerobabski-Masnec I, Situm M. Skin aging. Acta Clin PMID:23296650 Croat. 2010; 49:515–18. PMID:21830465 12. Xu S, Cai Y, Wei Y. mTOR Signaling from Cellular Senescence to Organismal Aging. Aging Dis. 2013; 2. Kohl E, Steinbauer J, Landthaler M, Szeimies RM. 5:263–73. Skin ageing. J Eur Acad Dermatol Venereol. 2011; https://doi.org/10.14336/AD.2014.0500263 25:873–84. PMID:25110610 https://doi.org/10.1111/j.1468-3083.2010.03963.x PMID:21261751 13. Hernandez-Segura A, Nehme J, Demaria M. 3. Ganceviciene R, Liakou AI, Theodoridis A, Hallmarks of Cellular Senescence. Trends Cell Biol. Makrantonaki E, Zouboulis CC. Skin anti-aging 2018; 28:436–53. strategies. Dermatoendocrinol. 2012; 4:308–19. https://doi.org/10.1016/j.tcb.2018.02.001 https://doi.org/10.4161/derm.22804 PMID:23467476 PMID:29477613 4. Uitto J. Understanding premature skin aging. N Engl J 14. Sharpless NE, Sherr CJ. Forging a signature of in vivo Med. 1997; 337:1463–65. senescence. Nat Rev Cancer. 2015; 15:397–408. https://doi.org/10.1056/NEJM199711133372011 https://doi.org/10.1038/nrc3960 PMID:9358147 PMID:26105537 5. Pandel R, Poljšak B, Godic A, Dahmane R. Skin 15. Lee SH, Lee JH, Lee HY, Min KJ. Sirtuin signaling in photoaging and the role of antioxidants in its cellular senescence and aging. BMB Rep. 2019; prevention. ISRN Dermatol. 2013; 2013:930164. 52:24–34. https://doi.org/10.1155/2013/930164 https://doi.org/10.5483/BMBRep.2019.52.1.290 PMID:24159392 PMID:30526767 www.aging-us.com 12319 AGING 16. Kupis W, Pałyga J, Tomal E, Niewiadomska E. The role https://doi.org/10.1038/nature01960 of sirtuins in cellular homeostasis. J Physiol Biochem. PMID:12939617 2016; 72:371–80. 25. Kaeberlein M, McDonagh T, Heltweg B, Hixon J, https://doi.org/10.1007/s13105-016-0492-6 Westman EA, Caldwell SD, Napper A, Curtis R, PMID:27154583 DiStefano PS, Fields S, Bedalov A, Kennedy BK. 17. Ghosh S, Zhou Z. SIRTain regulators of premature Substrate-specific activation of sirtuins by resveratrol. J senescence and accelerated aging. Protein Cell. 2015; Biol Chem. 2005; 280:17038–45. 6:322–33. https://doi.org/10.1074/jbc.M500655200 https://doi.org/10.1007/s13238-015-0149-1 PMID:15684413 PMID:25907989 26. Bai X, Yao L, Ma X, Xu X. Small Molecules as SIRT 18. Dai H, Sinclair DA, Ellis JL, Steegborn C. Sirtuin Modulators. Mini Rev Med Chem. 2018; 18:1151–57. activators and inhibitors: Promises, achievements, and https://doi.org/10.2174/138955751666616062009510 challenges. Pharmacol Ther. 2018; 188:140–54. 3 PMID:27334466 https://doi.org/10.1016/j.pharmthera.2018.03.004 27. Ebner F, Heller A, Rippke F, Tausch I. Topical use of PMID:29577959 dexpanthenol in skin disorders. Am J Clin Dermatol. 19. Kim J, Kang YG, Lee JY, Choi DH, Cho YU, Shin JM, Park 2002; 3:427–33. JS, Lee JH, Kim WG, Seo DB, Lee TR, Miyamoto Y, No https://doi.org/10.2165/00128071-200203060-00005 KT. The natural phytochemical dehydroabietic acid is PMID:12113650 an anti-aging reagent that mediates the direct 28. Chen MF, Yang TT, Yeh LR, Chung HH, Wen YJ, Lee WJ, activation of SIRT1. Mol Cell Endocrinol. 2015; Cheng JT. Activation of imidazoline I-2B receptors by 412:216–25. allantoin to increase glucose uptake into C₂C₁₂ cells. https://doi.org/10.1016/j.mce.2015.05.006 Horm Metab Res. 2012; 44:268–72. PMID:25976661 https://doi.org/10.1055/s-0032-1301898 20. Wahedi HM, Chae JK, Subedi L, Kang MC, Cho H, Kim S, PMID:22351479 Kim SY. NED416, a novel synthetic Sirt1 activator, 29. Arend N, Wertheimer C, Laubichler P, Wolf A, Kampik promotes cutaneous wound healing via the MAPK/Rho A, Kernt M. Idebenone Prevents Oxidative Stress, Cell pathway. Int J Mol Med. 2020; 46:149–58. Death and Senescence of Retinal Pigment Epithelium https://doi.org/10.3892/ijmm.2020.4564 Cells by Stabilizing BAX/Bcl-2 Ratio. Ophthalmologica. PMID:32319533 2015; 234:73–82. 