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. 2015 Dec 31;10(12):e0146124. doi: 10.1371/journal.pone.0146124

Fig 5. shRNA-mediated downregulation of either ALK5 or ALK1 inhibits TGFβ-induced chondrogenic differentiation of BMSCs.

Fig 5

Human fetal BMSCs (donor F1) were transduced with lentiviral ALK5-shRNA, ALK1-shRNA or empty vector as control (Ctrl vector) and pellet-cultured in chondrogenic medium containing TGFβ. Short hairpin RNA-mediated downregulation of ALK5 and ALK1 was determined by gene expression of TGFBR1 (A) and ACVRL1 (B) in pellets cultured for 1 or 7 days. The effect of ALK5-shRNA and ALK1-shRNA on TGFβ-induced chondrogenesis was evaluated by transcript analysis of ACAN (C) and COL2A1 (D) and by staining proteoglycans with Safranin O/Fast Green (E; upper panel) and collagen type II with immunohistochemistry (E; lower panel) in pellets cultured for 7 days. Representative images of consecutive pellet sections per condition are shown and the scale bar represents 500 μm. Gene expression was normalized to reference gene RPS27a and data are expressed as % relative to normalized mRNA levels in control vector-transduced pellets stimulated with TGFβ. Bars represent mean ± SD of quadruplet pellets from 1 representative experiment (out of 3), ns = not significant; **p<0.01; ***p<0.001.