Fig. 4: P38MAPK-dependent HDAC3 increase negatively regulates KLF5 expression in PV-IgG-treated keratinocytes.
From: Unbiased screening identifies regulators of cell-cell adhesion and treatment options in pemphigus
a Western blot analysis of HaCaT cells which were treated with Ctrl-IgG or PV-IgG for 24 h. Antibodies against KLF5, KLF5ac, and GAPDH were used. Representative Western blot images and quantifications of respective proteins (n = 6) are shown. Values were normalized to Ctrl-IgG. b Quantitative real-time PCR of mRNA extracted from HaCaT cells treated with IgG or PV-IgG. Four different primer pairs detecting KLF5 mRNA were used. Values were normalized to IgG control (n = 4, KLF5_1 p = 0.0334, KLF5_2 p = 0.0340, KLF5_3 p = 0.0301, KLF5_4 p = 0.0344). c Quantitative real-time PCR analysis of mRNA extracted from HaCaT cells stably transduced with sgNT or sgHDAC3. Primers detecting KLF5 mRNA were used. Values were normalized to sgNT (n = 6, KLF5 p = 0.0489) d, e Western Blot analysis of HaCaT cells stably transfected with sgHDAC3 treated with IgG or PV-IgG using DSG3, KLF5 and GAPDH antibodies. Values were normalized to Ctrl-IgG. Representative images are shown (n = 5). f ChIP-quantitative real-time PCR analysis of HaCaT cells using HDAC3 antibodies and primers detecting GAPDH, DSG3, and KLF5 promoters. (n = 5, GAPDH vs KLF5 p = 0.0019, DSG3 vs KLF5 p = 0.0028). g ChIP-quantitative real-time PCR analysis of HaCaT cells treated for 24 h with IgG or PV-IgG using HDAC3 antibodies and primers to detect the KLF5 promoter (n = 3, Ctrl-IgG vs PV-IgG, p = 0.0245). h Western blot analysis of HaCaT cell lysates treated with PV-IgG and DMSO or SB202190 using DSG3, KLF5, HDAC3, and GAPDH antibodies. Values were normalized to DSMO (n = 6, DSG3 p = 0.1276, KLF5 p = 0.039). Representative images are shown. Values expressed as mean with standard deviation (mean ± SD). One n represents one biological replicate. Source data are provided as a Source Data file. a–e, h were analyzed with two-sided one-sample t-tests. f was analyzed with with One-way-ANOVA, SIDAK correction. g was analyzed with two-sided Students t-test. p < 0.05*.
