Interleukin 21 Receptor Affects Adipogenesis of Human Adipose-Derived Stem/Stromal Cells

Figure 3

Silencing of the IL21R gene in ADSCs. Cells were transfected with siRNA of IL21R and a siRNA scramble as a control. (a) Relative quantification of IL21R RNA normalized by GAPDH using qRT-PCR after 24-hour transfection () (TAL 22, 27, and 32). (b) Western blot analysis to verify IL21R protein levels () (TAL22, 27, 32, and 36). Protein levels of GAPDH were used as a control in the load, representative images of the donor TAL22. (c) Mitochondrial probe JC-10 to measure mitochondrial membrane potential () in ADSCs with gene silencing using IL21R siRNA and scramble as control () (TAL23, 27, and 32). The JC-10 fluorescence ratio at basal () and uncoupling states () was used to measure . (d, e) Rhodamine 123 assay to measure after silencing of IL21R () (TAL23, 27, and 32). (d) Representative histogram with mitochondrial activity analysis. Cells without rhodamine (red curve) were used as the negative control. (e) Quantification of the relative fluorescence intensity means of rhodamine-stained cells. (f, g) Proliferation analysis using EdU incorporation in ADSCs after siRNA silencing () (TAL22, 28, and 32). (f) Representative histogram with proliferation analysis using EdU incorporation. Cells without EdU (red curve) were used as a negative control. (g) Percentage of EdU-positive cells. Mean with SEM; Student’s unpaired -test analysis: , ns: not significant.

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