Fig 2: The KLF5 repressive complex on TP53 is disrupted by stress.A Even in the presence of KLF5, p53 was induced by increasing UV stress in primary human esophageal keratinocytes, as shown by Western blot. β-actin served as a loading control. Western blots are representative of three independent biological experiments. B Both KLF5 and p53 were induced following exposure to 60 mj/cm2 of UV in primary human esophageal keratinocytes. β-actin served as a loading control. Western blots are representative of three independent biological experiments. C Apoptosis, assessed by flow cytometry with annexin V at different time points after 60 mj/cm2 of UV, was increased following KLF5 knockdown with shRNA in pTRIPZ-puro by doxycycline induction. D KLF5 knockdown with shRNA in pTRIPZ-puro also decreased cell viability, as assessed by trypan blue exclusion. E On Western blot, SIN3A levels decreased somewhat with UV stress, while HDAC2 levels were unchanged. Western blots are representative of three independent biological experiments. F By co-immunoprecipitation, SIN3A binding to KLF5 was abolished by UV stress, even as HDAC2 is still bound to KLF5. Western blots are representative of two independent biological experiments. G p53 protein levels increased on Western blot upon knockdown of HDAC2 with siRNA. β-actin served as a loading control. Western blots are representative of two independent biological experiments. H p53, as well as KLF5, was induced in primary human esophageal keratinocytes treated with the HDAC inhibitor trichostatin A (TSA). Western blots are representative of three independent biological experiments. I KLF5 acetylation at K369 increased with stress, as assessed by Western blot with an acetylation-specific KLF5 antibody. Western blots are representative of two independent biological experiments. J Plasmids containing KLF5-WTor KLF5-K369R, which incorporates a lysine to arginine mutation at amino acid 369 of KLF5, were utilized for experiments. K As demonstrated by co-immunoprecipitation following transfection in HEK293 cells, which have low levels of endogenous KLF5, the binding of SIN3A and KLF5 was not disrupted with KLF5-K369R.

Fig 3: Sin3A‐sensitive IL‐2 upregulation limits the differentiation of IL‐17A+ T cells A, B Cells were cultured as depicted in Fig 2 and then stimulated with PMA and Ionomycin for 4 h. A. Representative contour plots and B. percentages of IL‐2+ cells, N = 7.C RT‐PCR analysis of Il2 expression, N = 7.D, E Cells were cultured as depicted in Fig 2 in the absence or the presence of a neutralizing anti‐IL‐2 mAb. D. Bar plot depicting the frequency of Foxp3+, N = 5 and E. relative IL‐17A+ cells, N = 10.F Relative increase in IL‐17A+ Sin3A‐sufficient and ‐deficient cells with respect to the dose of anti‐IL‐2 mAb, N = 3.G Correlation of the relative increase in IL‐17A+ cells in the presence of anti‐IL‐2 antibody and the percentage of IL‐2+ cells in TAM‐treated cells. Data information: In (B–E) data are presented as mean (Bars) ± SD. Two‐tailed paired Student's t‐test. N = 9, 7, 5, 10 cultures per condition (NT or TAM) derived from individual mice, as indicated. In (F) each dot represents the mean ± SD of three independent determinations. Two‐tailed paired Student's t‐test.

Fig 4: CD4‐cre‐driven Sin3A deletion halts further CD4 and CD8 thymocyte development A, B Representative flow cytometry analysis of CD4+ and CD8+ thymocytes (A), and frequencies of DN, DP and SP cells in Sin3A+/+ and Sin3ACD4Δ mice (B). N = 5.C Sin3A intracellular levels in DN, DP, CD8SP, and CD4SP cells from Sin3A+/+ and Sin3ACD4Δ mice.D, E Representative FACS of splenic CD4+ and CD8+ cells (D) and frequencies (E). N = 6.F, G Representative dot plots of CD44 and CD62L levels of gated CD4+ viable T cells (F) and quantification (G). N = 6.H Representative dot plots of CD44 and Sin3A levels in Sin3A+/+ and Sin3ACD4Δ CD4+ T cells. Data information: In (B, E, G), data are presented as mean (Bars) ± SD. Two‐tailed unpaired Student's t‐test. Dots depict individual mice analyzed in independent experiments.

Fig 5: Sin3A levels are increased over the course of activation and differentiation A Representative flow cytometric analysis of Sin3A expression in wild‐type CD4+ T cells kept for 3 days in IL‐7 or differentiated to Th0, Th1, Th17 and Treg.B Kinetics of Sin3A upregulation in Th17 conditions.C Graphic representation of ex‐vivo deletion of Sin3A. CD4+ T cells were purified from secondary lymphoid organs derived from Sin3AF/F Rosa26‐CreERT2+/− mice, and kept in IL‐7 for 6 days in in the absence or the presence of 4‐hydroxy tamoxifen (TAM). Cells were then harvested and cultured for an additional 3 days in Th17 skewing conditions in the absence or the presence of TAM.D, E (D) Flow cytometric and (E) Western blot analyses of Sin3A levels. Data are also depicted in Fig 5B as part of the same source file. Source data are available online for this figure.