Fig. 3.

SIRT7 regulates doxorubicin induced cell death via p53. a Huh7.5 cells were treated with doxorubicin (DOX) for various time. Protein levels were evaluated by western blot by using antibodies as indicated. b Immunofluorescence for p53 (red) in cells treated with doxorubicin as in A. c Huh7.5 cells were transfected with luciferase reporter plasmid (p53-Luci) containing p53-binding consensus sequence, luciferase activity was measured in the absence or presence of doxorubicin treatment at various of time points. Values with different superscripts are significantly different from each other (p < 0.05, one way ANOVA). d Huh7.5 cells were untreated (UT) or treated with doxorubicin for 12 h. ChIP assay was performed with anti-p53 antibody. *P < 0.05, **P < 0.01 vs UT, Student’s t-test. e RT-PCR analysis of p53 target gene mRNA levels in cells untreated (UT) or treated with doxorubicin. *P < 0.05, vs UT, one way ANOVA. f Huh7.5 cells were treated with doxorubicin for various of time or dose as indicated, p53 were immunoprecipitated from cell lysates and acetylation levels of p53 were evaluated by western blot. g and h Huh7.5 cells were treated with doxorubicin in the absence (UT) or presence DMSO (Con), Sirtinol (20 μM), C646 (25 μM) or RSV (100 μM) for 36 h, protein levels were evaluated by western blot (g) and cell death were measured by caspase3/7 activity (h). Values with different superscripts are significantly different from each other (p < 0.05, one way ANOVA). i Huh7.5 cells were treated with scramble (shTRC) or shRNA targeting p53 (shTP53) for 72 h, p53 levels were evaluated by western blot. j Cells in I were untreated (UT) or treated with doxorubicin for 36 h and cell death were evaluated by TUNEL assay. *P < 0.05 vs shTRC/DOX, Student’s t-test. k Cells in I were transfected with empty vector (EV), SIRT7 or inactive SIRT7 H187Y for 24 h followed by doxorubicin treatment for 36 h, Cell death were evaluated by TUNEL assay. **P < 0.01 vs EV, Student’s t-test. l Hep3B cells were transfected with empty vector (EV), SIRT7 or inactive SIRT7 H187Y for 24 h, cell were left untreated (Control) or treated with doxorubicin for 36 h. Cell death were evaluated by caspase3/7 activity and TUNEL assay. Values with different superscripts are significantly different from each other (p < 0.05, one way ANOVA). Data are representative of three independent experiments. Graphs show mean ± SEM of at least three independent experiments