Fig. 3

HDAC5 is indispensable for deacetylation of SOX9 in MCF-7 TAMR cells. a Western blot of HDAC5 protein from shNC and shHDAC5 TAMR MCF-7 cells. b Western blot of SOX9 protein in cytoplasmic and nuclear fractions from shNC and shHDAC5 MCF-7 cells. c Representative confocal images of immunofluorescence for SOX9 locations in shNC and shHDAC5 TAMR MCF-7 cells. d Immunoprecipitation in shNC and shHDAC5 MCF-7 cells with anti-SOX9 followed by immunoblotting with an antibody against acetylation proteins. e MTT assay of growth rates in shNC or shHDAC5 TAMR MCF-7 cells. f MTT assay of growth rates of TAMR MCF-7 cells treated with DMSO or HDAC4/HDAC5 inhibitor LMK-235 (12 nM). g Flow cytometry of ALDH⁺ cells following HDAC5 knocking down in TAMR MCF-7 cells. h Western blot analysis for CD44, ALDH1A1 and SOX2 proteins in TAMR MCF-7 cells with shNC and shHDAC5. i MTT assay of growth rates of MCF-7 transfected with HDAC5 or vector control and treated with tamoxifen (1 μM). j Bioinformatics analysis of HDAC5 expression in the public dataset (GEO: GSE9574) of healthy control and patients with ER⁺ breast cancer (control n = 14, breast cancer n = 14). k Cox regression to quantify hazard ratio (HR) and the corresponding confidence interval (CI) between overall survival (OS) and SOX9, HDAC5 expression in human breast cancer tissues (N = 1178, HR = 0.91, 95% CI: 0.82–1.0, P = 0.0409). Data are representative of means ± SEM of three independent experiments (unpaired t-test, *p < 0.05, **p < 0.01)