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. 2013 Oct 2;8(10):e74799. doi: 10.1371/journal.pone.0074799

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© 2013 Kazantseva et al

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A) TAF4 down-regulation slows down adipogenesis in hMSCs. Oil-Red-O staining of lipid droplets of TAF4 or control siRNA treated and adipogenesis stimulated hMSCs at day 6 post-induction. siRNAs were transfected 24 h prior to the stimulation of differentiation of hMSCs. Scale bar, 40 µm (top left). Western blot analysis reveals reduced expression of adipogenic markers, namely PPARG and ADIPOQ at day 5 after induction of differentiation of TAF4 siRNA treated hMSCs along adipocyte pathway (right panel). Real-time PCR analysis of the expression of adipocyte markers in hMSCs at different time points of post-transfection and differentiation. The expression is normalized to control siRNA treatments (middle panel). RT-PCR analysis of TAF4 ASVs expression using TAF4_v1b specific primers in TAF4 or control siRNA transfected and towards adipogenesis stimulated hMSCs at day 5 post-transfection. GAPDH mRNA was analyzed for gel loading normalization (left bottom). (B) RNAi of hTAF4-TAFH inhibits osteogenic differentiation of hMSCs. Alkaline phosphatase staining of TAF4 or control siRNA exposed and osteogenesis stimulated hMSCs at day 6 post-induction. siRNAs were transfected 24 h prior to the stimulation of hMSC differentiation. Scale bar, 40 µm (top left). Western blot analysis reveals reduced expression of osteogenic markers RUNX2 and OPN (right panel). Real-time PCR analysis of the expression of osteogenic markers in hMSCs at different time points post-transfection and differentiation. The expression is normalized to negative control siRNA treatment (middle panel). RT-PCR analysis of TAF4 ASVs expression using TAF4_v1b specific primers in TAF4 or control siRNA treated and stimulated to osteogenic differentiation hMSCs at day 5 post-transfection. GAPDH mRNA was analyzed for gel loading normalization (left bottom). (C) RNAi of hTAF4-TAFH accelerates chondrogenic differentiation of hMSCs. Immunofluorescence staining analysis of TAF4 or control siRNA treated hMSCs upon chondrogenic stimulation at day 6 post-treatment reveals induced expression of COL2A1 and SOX9. siRNAs were transfected 24 h prior to stimulation of differentiation of hMSCs. Scale bar, 40 µm (top left). Western blot analysis shows increased expression of chondrogenic marker genes in TAF4 siRNA treated hMSCs. COL2A1 expression was upregulated at day 2 post-differentiation; SOX9 and MMP13 level of expression were increased at 8 days after siRNA treatments (right panel). Real-time PCR analysis of the expression of chondrogenic markers in hMSCs at different time points post-transfection and differentiation. The expression is normalized to control siRNA treatment (middle panel). RT-PCR analysis of TAF4 ASV expression using TAF4_v1b specific primers in TAF4 or control siRNA treated hMSCs upon stimulation to chondrogenic differentiation at day 5 post-transfection. GAPDH mRNA was analyzed for gel loading normalization (left bottom). Effects of RNAi of hTAF4-TAFH on activation of TP53^Ser15 were observed in each differentiation study using immunoblot analysis. Real-time PCR differences were found to be statistically significant with P<0.005.