21. Krueger JG, Suárez-Fariñas M, Cueto I, Khacherian A, https://doi.org/10.1159/000381726 PMID:26044821 Matheson R, Parish LC, Leonardi C, Shortino D, Gupta 30. Choi YJ, Kim DH, Lee EK, Kim JM, Ha YM, Kim ND, Jung A, Haddad J, Vlasuk GP, Jacobson EW. A Randomized, JH, Choi JS, Yu BP, Chung HY. Attenuation of age- Placebo-Controlled Study of SRT2104, a SIRT1 related changes in FOXO3a activity and the PI3K/Akt Activator, in Patients with Moderate to Severe pathway by short-term feeding of ferulate. Age Psoriasis. PLoS One. 2015; 10:e0142081. (Dordr). 2012; 34:317–27. https://doi.org/10.1371/journal.pone.0142081 https://doi.org/10.1007/s11357-011-9235-3 PMID:26556603 PMID:21468671 22. Walle T. Bioavailability of resveratrol. Ann N Y Acad Sci. 31. Chen WL, Kang CH, Wang SG, Lee HM. α-Lipoic acid 2011; 1215:9–15. regulates lipid metabolism through induction of sirtuin https://doi.org/10.1111/j.1749-6632.2010.05842.x 1 (SIRT1) and activation of AMP-activated protein PMID:21261636 kinase. Diabetologia. 2012; 55:1824–35. 23. Scalia S, Trotta V, Iannuccelli V, Bianchi A. https://doi.org/10.1007/s00125-012-2530-4 Enhancement of in vivo human skin penetration of PMID:22456698 resveratrol by chitosan-coated lipid microparticles. 32. Doan KV, Ko CM, Kinyua AW, Yang DJ, Choi YH, Oh IY, Colloids Surf B Biointerfaces. 2015; 135:42–49. Nguyen NM, Ko A, Choi JW, Jeong Y, Jung MH, Cho https://doi.org/10.1016/j.colsurfb.2015.07.043 WG, Xu S, et al. Gallic acid regulates body weight and PMID:26241915 glucose homeostasis through AMPK activation. 24. Howitz KT, Bitterman KJ, Cohen HY, Lamming DW, Lavu Endocrinology. 2015; 156:157–68. S, Wood JG, Zipkin RE, Chung P, Kisielewski A, Zhang LL, https://doi.org/10.1210/en.2014-1354 Scherer B, Sinclair DA. Small molecule activators of PMID:25356824 sirtuins extend Saccharomyces cerevisiae lifespan. 33. Ito T, Yoshikawa N, Inui T, Miyazaki N, Schaffer SW, Nature. 2003; 425:191–96. Azuma J. Tissue depletion of taurine accelerates www.aging-us.com 12320 AGING skeletal muscle senescence and leads to early death in undergoing aging in vitro. Rejuvenation Res. 2005; mice. PLoS One. 2014; 9:e107409. 8:46–57. https://doi.org/10.1371/journal.pone.0107409 https://doi.org/10.1089/rej.2005.8.46 PMID:15798374 PMID:25229346 41. McFarland GA, Holliday R. Retardation of the 34. Hawley SA, Fullerton MD, Ross FA, Schertzer JD, senescence of cultured human diploid fibroblasts by Chevtzoff C, Walker KJ, Peggie MW, Zibrova D, Green carnosine. Exp Cell Res. 1994; 212:167–75. KA, Mustard KJ, Kemp BE, Sakamoto K, Steinberg GR, https://doi.org/10.1006/excr.1994.1132 Hardie DG. The ancient drug salicylate directly PMID:8187813 activates AMP-activated protein kinase. Science. 2012; 42. Uddin AN, Labuda I, Burns FJ. A novel mechanism of 336:918–22. filaggrin induction and sunburn prevention by β- https://doi.org/10.1126/science.1215327 damascenone in Skh-1 mice. Toxicol Appl Pharmacol. PMID:22517326 2012; 265:335–41. 