Figure 4

Knockdown of ZEB2 partially blocks TGF-β signaling in TERT-siSFRP1 cells. Stealth RNAi™ siRNA negative controls (62.5 nM) were transfected into TERT-pSUPER and TERT-siSFRP1 cells [TERT-pSUPER (control) and TERT-siSFRP1 (control)] and ZEB2 Stealth RNAi™ siRNA (62.5 nM) was transfected into TERT-siSFRP1 cells [TERT-siSFRP1 (siZEB2)]. Forty-eight hours after transfection, total RNA was isolated from each cell line for real-time PCR analysis. The levels of ZEB2 mRNA (A) β₃ Integrin mRNA and PAI-1 mRNA (C) were normalized to amplification of β-actin mRNA, which was performed in parallel wells for each cell line. Bars represent mean ± SEM and are expressed as relative expression of TERT-pSUPER (control) cells. *p < 0.05, ***p < 0.001 (significantly different from TERT-pSUPER (control) cell line using Bonferroni's t-test following 1-way ANOVA). (B) TERT-siSFRP1 cells were transfected with either a Stealth RNAi™ siRNA negative control (62.5 nM) or ZEB2 Stealth RNAi™ siRNA (62.5 nM) and CAGA-Luc plus CMV-Renilla reporter vectors. The relative luciferase activity was measured after an overnight incubation in the presence or absence of the 2.5 ng/ml TGF-β1. Bars represent mean ± SEM of relative luciferase activity (firefly luciferase activity/renilla luciferase activity) normalized to the relative luciferase activity in TERT-siSFRP1 (control) cells treated with vehicle. ***p < 0.001 [significantly different from corresponding TERT-siSFRP1 (control) cell line using students's t-test]