TGF-β isoforms promote XIAP gene expression via Smad and PI3-K pathways. A-C) Before KLE cells were treated with the indicated recombinant TGF-β isoforms (10 ng/mL) or with vehicle for 24 h, they were pre-treated with 50 μM PI3-K inhibitor LY294002 or vehicle for 1 h (A), 10 μM MEK1/ERK pathway inhibitor PD98059 or vehicle for 1 h (B) or Smad4 siRNA or control (scrambled) siRNA for 24 h (C). Levels of XIAP mRNA were determined using RT-PCR and results from densitometric analysis are presented. In all experiments, β-actin or GAPDH were used as loading controls. Levels of Smad4 protein, phosphorylated ERK (P-ERK) or phosphorylated Akt (P-Akt) were determined to monitor the efficiency of Smad4 RNAi, MEK/ERK pathway inhibition or PI3-K inhibition, respectively. Graphs represent mean ± SE of three independent experiments. *p < 0.05. D-E) KLE and HeLa cells were treated with control (scrambled) or Smad4 siRNA for 24 h and levels of XIAP mRNA (D) and protein (E) were determined using RT-PCR (D) and western blot analysis (E). β-actin and GAPDH were used as loading controls.