35. Manna K, Khan A, Kr Das D, Bandhu Kesh S, Das U, https://doi.org/10.1016/j.taap.2012.08.035 Ghosh S, Sharma Dey R, Das Saha K, Chakraborty A, PMID:22982537 Chattopadhyay S, Dey S, Chattopadhyay D. Protective effect of coconut water concentrate and its active 43. Go EK, Jung KJ, Kim JY, Yu BP, Chung HY. Betaine component shikimic acid against hydroperoxide suppresses proinflammatory signaling during aging: mediated oxidative stress through suppression of NF- the involvement of nuclear factor-kappaB via nuclear κB and activation of Nrf2 pathway. J Ethnopharmacol. factor-inducing kinase/IkappaB kinase and mitogen- 2014; 155:132–46. activated protein kinases. J Gerontol A Biol Sci Med Sci. https://doi.org/10.1016/j.jep.2014.04.046 2005; 60:1252–64. PMID:24835026 https://doi.org/10.1093/gerona/60.10.1252 PMID:16282556 36. Buenger J, Driller H. Ectoin: an effective natural substance to prevent UVA-induced premature 44. Hsu CC, Cheng CH, Hsu CL, Lee WJ, Huang SC, Huang photoaging. Skin Pharmacol Physiol. 2004; 17:232–37. YC. Role of vitamin B6 status on antioxidant defenses, https://doi.org/10.1159/000080216 glutathione, and related enzyme activities in mice with PMID:15452409 homocysteine-induced oxidative stress. Food Nutr Res. 2015; 59:25702. 37. Nakagawa-Yagi Y, Sato Y, Matsumoto E, Nakatsuka S, https://doi.org/10.3402/fnr.v59.25702 Sakaki T, Muramatsu Y, Hara T, Aigaki T. PMID:25933612 Pharmacological modulation of histone demethylase activity by a small molecule isolated from subcritical 45. Kostyuk VA, Potapovich AI, Lulli D, Stancato A, De Luca water extracts of Sasa senanensis leaves prolongs the C, Pastore S, Korkina L. Modulation of human lifespan of Drosophila melanogaster. BMC keratinocyte responses to solar UV by plant Complement Altern Med. 2012; 12:101. polyphenols as a basis for chemoprevention of non- https://doi.org/10.1186/1472-6882-12-101 melanoma skin cancers. Curr Med Chem. 2013; PMID:22809229 20:869–79. PMID:23210792 38. Lu CY, Yang YC, Li CC, Liu KL, Lii CK, Chen HW. Andrographolide inhibits TNFα-induced ICAM-1 46. Masaki H, Atsumi T, Sakurai H. Protective activity of expression via suppression of NADPH oxidase hamamelitannin on cell damage of murine skin activation and induction of HO-1 and GCLM expression fibroblasts induced by UVB irradiation. J Dermatol Sci. through the PI3K/Akt/Nrf2 and PI3K/Akt/AP-1 1995; 10:25–34. pathways in human endothelial cells. Biochem https://doi.org/10.1016/0923-1811(95)93711-9 Pharmacol. 2014; 91:40–50. PMID:7577835 https://doi.org/10.1016/j.bcp.2014.06.024 47. Xiang L, Sun K, Lu J, Weng Y, Taoka A, Sakagami Y, Qi J. PMID:24998495 Anti-aging effects of phloridzin, an apple polyphenol, 39. Wanitphakdeedecha R, Meeprathom W, Manuskiatti on yeast via the SOD and Sir2 genes. Biosci Biotechnol W. Efficacy and safety of 0.1% kinetin cream in the Biochem. 2011; 75:854–58. treatment of photoaging skin. Indian J Dermatol https://doi.org/10.1271/bbb.100774 Venereol Leprol. 2015; 81:547. PMID:21597195 https://doi.org/10.4103/0378-6323.157446 48. Lee J, Cho JY, Lee SY, Lee KW, Lee J, Song JY. Vanillin PMID:25994881 protects human keratinocyte stem cells against 40. Rattan SI, Sodagam L. Gerontomodulatory and youth- ultraviolet B irradiation. Food Chem Toxicol. 2014; preserving effects of zeatin on human skin fibroblasts 63:30–37. www.aging-us.com 12321 AGING https://doi.org/10.1016/j.fct.2013.10.031 Thomas RJ, Kates M, Jones S, et al. Discovery of indoles PMID:24184596 as potent and selective inhibitors of the deacetylase SIRT1. J Med Chem. 2005; 48:8045–54. 