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sc-12-0871_sm_SupplFigure1.TIF211KSupplemental Figure 1. c-Cbl regulates STAT5 ubiquitination in murine MSC. (A) representative Western blot and quantification of multiple blots from separate experiments (B) showing the effects of sh-c-Cbl1 and sh-c-Cbl2 on c-Cbl and Cbl-b protein levels in murine C3H10T1/2 cells compared to empty vector (EV). *P<0.05 vs EV. (C) Cell lysates from C3H10T1/2 cells transduced with sh-c-Cbl1, sh-c-Cbl2 or the empty vector were immunoprecipitated with anti-STAT5 antibody and immuno-precipitates were analysed by immunoblotting (IB) with STAT5 or ubiquitin antibodies (C). Total lysates and IgG immunoprecipitates were used as controls.
sc-12-0871_sm_SupplFigure2.TIF83KSupplemental Figure 2. c-Cbl silencing does not modulate total STAT3 in human MSC. Representative Western blot (A) and quantification of multiple blots (B) showing that c-Cbl silencing in human F/STRO-1+A cells slightly increased phospho-STAT3 but had no effect on total STAT3 protein levels. *P<0.05 vs sh-Control.
sc-12-0871_sm_SupplFigure3.TIF168KSupplemental Figure 3. c-Cbl regulates STAT5 activity in human MSC. STAT5 RE-LUC reporter assay in F/STRO-1+A cells (A) showing the effect of c-Cbl silencing on STAT5 transcriptional activity in the presence or absence of the STAT5 inhibitor (75 μM). Cell lysates from F/STRO-1+A cells transduced with sh-c-Cbl RNA or sh-Control and cultured in the presence or absence of the STAT5 inhibitor (75 μM) were immuno-precipitated (IP) with anti-RUNX2 antibody and immuno-precipitates were analysed by immuno-blotting (IB) with STAT5 antibody. Total lysates and IgG immuno-precipitates were used as controls. a: P<0.05 vs the respective sh-Control; b: P<0.05 vs corresponding untreated cells.
sc-12-0871_sm_SupplFigure4.TIF235KSupplemental Figure 4. Functional role of STAT5 in osteogenic differentiation induced by c-Cbl silencing in murine MSC. (A) Effect of the STAT5 inhibitor (75 μM) on alkaline phosphatase (ALP) staining (A), matrix mineralization (B) and osteoblast gene expression (C) induced by sh-c-Cbl1 and sh-c-Cbl2 in murine C3H10T1/2 cells. a: P<0.05 vs EV; b: P<0.05 vs corresponding untreated cells.
sc-12-0871_sm_SupplFigure5.TIF92KSupplemental Figure 5. Effect of STAT5 inhibition on phenotypic gene expression in human MSC. Effect of the STAT5 inhibitor (75 μM) on the expression of late osteoblast marker genes (A), phenotypic chondrocyte genes (B) and adipocyte genes (C) and in c-Cbl silenced human F/STRO+A cells. a: P<0.05 vs sh-Control; b: P<0.05 vs treated sh-Control.
sc-12-0871_sm_SupplFigure6.TIF94KSupplemental Figure 6. IGF-1 neutralization abrogates osteoblast gene expression induced by c-Cbl silencing in human MSC. Effect of IGF-1 neutralizing antibody (Ab) on osteoblast gene expression induced by c-Cbl silencing in human F/STRO+A cells. (A). Effect of STAT5 inhibitor (75 μM) and IGF-1 neutralizing antibody on the expression of the Wntresponsive gene c-Myc induced by c-Cbl silencing in human F/STRO+A cells (B). a P<0.05 vs sh-Control; b P<0.05 vs corresponding untreated cells.
sc-12-0871_sm_SupplTable1.pdf21KSupplemental Table 1
sc-12-0871_sm_SupplTable2.pdf68KSupplemental Table 2

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