Fig 2: HDAC1 knockdown reduces expression of key stemness and cell fate factors.(A) Immunoblots showing increase in H3K4/19ac and H3K27ac after HDAC1 silencing in BT145 and BT187 (shH1_A, shHDAC1_A; shH1B, shHDAC1_B) (n = 3). (B) Representative immunoblots of p53-WT (BT145), p53-mutant (BT187) and p53-WT cells overexpressing p53-DN (BT145 + p53-DN) hGSCs after acute silencing of HDAC1 probed for various markers (n = 3). Black arrow indicates expression of WT EGFR. (C–E) Quantification of expression of proteins (normalized to Vinculin) after HDAC1 knockdown from immunoblots using p53-WT (C), p53-mutant (D), and p53-WT cells overexpressing p53-DN (E). For each cell line, the data are compiled from at least 3 independent experiments for each shRNA. Error bars indicate SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. P values were determined using the 2-way ANOVA with Tukey’s multiple comparisons test.

Fig 3: Impaired transcription of p21 downstream of p53 stabilization upon IR in mutant cells.(A) Western Blot analysis of lysates from wildtype (+/Y) and Tspyl2 m/Y mutant (m/Y) MEFs collected at indicated time points post 10 Gy IR to acetyl-p53 at Lys379, total p53 and p21. Actin served as a loading control. (B) qPCR analysis in MEFs (left panel) and thymocytes (right panel) after IR at 10 Gy. Transcription levels of indicated checkpoint genes were normalized by Hprt. The fold induction of individual gene is expressed as mean value ± SEM when compared to its untreated control (n = 5 per group). Data points showing asterisk indicate statistical significance between wildtype and mutants as revealed by Bonferroni post-test after 2-way ANOVA (*: p<0.05, **: p<0.01).

Fig 4: Accumulation of polyUb-p53 during Cd exposure is independent of proteasomes.a–d Cells were treated with 23 µM Cd for 6 h and 12 h. Immunoblotting was performed on lysates for Ub, and 600 µg of remaining protein was used for IP analysis with a p53 antibody and mouse IgG and then immunoblotted for Ub (a, b), and vice versa (c, d). e, f MES13 cells were pretreated with increasing concentrations of MG132 (0.5–5 µM) for 2 h and then treated with Cd (23 µM) for 12 h, harvested, lysed, and immunoblotted for the indicated proteins. ß-Actin was used as the loading control. The levels of HMW-p53 and monomer-p53 were quantified by densitometry and normalized to those of ß-actin. The data are presented as the mean ± SD (n = 3). *P < 0.05; **P < 0.005; ***P < 0.0005.

Fig 5: NED416 increases Sirt1 expression and activity in skin cells. (A) NED416 significantly enhanced the enzymatic activity of Sirt1 in a concentration-dependent manner. (B) Acetylation of p53 after NED416 treatment. (C) NHDF cells treated with different concentrations of NED416 for 24 h showed a dose-dependent increase in Sirt1 expression in western blot analysis. (D) Quantification of Sirt1 expression in NHDF cells following NED416 treatment. (E) HaCaT cells treated with different concentrations of NED416 for 24 h exhibited a dose-dependent increase in the expression of Sirt1 in western blot analysis. (F) Quantification of Sirt1 expression in HaCaT cells following NED416 treatment. Data are presented as the mean ± SEM. *P<0.05, ***P<0.001 vs. 0 µM. NED416, (E)-3-(2,4-dichlorophenyl)-N-phenylacrylamide; Sirt1, sirtuin 1; NHDF, normal human dermal fibroblasts; resv., resveratrol.