Fig. 3

HDAC9 regulated the autophagy in BMMSCs by directly binding to the promoters of autophagy-related genes. To evaluate the role of HDAC9 in regulating autophagy, young and aged BMMSCs cultured in vitro and aged BMMSCs were transfected with Nc siRNA or HDAC9 siRNA. a LC3 was measured by immunofluorescence staining in young and aged BMMSCs, and those cells treated with CQ. Scale bars = 50 μm. b Autophagy-related proteins were detected in young and aged BMMSCs, and those cells treated with CQ by western blotting. c LC3 was measured by immunofluorescence staining in aged BMMSCs transfected with Nc siRNA or HDAC9 siRNA, and those cells treated with CQ. Scale bar = 50 μm. d Autophagy-related proteins were detected in aged BMMSCs transfected with Nc siRNA or HDAC9 siRNA, and those cells treated with CQ by western blotting. e, f The chromatin immunoprecipitation (ChIP) assay was performed to investigate whether HDAC9 could bind with the promoters of autophagy-related genes. Chromatin was isolated from young and aged BMMSCs (e) and aged BMMSCs transfected with Nc siRNA or HDAC9 siRNA (f), and incubated with HDAC9, acetylated-histone H3K9 (H3K9ac), and IgG antibodies. An IgG antibody was used as a negative control. Immunoprecipitation with specific HDAC9 or H3K9ac antibody. Data are presented as the mean ± SD of triplicate samples. *P < 0.05, **P < 0.01, ***P < 0.001. a, c One-way analysis of variance (ANOVA). e, f Unpaired two-tailed Student’s t test