Fig. 5

ActA signaling to Smad2/3 in U2OS cells is mediated via ALK4 and ACVR2A. Cells were transfected with siRNA to ACVR2A, ALK4, or scrambled siRNA (control). After 24 h, they were taken either for signaling studies (a, b) or for RT-qPCR determination of the mRNA levels of ACVR2A and ALK4 (c). a A representative experiment. For the signaling studies, cells were starved (2 h, 1% serum) and stimulated (30 min, 37 °C) with ActA (4 nM), or left in starvation medium (control). Cells were lysed, subjected to SDS-PAGE, and immunoblotted for pSmad2/3, tSmad2/3 and β-actin. b Quantification of ActA signaling to Smad2/3. The bands were visualized by ECL and quantified by densitometry (see “Methods”). Data are mean ± SEM of the pSmad2/3 over β-actin ratio of 4 independent experiments. The value obtained for ActA-stimulated cells transfected with siScrambled RNA was taken as 1. pSmad2/3 formation in response to ActA in U2OS cells was almost fully abrogated by knockdown of ALK4 and was significantly reduced by knocking down ACVR2A (or ACVR2B; Additional file 1: Fig. S7a, b). c RT-qPCR quantification of siRNA-mediated knockdown of ALK4 or ACVR2A. Data were normalized using GAPDH as the housekeeping gene, taking the level of the respective mRNA in cells transfected with siScrambled as 1. The transcript expression levels of ACVR2A and ALK4, taken from the Cancer Cell Line Encyclopedia [65] are given in Additional file 2: Table S1. The results are the mean ± SEM of three independent experiments, each conducted in triplicate. Asterisks show significant differences between the pairs indicated by the brackets, using one-way ANOVA and Bonferroni post hoc test (for the signaling studies; panel b) or Student’s two-tailed t test (RT-qPCR, panel c). *, P < 0.02; **, P < 0.003; ***, P < 10⁻⁴