Figure 1.
Effects of NaB on MSC proliferation and differentiation.
(A) MSCs were harvested at 24, 48 and 72 h after the addtion of NaB and the proliferation of the treated MSCs was measured with a CCK-8 assay at the indicated time points. OD, optical density. *, P<0.05, ***, P<0.001 vs. the OD value of MSCs treated with 0 mmol/L NaB at each time points. (B) Relative expressions of the SMC specific genes (α-SMA, calponin and SM-MHC) in MSCs treated with the indicated concentration of NaB for 48 h. β-actin was used as the internal standard. *, P<0.05, **, P<0.01, ***, P<0.001 relative to 0 mmol/L NaB treated MSCs.
Figure 2.
Immunofluorescence analysis of MSC specific protein expression in NaB-treated MSCs.
MSCs were treated with 1 mmol/L NaB for 48 h, stained with FITC-conjugated anti-α-SMA, calponin or SM-MHC antibodies, and observed under a fluorescence microscope. The untreated MSCs were used as negative control and the primary SMCs were used as positive control. The isotype antibody was used as a background control. DAPI was used to stain the nuclei. Scale bar = 25 µm.
Figure 3.
NaB induces SMC specific gene expression in MSCs co-cultured with SMCs.
MSCs were co-cultured with SMCs in a transwell chamber with SMCs in the insert chamber and MSCs in the lower chamber. The MSCs were pretreated with 0, 0.5, 1.0 and 1.5 mM NaB before co-culturing. The MSCs in the co-culture system were harvested, and the expression of the SMC specific genes α-SMA, calponin and SM-MHC was determined by quantitative real-time RT-PCR (A) and Western blot (B). *, P<0.05, **, P<0.01 vs. all other time points in the same NaB concentration group; ▴, P<0.01 vs. all other time points in all NaB concentration groups. (C) The co-cultured MSCs were stimulated with 1 mmol/L NaB for 48 h, stained with FITC-conjugated anti-α-SMA, calponin or SM-MHC antibodies, and observed under a fluorescence microscope. DAPI was used to stain the cell nuclei. The isotype antibody was used as a background control. Scale bar = 25 µm.
Figure 4.
Histone acetylation modifications in co-cultured MSCs treated with NaB.
MSCs were co-cultured with SMCs in a transwell chamber for 48 h. The MSCs were pretreated with 1.0 mM NaB before co-culturing. The MSCs in the co-culture system were harvested, and the genomic DNA was isolated and sonicated for a ChIP assay with antibodies against acetyl-histone H3K9, acetyl-histone H4 and normal rat IgG. The specific DNA fragments retrieved in the pull-down were further used for qPCR assays. The qPCR primers were designed to target the promoter of each gene. Values were given as folds of enrichment relative to the IgG control. Data are expressed as the mean ± SD of three biological replicates.*, P<0.05, **P<0.01, ***P<0.001 compared to the untreated MSCs. #, P<0.05, ##P<0.01, ###P<0.001 compared to the co-cultured MSCs.
Figure 5.
Effects of NaB on HDAC1/2 expression and recruitment in MSCs.
(A) Immunofluorescence analysis of HDAC1 and HDAC2 expression in MSCs treated with NaB (1 mM for 48 h). (B) Western blot assay to determine the expression of HDAC1 and HDAC2 in MSCs treated with 1 mM NaB for 48 h. (C) ChIP-qPCR assay to determine the recruitment of HDAC1 and HDAC2 to the α-SMA, calponin and SM-MHC promoters in MSCs treated with 1 mM NaB for 48 h. The ChIP assay was conducted with ChIP grade anti-HDAC1, HDAC2 and normal rat IgG antibodies, which were incubated with the sonicated supernatants of MSCs treated with 1 mM NaB. The isolated DNA fragments were analyzed by qPCR to determine the presence of the promoter regions of the α-SMA, calponin and SM-MHC genes. Values were given as fold changes normalized with normal rat IgG control. **, P<0.01 compared to the untreated MSCs.
Figure 6.
Measurement of intracellular calcium levels.
(A) The traces show the carbachol- (10 mM) and KCl (100 mM)-induced Ca2+ response in MSCs, SMCs, MSCs treated with 1 mM NaB for 48 h, and NaB-pretreated (1 mM for 48 h) MSCs co-cultured with SMCs for 3 d. F/F0 denotes the relative change in Fluo-8 intensity, F0 is the baseline average and F is the absolute fluorescence value in an area of interest during treatment. Forty-five percent means that 45 of one hundred cells exhibited a response. (B) The amplitude of the Ca2+ peak in response to carbachol (10 mM) or KCl (100 mM) in MSCs, SMCs, MSCs treated with NaB, and NaB-pretreated MSCs co-cultured with SMCs. *P<0.05 vs. MSCs, NaB+MSCs and co-cultured MSCs; **P<0.01 vs. MSCs; ▴P<0.01 vs. all other gourps.