Supplementary Figure 1 Analysis of the expressions of HDACs family members in heart, liver and lung from young and aged mice. (A-C) Expressions of HDACs were examined in heart (A), liver (B) and lung (C) from young mice compared with those from aged mice by qRT-PCR. (D) The proteins of HDAC9 and acetylation levels of H3K9 were examined in heart, liver and lung from young and aged mice. The data are presented as the means ± s.d. of each independent experiment performed in triplicate. *P < 0.05, **P < 0.01, ***P < 0.001.
Supplementary Figure 2 HDACs inhibitors treatment promoted osteogenic differentiation and inhibited adipogenic differentiation in BMMSCs. (A, B) BMMSCs were treated with different dose of HDAC9 inhibitors (TSA or NaB), and the expression of HDAC9 and the levels of acetylation of H3K9 were detected by western blotting. (C) Expressions of the senescence-related proteins p53 and p-p53 in BMMSCs cultured in vitro from aged mice were examined by western blotting. (D) Expressions of the senescence-related proteins p53 and p-p53 in BMMSCs cultured in vitro from young mice were examined by western blotting. Scale bars = 100 μm. The data are presented as the means ± s.d. of each independent experiment performed in triplicate.
Supplementary Figure 3 The silence efficiency of HDAC9. (A) The expression of HDAC9 mRNA were examined by qRT-PCR in aged BMMSCs after transfected with an HDAC9 siRNA for 48 hours. (B) Protein level of HDAC9 was examined by western blotting in aged BMMSCs after transfection with an HDAC9 siRNA for 48 hours. (C) The level of acetylation of H3K9 was examined by western blotting in aged BMMSCs after transfection with an HDAC9 siRNA for 48 hours. (D) HDAC9 mRNA expression was measured by qRT-PCR 3days and 7days after siHDAC9 transfection in aged BMMSCs. (E) HDAC9 protein expression was measured by western blotting 3days and 7days after siHDAC9 transfection in aged BMMSCs. The data are presented as the means ± s.d. of each independent experiment performed in triplicate. **P < 0.01.
Supplementary Figure 4 Inhibition of HDAC9 promoted osteogenic differentiation and inhibited adipogenic differentiation of BMMSCs from young mice. (A) Alizarin Red staining was performed and osteogenesis-related proteins were detected in young BMMSCs treated with an
HDAC9 siRNA. (B) Oil Red O staining were performed and adipogenesis-related proteins were performed in young BMMSCs treated with an HDAC9 siRNA. Scale bars = 100 μm. The data are presented as the means ± s.d. of each independent experiment performed in triplicate. *P < 0.05.
Supplementary Figure 5 Autophagy activity decreased in aged BMMSCs cultured in vitro compared with those cells from young mice. (A) Transmission electron microscopy (TEM) was used to detect autophagosomes in young and aged BMMSCs and cells treated with chloroquine (CQ). Scale bars = 1 μm. (B) LC3 was measured by immunofluorescence staining in young and aged BMMSCs, and cells treated with CQ. Scale bars = 50 μm. (C) Autophagy related proteins were detected in young and aged BMMSCs, and cells treated with CQ by western blotting. The data are presented as the means ± s.d. of each independent experiment performed in triplicate. *P < 0.05, *** P < 0.001.
Supplementary Figure 6 HDAC9 siRNA treatment decreased the levels of acetylation of H3K9 in aged BMMSCs cultured in vitro. To evaluate the effect of HDAC9 removing acetyl groups from H3K9, the expression of HDAC9 and the levels of acetylation of H3K9 were examined by western blotting in aged BMMSCs transfected with an HDAC9 siRNA and those cells treated with CQ. The data are presented as the means ± s.d. of each independent experiment performed in triplicate.
Supplementary Figure 7 The chromatin immunoprecipitation (ChIP) assay was performed to examine the binding of HDAC9 to the promoters of autophagy-related genes. (A, B) Chromatin were isolated from young and aged BMMSCs, and incubated with HDAC9 antibody, acetylated-histone H3K9 (H3K9ac) antibody and IgG (as a negative control). Lane 1, immunoprecipitation with IgG. Lane 2, immunoprecipitation with specific HDAC9 (A) or H3K9ac (B) antibody. The data are presented as the means ± s.d. of each independent experiment performed in triplicate. *P < 0.05, **P < 0.01, ***P < 0.001.
Supplementary Figure 8 The silence efficiency of BECN1 in aged BMMSCs cultured in vitro. Protein expression of Beclin1 was examined by western blotting in aged BMMSCs transfected with a BECN1 siRNA 48 hours later. The data are presented as the means ± s.d. of each independent experiment performed in triplicate.