49. Sharma S, Kaur IP. Development and evaluation of https://doi.org/10.1021/jm050522v PMID:16335928 sesamol as an antiaging agent. Int J Dermatol. 2006; 45:200–08. 58. Parenti MD, Grozio A, Bauer I, Galeno L, Damonte P, https://doi.org/10.1111/j.1365-4632.2004.02537.x Millo E, Sociali G, Franceschi C, Ballestrero A, PMID:16533216 Bruzzone S, Del Rio A, Nencioni A. Discovery of novel and selective SIRT6 inhibitors. J Med Chem. 50. Nguyen DT, Le TH, Bui TT. Antioxidant 2014; 57:4796–804. activities of thiosemicarbazones from substituted https://doi.org/10.1021/jm500487d PMID:24785705 benzaldehydes and N-(tetra-O-acetyl-β-D- galactopyranosyl)thiosemicarbazide. Eur J Med 59. Xie J, Zhang X, Zhang L. Negative regulation of Chem. 2013; 60:199–207. inflammation by SIRT1. Pharmacol Res. 2013; https://doi.org/10.1016/j.ejmech.2012.10.004 67:60–67. PMID:23291121 https://doi.org/10.1016/j.phrs.2012.10.010 PMID:23098819 51. Ham JR, Lee HI, Choi RY, Sim MO, Seo KI, Lee MK. Anti- steatotic and anti-inflammatory roles of syringic acid in 60. Li Y, Tollefsbol TO. p16(INK4a) suppression by glucose high-fat diet-induced obese mice. Food Funct. 2016; restriction contributes to human cellular lifespan 7:689–97. extension through SIRT1-mediated epigenetic and https://doi.org/10.1039/c5fo01329a PMID:26838182 genetic mechanisms. PLoS One. 2011; 6:e17421. 52. Huang GJ, Pan CH, Wu CH. Sclareol exhibits anti- https://doi.org/10.1371/journal.pone.0017421 PMID:21390332 inflammatory activity in both lipopolysaccharide- stimulated macrophages and the λ-carrageenan- 61. Pluquet O, Pourtier A, Abbadie C. The unfolded protein induced paw edema model. J Nat Prod. 2012; response and cellular senescence. A review in the 75:54–59. theme: cellular mechanisms of endoplasmic reticulum https://doi.org/10.1021/np200512a PMID:22250858 stress signaling in health and disease. Am J Physiol Cell 53. Zhao L, Ye J, Wu GT, Peng XJ, Xia PF, Ren Y. Physiol. 2015; 308:C415–25. https://doi.org/10.1152/ajpcell.00334.2014 Gentiopicroside prevents interleukin-1 beta induced PMID:25540175 inflammation response in rat articular chondrocyte. J Ethnopharmacol. 2015; 172:100–07. 62. Abbadie C, Pluquet O. Unfolded Protein Response https://doi.org/10.1016/j.jep.2015.06.031 (UPR) Controls Major Senescence Hallmarks. Trends PMID:26116164 Biochem Sci. 2020; 45:371–74. 54. Gao Y, Fang L, Liu F, Zong C, Cai R, Chen X, Qi Y. https://doi.org/10.1016/j.tibs.2020.02.005 Suppressive effects of irisflorentin on LPS-induced PMID:32311331 inflammatory responses in RAW 264.7 macrophages. 63. Martínez-Rovira I, Seksek O, Dokic I, Brons S, Abdollahi Exp Biol Med (Maywood). 2014; 239:1018–24. A, Yousef I. Study of the intracellular nanoparticle- https://doi.org/10.1177/1535370214530081 based radiosensitization mechanisms in F98 glioma PMID:24740875 cells treated with charged particle therapy through 55. You W, Zheng W, Weiss S, Chua KF, Steegborn C. synchrotron-based infrared microspectroscopy. Structural basis for the activation and inhibition of Analyst. 2020; 145:2345–56. Sirtuin 6 by quercetin and its derivatives. Sci Rep. 2019; https://doi.org/10.1039/c9an02350j 9:19176. PMID:31993615 https://doi.org/10.1038/s41598-019-55654-1 64. Gault N, Rigaud O, Poncy JL, Lefaix JL. Infrared PMID:31844103 microspectroscopy study of gamma-irradiated and 56. Röck K, Tigges J, Sass S, Schütze A, Florea AM, Fender H2O2-treated human cells. Int J Radiat Biol. 2005; AC, Theis FJ, Krutmann J, Boege F, Fritsche E, 81:767–79. Reifenberger G, Fischer JW. miR-23a-3p causes cellular https://doi.org/10.1080/09553000500515368 senescence by targeting hyaluronan synthase 2: PMID:16449084 possible implication for skin aging. J Invest Dermatol. 