Figure 7. Effects of specific PI3K p110α gene suppression by siRNA.

Ex vivo human lung fibroblasts (from three different donors) were kept resting or transfected with small interfering RNAs (siRNAs) specifically targeting p110α (at the concentrations of 20 and 50 nM) or with negative control (50 nM), for 24 hrs in serum-free RPMI medium, afterward they were kept resting or stimulated with TGF-β (10 ng/ml) for 24 hrs in 2% FBS medium. A) Representative western blot analysis of p110α, phosphorylated (Ser⁴⁷³) Akt and GAPDH expression. with means of p110α-/GAPDH and pAKT/α-GAPDH ratios calculated in three separate experiments B) Results of cell counting by optical microscopy. Medians and standard deviations of at least three separate counts in triplicate wells for three separate experiments are reported. C) Representative Western blot analysis of α-SMA expression. and pertinent graph with means of α-SMA/GAPDH ratios calculated in three separate experiments. D) Total soluble collagen levels in one ml of culture medium, measured by the Sircol Soluble Collagen Assay, were reported as micrograms for ml. Statistical significance across treatment groups was determined using the one-way ANOVA. A P value <0.05, which indicates a significant difference, is designated with an asterisk.