Research Paper Volume 13, Issue 23 pp 25055—25071

Figure 2. IGFBP-6 induces CAF differentiation in mesenchymal stem cells. (A) HS5 cells exposed to 200 ng/mL of IGFBP-6 for 24h and 48h were lysed and subjected to immunoblotting using specific antibodies against α-SMA, FAP1, and TGF-β. Protein content was normalized to the housekeeping protein β-actin. The entire assay was made in triplicate, a representative one is shown. Signals from immunodetected bands were semi-quantified by densitometry. (B–D) Statistical analysis of data revealed that α-SMA expression was significantly increased in the HS5 cells IGFBP-6- induced for 24h and 48h (B), FAP1 expression was significantly increased in the HS5 cells IGFBP-6- induced for 24h (C) TGF-β expression was significantly increased in the HS5 cells IGFBP-6- induced for 24h and 48h (D). Data are presented as means ± sem. **p < 0.01 and ***p < 0.001 vs. untreated. (E) qPCR results were obtained for TGF-β in HS5 cells exposed to 200 ng/mL of IGFBP-6 for 24h and 48h. Relative mRNA expression level normalized with β-actin by using a comparative 2-ΔΔCt method. **p < 0.01.