65. Holman HY, Martin MC, Blakely EA, Bjornstad K, 2015; 135:369–77. McKinney WR. IR spectroscopic characteristics of cell https://doi.org/10.1038/jid.2014.422 PMID:25264594 cycle and cell death probed by synchrotron radiation 57. Napper AD, Hixon J, McDonagh T, Keavey K, Pons JF, based Fourier transform IR spectromicroscopy. Barker J, Yau WT, Amouzegh P, Flegg A, Hamelin E, Biopolymers. 2000; 57:329–35. www.aging-us.com 12322 AGING https://doi.org/10.1002/1097-0282(2000)57:6<329:: p21(CIP1), but not p16(INK4a). Mol Cell. 2004; AID-BIP20>3.0.CO;2-2 PMID:11054652 14:501–13. https://doi.org/10.1016/s1097-2765(04)00256-4 66. Bozkurt O, Severcan M, Severcan F. Diabetes induces PMID:15149599 compositional, structural and functional alterations on rat skeletal soleus muscle revealed by FTIR 75. Hernandez-Segura A, de Jong TV, Melov S, Guryev V, spectroscopy: a comparative study with EDL muscle. Campisi J, Demaria M. Unmasking Transcriptional Analyst. 2010; 135:3110–19. Heterogeneity in Senescent Cells. Curr Biol. 2017; https://doi.org/10.1039/c0an00542h PMID:20967384 27:2652–60.e4. https://doi.org/10.1016/j.cub.2017.07.033 67. Hetz C. The unfolded protein response: controlling cell PMID:28844647 fate decisions under ER stress and beyond. Nat Rev Mol Cell Biol. 2012; 13:89–102. 76. Margariti A, Li H, Chen T, Martin D, Vizcay-Barrena G, https://doi.org/10.1038/nrm3270 PMID:22251901 Alam S, Karamariti E, Xiao Q, Zampetaki A, Zhang Z, 68. Rabelo TK, Zeidán-Chuliá F, Caregnato FF, Schnorr CE, Wang W, Jiang Z, Gao C, et al. XBP1 mRNA splicing triggers an autophagic response in endothelial cells Gasparotto J, Serafini MR, de Souza Araújo AA, through BECLIN-1 transcriptional activation. J Biol Quintans-Junior LJ, Moreira JC, Gelain DP. In Vitro Chem. 2013; 288:859–72. Neuroprotective Effect of Shikimic Acid Against Hydrogen Peroxide-Induced Oxidative Stress. J Mol https://doi.org/10.1074/jbc.M112.412783 PMID:23184933 Neurosci. 2015; 56:956–65. https://doi.org/10.1007/s12031-015-0559-9 77. Debacq-Chainiaux F, Erusalimsky JD, Campisi J, PMID:25862258 Toussaint O. Protocols to detect senescence-associated beta-galactosidase (SA-betagal) activity, a biomarker of 69. Raciti M, Lotti LV, Valia S, Pulcinelli FM, Di Renzo L. senescent cells in culture and in vivo. Nat Protoc. 2009; JNK2 is activated during ER stress and promotes cell survival. Cell Death Dis. 2012; 3:e429. 4:1798–806. https://doi.org/10.1038/nprot.2009.191 https://doi.org/10.1038/cddis.2012.167 PMID:20010931 PMID:23171849 70. Kim H, Moon SY, Kim JS, Baek CH, Kim M, Min JY, Lee 78. Vichai V, Kirtikara K. Sulforhodamine B colorimetric assay for cytotoxicity screening. Nat Protoc. 2006; SK. Activation of AMP-activated protein kinase inhibits 1:1112–16. ER stress and renal fibrosis. Am J Physiol Renal Physiol. 2015; 308:F226–36. https://doi.org/10.1038/nprot.2006.179 PMID:17406391 https://doi.org/10.1152/ajprenal.00495.2014 PMID:25428127 79. Konjhodzic-Prcic A, Jakupovic S, Hasic-Brankovic L, 71. Mir R, Jallu S, Singh TP. The shikimate pathway: review Vukovic A. In vitro comparison of cytotoxicity of four root canal sealers on human gingival fibroblasts. Med of amino acid sequence, function and three- Arch. 2015; 69:24–27. dimensional structures of the enzymes. Crit Rev Microbiol. 