Figure 4
HDAC1 inhibition promoted CMS-induced osteogenic differentiation in human BMSCs. (a) qRT-PCR analysis and (b, c) western blotting analysis of JAG1, ALP, COL1a1, and OCN in BMSCs after transfected with specific siRNA-JAG1 or its corresponding scrambled control (siRNA-NC) under 10% CMS for 3 weeks. Two specific siRNAs for JAG1 were used, and then after the confirmation of knockdown efficiency, siRNA1 was used for the subsequent experiments. (d) Alizarin red staining of BMSCs after transfected with specific siRNA-JAG1 or its corresponding scrambled control or JAG1-overexpressing vector under 10% CMS for 3 weeks. (e) qRT-PCR analysis of HDAC1, ALP, COL1a1, and OCN in BMSCs after transfected with specific siRNA-HDAC1 or its corresponding scrambled control (siRNA-NC) under 10% CMS for 3 weeks. Two specific siRNAs for HDAC1 were used, and then after the confirmation of knockdown efficiency, siRNA1 was used for the subsequent experiments. (f) The knockdown efficiency of HDAC1-specific siRNA (siRNA-HDAC1) was confirmed by comparison to a scrambled control siRNA (siRNA-NC). (g) Representative images of ALP staining (including quantitative analysis) of BMSCs after transfected with specific siRNA-HDAC1 or its corresponding scrambled control or HDAC1-overexpressing vector under 10% CMS for 3 weeks. (h) qRT-PCR analysis of JAG1, HES1, and HEY1 mRNA levels and (i) western blotting analysis of BMSCs after transfected with specific siRNA1-HDAC1 under 10% CMS for 3 weeks. (j) Alizarin red staining of BMSCs after co-transfected with specific siRNA-HDAC1 with inhibitor of Notch signaling transduction (10 nM RO4929097) under 10% CMS for 3 weeks. GAPDH was used as an internal control. All results are representative of at least three independent experiments. All the staining data were confirmed by three repeated tests. Data were mean±S.D., *P<0.01. All P-values are based on Student's t-test
