Fig 2: ARN3236 and ARN3261 decrease phosphorylation of HDAC4/5/7.Phosphorylation level of HDAC4/5/7. Twenty ovarian cancer and 2 TNBC cell lines were treated with ARN3236 (4 µM) or ARN3261 (4 µM) for 24 hours. Representative image is from 1 independent experiment. Experiments were repeated twice with similar results.

Fig 3: LMK-235 inhibits ESCC cells proliferation. (A) Growth curves of the five ESCC cell lines treated with LMK-235 for 48 hr. Absorbance at OD450 nm are normalized to vehicle control (0.1% DMSO; n = 5 wells/dose point). IC50 of KYSE150 is marked as the red point. (B) EdU incorporation assay of KYSE150 treated with LMK-235 (0.824 μM) for 48 hr. Scale bar = 50 μm. (C) The expressions of H3K27ac, H3K14ac, H3K9ac, HDAC4, and HDAC5 are determined by western blot in KYSE150 treated as (B). Histone H3 is used as the loading control. (D, E). Cell cycle distribution (D) and the percentage of Annexin V+ cells (E) of KYSE150 treated as (B), analyzed by flow cytometry. (A–E) Error bar denotes SEM of three replicates.

Fig 4: 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)

Fig 5: ZIP4 upregulated Class IIa HDACs. (A,B). ZIP4-OE up regulated the expression of HDAC4, 5, and 7; ZIP4-KO and NOTCH3-KO downregulated the expression HDAC4, 5, and 7 in PE04 cells, with little or no effect on other classes of HDACs. (C). Summary of quantified Western blot results from > three independent experiments for ZIP4, HDAC4, 5, and 7. (D). PEA2 cells expressed relatively higher levels of HDAC4, 5, and 7 than PEA1 cells; ZIP4-KD reduced expression levels of HDAC4, 5, and 7 in PEA2 cells. (E). Summary of quantified Western analyses results from > three independent experiments. * p < 0.05; ** p < 0, 01; *** p < 0.001.