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. 2017 May 11;8:479. doi: 10.3389/fimmu.2017.00479

Figure 7.

Figure 7

TAK1 plays divergent roles in regulating TNFα + TGFβ1 cooperatively-induced CCL2/CXCL8 and Cox-2 expression in MSCs. (A) Human BM-derived MSCs were stimulated with vehicle (“--”), TNFα (50 ng/ml), TGFβ1 (10 ng/ml), or TNFα + TGFβ1 (same concentrations) for 10 min (FBS-free medium). Tα + Tβ = TNFα + TGFβ1. TAK1 expression was determined by WB; GAPDH was used as a loading control. The findings are representatives of n = 3 independent experiments, performed with MSCs of three different donors, which have shown similar results. (B,C) The effects of TAK1 downregulation on MSC signaling and functions. siRNA concentrations were determined by preliminary titration analysis (data not shown). (B) Signaling: human BM-derived MSCs were transiently transfected with control siRNA (“CTRL”, 50 nM) or with siRNA to TAK1 (50 nM; efficacy of TAK1 downregulation is demonstrated in Figure S6A1 in Supplementary Material). Following siRNA transfection, the cells were stimulated with vehicle (“--”) or with TNFα (50 ng/ml) + TGFβ1 (10 ng/ml) for 10 min (0.5% FBS-containing medium). Tα + Tβ = TNFα + TGFβ1. (B1) The expression levels of IκBα were determined by WB; GADPH was used as loading control. (B2) p65 and Smad3 activation levels were determined as described in Figure 5. p38 activation levels are demonstrated in Figure S6A2 in Supplementary Material. (C) Function: human BM-derived MSCs were transiently transfected with control siRNA (“CTRL”, 50 nM) or with siRNA to TAK1 (50 nM; efficacies of TAK1 downregulation are demonstrated in Figures S6B1,B2 in Supplementary Material). Following siRNA transfection, the cells were stimulated with vehicle (“--”) or with TNFα (50 ng/ml) + TGFβ1 (10 ng/ml) for 24 h (0.5% FBS-containing medium). Tα + Tβ = TNFα + TGFβ1. The impact of TAK1 siRNA on the expression of CCL2 (C1), CXCL8 (C2) and Cox-2 (C3) was determined as described in Figure 5. NS, not significant. In all panels, the findings are representatives of n = 3 independent experiments, performed with MSCs of two different donors, which have shown similar results. Similar findings, obtained in MSCs from another donor, are demonstrated in Figure S7 in Supplementary Material (each panel with its corresponding validation of downregulation efficacy).