2015; 41:172–89. https://doi.org/10.5455/medarh.2015.69.24-27 https://doi.org/10.3109/1040841X.2013.813901 PMID:25870472 PMID:23919299 80. Escobar-García M, Rodríguez-Contreras K, Ruiz- 72. Estévez AM, Estévez RJ. A short overview on the Rodríguez S, Pierdant-Pérez M, Cerda-Cristerna B, medicinal chemistry of (-)-shikimic acid. Mini Rev Med Pozos-Guillén A. Eugenol Toxicity in Human Dental Chem. 2012; 12:1443–54. Pulp Fibroblasts of Primary Teeth. J Clin Pediatr Dent. https://doi.org/10.2174/138955712803832735 2016; 40:312–18. PMID:22827174 https://doi.org/10.17796/1053-4628-40.4.312 PMID:27471810 73. Meng Y, Ren Z, Xu F, Zhou X, Song C, Wang VY, Liu W, Lu L, Thomson JA, Chen G. Nicotinamide Promotes Cell 81. Yang B, Ji C, Kang J, Chen W, Bi Z, Wan Y. Trans-Zeatin Survival and Differentiation as Kinase Inhibitor in inhibits UVB-induced matrix metalloproteinase-1 Human Pluripotent Stem Cells. Stem Cell Reports. expression via MAP kinase signaling in human skin 2018; 11:1347–56. fibroblasts. Int J Mol Med. 2009; 23:555–60. https://doi.org/10.1016/j.stemcr.2018.10.023 https://doi.org/10.3892/ijmm_00000164 PMID:30503259 PMID:19288033 74. Herbig U, Jobling WA, Chen BP, Chen DJ, Sedivy JM. 82. Noren Hooten N, Evans MK. Techniques to Induce and Telomere shortening triggers senescence of human Quantify Cellular Senescence. J Vis Exp. 2017; cells through a pathway involving ATM, p53, and 123:55533. www.aging-us.com 12323 AGING https://doi.org/10.3791/55533 PMID:28518126 A, Štajdohar M, Umek L, Žagar L, et al. Orange: Data Mining Toolbox in Python. Journal of Machine Learning 83. Fang J, Ianni A, Smolka C, Vakhrusheva O, Nolte H, Research. 2013; 14:2349–53. Krüger M, Wietelmann A, Simonet NG, Adrian-Segarra JM, Vaquero A, Braun T, Bober E. Sirt7 promotes 85. Ghimire H, Venkataramani M, Bian Z, Liu Y, Perera adipogenesis in the mouse by inhibiting autocatalytic AG. ATR-FTIR spectral discrimination between activation of Sirt1. Proc Natl Acad Sci USA. 2017; normal and tumorous mouse models of lymphoma 114:E8352–61. and melanoma from serum samples. Sci Rep. 2017; https://doi.org/10.1073/pnas.1706945114 7:16993. PMID:28923965 https://doi.org/10.1038/s41598-017-17027-4 PMID:29209060 84. Demšar J, Curk T, Erjavec A, Gorup V, Hočevar T, Milutinovič M, Možina M, Polajnar M, Toplak M, Starič www.aging-us.com 12324 AGING SUPPLEMENTARY MATERIALS Supplementary Methods Senescence induction by UVB irradiation For the non-irradiation control conditions, HDF were plated at a density of 6×104 cells/plate in 6-well HDF were plated at a density of 6×104 cells/plate in 6- plates. After 24h, culture medium was removed and well plates. After 24h, culture medium was removed replaced by fresh cell culture medium containing the and replaced with fresh cell culture medium containing compounds at different concentrations. After 24h, the compounds at different concentrations. After 24h, cells were trypsinized and reseeded at 6×104 cell culture medium was removed, and cells were cells/plate in fresh cell culture medium containing the irradiated at 25 mJ/cm2 of UVB light (312 nm) in a Bio- compound in 6-well plates. After 3 days, cells were LINK Crosslinker BLX-312/365 (Witec ag) in a small trypsinized again and reseeded at 8×104 cells/plate in amount of PBS. PBS was then removed and fresh cell fresh cell culture medium containing the compound in culture medium with the compound was added to each 6-well plates. After 2 days, cells