Autocrine and paracrine TGF-β signaling regulates XIAP gene expression in a Smad-dependent manner. A-B) HeLa human cervical cancer cell line and KLE human endometrial carcinoma cell line were treated with the indicated recombinant TGF-β isoforms (10 ng/mL) or with vehicle, for 24 h. XIAP protein levels in treated HeLa cells were determined using western blot (A); XIAP mRNA levels in treated KLE and HeLa cells were determined using RT-PCR, and results from densitometric analysis are presented (B). C-D) KLE and HeLa cells were treated with 2 μg/mL anti-TGF-β neutralizing antibody or isotypic control antibody for 24 h, and XIAP mRNA levels were determined using RT-PCR (C) whereas XIAP protein levels were determined using western blot (D). E-F) KLE and HeLa cells were treated with 50 μM ALK5 inhibitor SB431543 or vehicle for 24 h, and XIAP mRNA levels were determined using RT-PCR (E) whereas XIAP protein levels were determined using western blot (F). In D,F), levels of phosphorylated Smad2 (P-Smad2) were determined to monitor the efficiency of TGF-β pathway inhibition. In all experiments, β-actin or GAPDH were used as loading controls. Graphs represent mean ± SE of three independent experiments. * p < 0.05 compared to control-treated cells.