Figure 5

ICG-001 suppresses TGF-β1-mediated myofibroblast differentiation of LR-MSCs by a Smad-independent mechanism. (A–D) Human LR-MSCs were treated with ICG-001 at various concentrations in the presence or absence of hTGF-β1 (10 ng/ml) for 24 h. (A) The protein levels of α-SMA, collagen I and fibronectin were examined by Western blot. Representative gel electrophoresis bands are shown, and the expression levels of proteins were quantified by densitometry and normalized to the expression of β-actin. Densitometry data are shown as mean ± SD. *P < 0.05 versus hTGF-β1 alone. (B) The expression of α-SMA and fibronectin was detected by immunofluorescence assay. Nuclei were stained with DAPI (blue). (C) Changes of α-SMA and COL1α2 transcripts were analyzed by Q-PCR. Results are shown as mean ± SD. *P < 0.05 versus hTGF-β1 alone. (D) The expression of p-Smad2, p-Smad3 and Smad2/3 was determined using Western blot. Representative gel electrophoresis bands are shown, and the expression levels of proteins were quantified by densitometry and normalized to the expression of smad2/3. Densitometry data are shown as mean ± SD. (E,F) Mouse LR-MSCs were pretreated with ICG-001 at 5 μM for 1 h, followed by the addition of TGF-β1 into the culture medium to a final concentration of 10 ng/ml and incubation for 24 h. (E) The protein levels of α-SMA, collagen I and fibronectin were examined by Western blot. Representative gel electrophoresis bands are shown, and the expression levels of proteins were quantified by densitometry and normalized to the expression of β-actin. Densitometry data are shown as mean ± SD. *P < 0.05 versus TGF-β1 alone. (F) The expression of α-SMA and collagen I was assessed by immunofluorescence assay. Nuclei were stained with DAPI (blue).