Fig. 7: MET or SPRY2 knockdown causes similar alterations in MAP Kinase pathway activity in RMS cells.

RMS cells were cultured for 144-h post transfection with MET, SPRY2, or control siRNA and lysates were separated by SDS-PAGE and analyzed by western blotting for MAP kinase pathway activation. Representative blots for levels of phosphorylated and total ERK1/2 (upper panels), as well as phosphorylated and total p38 (lower panels) detected using specific antibodies in RD (a) and SJRH30 (c) cells are shown. The ratios of pERK1/2 to T-ERK1/2 and p38 to total p38 were measured by densitometry (b, d). ERK activation is significantly reduced in both MET and SPRY2 silenced RD (b, upper panel) and SJRH30 (d, upper panel) cells when compared to control cells. However, phosphorylation of p38 is unchanged in RD cells (b, lower panel) but significantly reduced in SJRH30 cells (d, lower panel) between MET/SPRY2 and control siRNA transfections. Representative bright field micrographs of wound closure assays using SJRH30 cells, imaged at specific time points after incubation with (+) or without (–) the MEK/ERK inhibitor U0126 (e). Bar graphs show that wound closure in the monolayers of SJRH30 is significantly decreased in U0126 treated cells compared to untreated cells (f). Representative western blots for p-ERK1/2 and total-ERK1/2 using cell lysates prepared at the end of the wound closure are shown (g). Densitometry shows that p-ERK1/2 levels are significantly lower in U0126 treated SJRH30 cells when compared to untreated cells at the end of scratch assay (h). The graphical data are presented as mean ± SEM of a minimum of three independent knockdown experiments. Scale bar in e is 100 µm