were processed well. After 3 days, cells were irradiated again under the according to the qPCR, β-Gal staining, apoptosis, cell same conditions. After 2 days, cells were processed cycle or immunofluorescence protocols. according to the qPCR, β-Gal staining, apoptosis, cell cycle or immunofluorescence protocols. www.aging-us.com 12325 AGING Supplementary Figures Supplementary Figure 1. Sirtuin activity assay results. (A) Quantification of H4K16ac levels and (B) H3K9ac levels from (n=3) Western- blots as in Figure cells treated with the following compounds during 24h: Idebenone (0.5, 1, 2.5, 5 μM), Ferulic acid (50, 100, 200, 500 μM), Lipoic acid (50, 100, 200, 500 μM), Quercetin (50, 100, 200, 250 μM), Phloretin (10, 25, 50, 75 μM), 3,4-dihydroxybenzaldehyde (3,4-dhb, 0.5, 0.75, 1, 2 mM), Salicylic acid (0.5, 0.75, 1, 2.5 mM), Vanillin (0.5, 0.75, 1, 2.5 mM), Zeatin (0.25, 0.5, 0.75, 1 mM), Kinetin (0.5, 0.75, 1, 2 mM), Betaine (5, 10, 25, 50 mM), Salicylaldehyde thiosemicarbazone (50, 100, 250, 500 μM), Sclareol (5, 10, 20, 40 μM), Gentiopicrin (250, 500, 750, 1000 μM), Gallic acid (25, 50, 75, 100 μM), Taurine (5, 10, 25, 50 mM), Allantoin (5, 10, 25, 50 mM), Syringic acid (0.5, 1, 2.5, 5 mM), Andrographolide (1, 5, 10, 20 μM), Sesamol (100, 250, 500, 1000 μM), Dexpanthenol (10, 25, 50, 100 mM), Ectoine (50, 100, 250, 300 mM), Hamamelitannin (25, 50, 75, 100 μM), Irisflorentin (10, 25, 50, 75 μM), Carnosine (1, 5, 10, 25 mM), Pyridoxine (0.5, 1, 5, 10 mM), Verbascoside (10, 25, 50, 75 μM) and Damascenone (1, 10, 25, 50 μM). The data is grouped in 5 groups including no compound (-) and the four concentrations indicated above for each compound. H4K16 and H3K9 acetylation levels were normalized to H4 and H3 respectively. For each drug all values are represented relative to the condition without compound. Student T-test were performed comparing these values with no compound conditions. *p<0.05, **p<0.01, ***p<0.001. www.aging-us.com 12326 AGING Supplementary Figure 2. Senescence assay results from cells treated with (A) Resveratrol or (B) Quercetin, including SA-β-Gal staining (phase-contrast image in the left column and bright-field image in the right column ) and relative HAS2 mRNA levels (n=3) compared with control conditions without UVB treatment in absence of shikimic acid. In the case of resveratrol, a quantification (n=3) of SA-β-Gal is also included. Student T-test, *p<0.05, **p<0.01, ***p<0.001. www.aging-us.com 12327 AGING Supplementary Figure 3. Senescence assay results from cells treated with (A) Dexpanthenol, (B) Andrographolide, (C) Sesamol or (D) 3,4- dihydroxybenzaldehyde, including SA-β-Gal staining (phase-contrast image in the left column and bright-field image in the right column) and relative HAS2 mRNA levels (n=3) compared with control conditions without UVB treatment in absence of shikimic acid. Student T-test, *p<0.05, **p<0.01, ***p<0.001. www.aging-us.com 12328 AGING Supplementary Figure 4. Senescence assay results from cells treated with or without Shikimic acid (10, 25 and 50 mM) plus (A) AGK2 2 μM or (B) OSS128167 200 μM, including SA-β-Gal staining (phase-contrast image in the left column and bright-field image in the right column) and relative HAS2 mRNA levels (n=3) compared with control conditions without UVB treatment in absence of shikimic acid. Student T-test, *p<0.05, **p<0.01, ***p<0.001. (C) mRNA levels of SIRT1, SIRT2, SIRT6 in the siRNA-mediated downregulation experiments