FIG. 1.

RIN1 is a positive regulator of TGF-β signaling. (A) HeLa cells were transiently transfected with 3TP-Lux and either vector (ctr) or RIN1 and then incubated without or with TGF-β (5 ng/ml) for 18 h. Increased amounts of transduced RIN1 expression construct (wedge) were used to demonstrate concentration dependence. (Right side) Baseline luciferase activity (no added TGF-β) increased with the amount of transduced RIN1. (B) HeLa cells were transduced with ctr or RIN1 lentivirus. Stable, selected populations were then transfected with SBE-Lux and stimulated (or not) by TGF-β (5 ng/ml) for 18 h. Immunoblots for RIN1 and TUBB (normalization control) are shown above the graph. *, P = 0.02. (C) MDA-MB-231 cells were transduced with ctr or RIN1 lentivirus. Stable, selected populations were transfected with 3TP-Lux and stimulated (or not) by TGF-β (5 ng/ml) for 5 h. Immunoblots for RIN1 and TUBB (normalization control) are shown above the graph. *, P = 0.01. (D) MDA-MB-231 cells were transduced with shRNA lentivirus (ctr or RIN1). Stable, selected populations were transfected with SBE-Lux and stimulated (or not) by TGF-β (5 ng/ml, 18 h). Immunoblots for RIN1 and TUBB (normalization control) are shown above graph. *, P = 0.02. (A to D) Luciferase activities were normalized to Renilla luciferase activity and plotted as means ± SDs of triplicates from a representative of at least two independent experiments. (E) RIN1 silencing dampens SMAD2 nuclear localization in response to TGF-β. (Left) MDA-MB-231 cells transduced with ctr or RIN1 shRNA lentivirus (same as panel D) were induced with TGF-β (5 ng/ml, 15 min) and then fixed and stained with anti-SMAD2/3 and DAPI. (Right) Nuclear SMAD staining after 5 or 15 min of TGF-β treatment (mean of 10 fields counted; *, P < 0.001).