Research Paper Volume 13, Issue 9 pp 12308—12333

Shikimic acid protects skin cells from UV-induced senescence through activation of the NAD+-dependent deacetylase SIRT1

(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, *pF) 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

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.