shown in Figure 3E. Scr: Scramble siRNA. Student T-test, ***p<0.001. www.aging-us.com 12329 AGING Supplementary Figure 5. (A) Western Blot analysis of SIRT1, SIRT2, SIRT6 and SIRT7 protein levels in non-treated cells and cells treated with Shikimic acid (5, 10, 25 and 50 mM). (B) Relative mRNA levels and (C) relative protein levels quantification (n=3) of SIRT1, SIRT2, SIRT6 and SIRT7 in HDF treated with Shikimic acid at the indicated doses for 24h. (D) A representative Western-Blot of SIRT1 in vitro activity assay at the indicated doses of Shikimic acid and quantified in Figure 3H. FLAG-p53 was affinity purified with FLAG-resin and monitored with anti-FLAG anti body. The levels of H3K9 ac/H3, H4K16 ac /H4 and p53K382 ac/p53 are shown. Student T-test, *p<0.05, **p<0.01, ***p<0.001. www.aging-us.com 12330 AGING Supplementary Figure 6. Quantification (n=3) of the percentage of cells in each cell cycle phase and polyploidies in (A) non-irradiated cells treated with Shikimic acid at the indicated doses and (B) UVB-irradiated cells treated with Shikimic acid at the indicated doses, following the senescence induction method described in Figure 2A. A representative experiment of n=3 experiments is shown in Figure 4D. (C) Relative p21 mRNA levels (n=3) in UVB-irradiated cells treated with Shikimic acid at the indicated doses, following the senescence induction method described in Figure 2A. (D) Second (PC-2) and third (PC-3) principal components in the fingerprint region (1800–950 cm−1) from the samples mentioned in Figure 4G. Student T-test, *p<0.05, **p<0.01, ***p<0.001. www.aging-us.com 12331 AGING Supplementary Figure 7. (A) mRNA levels of BiP in the siRNA-mediated downregulation experiments shown in Figure 5C. Scr: Scramble siRNA. (B) non-irradiated A representative Western-Blot of LC3-I/II and Tubulin of the quantifications shown in Figure 5E. Student T-test, ***p<0.001. www.aging-us.com 12332 AGING Supplementary Tables Supplementary Table 1. List of primers for qPCR analysis. Gene Forward primer Reverse primer β-actin 5′-ACTGGAACGGTGAAGGTGACA-3′ 5′-ATGGCAAGGGACTTCCTGTAAC-3′ HAS2 Bio Rad Bio Rad IL-6 Bio Rad Bio Rad p16 Bio Rad Bio Rad p21 5′-CTGGAGACTCTCAGGGTCGAA-3′ 5′-CCAGGACTGCAGGCTTCC T-3′ SIRT1 5’-TGGGTACCGAGATAACCTTCT-3’ 5’-TGTTCGAGGATCTGTGCCAA-3’ SIRT2 Bio Rad Bio Rad SIRT6 5’-GCAGTCTTCCAGTGTGGTGT-3’ 5’-AAGGTGGTGTCGAACTTGGG-3’ SIRT7 5’-ACTTGGTCGTCTACACAGGC-3’ 5’-CAGCACTAACGCTTCTCCCT-3’ sXBP1 5’-CTGAGTCCGCAGCAGGTGCA-3’ 5’-GGTCCAAGTTGTCCAGAATGCCCAA-3’ uXBP1 5’-ACTCAGACTACGTGCACCTC-3’ 5’-GTCAATACCGCCAGAATCCA-3’ Supplementary Table 2. List of antibodies for western blot analysis. Antibody Dilution Purchase information Anti-H3K9ac (rabbit) 1:1000 Cell Signaling (#9649) Anti-H3 (rabbit) 1:10000 Cell Signaling (#9715) Anti-H4K16ac (rabbit) 1:1000 Millipore (#07-329) Anti-H4 (rabbit) 1:10000 Abcam (#ab10158) Anti-SIRT1 (mouse) 1:1000 Abcam (#ab7343) Anti-SIRT2 (rabbit) 1:1000 Abcam (#ab51023) Anti-SIRT6 (rabbit) 1:1000 Abcam (#ab62739) Anti-SIRT7 (rabbit) 1:1000 Cell Signaling (#5360S) Anti-Tubulin (mouse) 1:20000 Sigma-Aldrich (#T5168) Anti-LC3B (rabbit) 1:500 Cell Signaling (#2775S) Anti-p62 (rabbit) 1:1000 Abcam (#ab91526) Anti-BiP (rabbit) 1:1000 Cell Signaling (#3177S) Anti-IgG rabbit (donkey) 1:3000 Tebu bio (#611-7302) Anti-IgG mouse (goat) 1:10000 Sigma-Aldrich (#A9917) Anti-p53K382ac (rabbit) 1:1000 Abcam (ab75754) Anti-FLAG (rabbit) 1:1000 Sigma-Aldrich (#F7425) www.aging-us.com 12333 AGING