Fig. 7
EPH-A2 and EPH-B2 knocking down by RNA interfering affects ERMS cell viability, cell cycle distribution, activation of signal transduction pathways and radiosensitivity. a EPH-A2 and EPH-B2 protein expression levels measured by Western blotting at 72 h in RD and TE671 cells after EPH-A2 (EPH-A2siRNA) and/or EPH-B2 (EPH-B2siRNA) silencing in comparison to samples transfected with non-targeting control siRNA (CTRsiRNA), arbitrarily set at 1. Images show representative Western blots of three independent experiments; α-Tubulin was used as loading control. b Viability of RD and TE671 cells 72 h post-transfection with EPH-A2 and/or EPH-B2 siRNAs was calculated with respect to control cells (CTRsiRNA) by using trypan blue exclusion staining. Results represent the mean value of three independent experiments ± SD. Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001 vs CTRsiRNA, $$$ p < 0.001 vs EPH-A2siRNA, ### p < 0.001 vs EPH-B2siRNA. c FACS analysis performed on ERMS cells silenced with EPH-A2, EPH-B2 or CTR siRNAs. Histograms show the distribution of cell populations in each phase of the cell cycle. Results represent the mean values of three independent experiments (upper panel). Cell lysates were analysed by immunoblotting with specific antibodies for indicated proteins; α-Tubulin expression shows the loading of samples. Representative of three independent experiments (lower panel). d Cell lysates from RD and TE671 cells were analysed by immunoblotting with specific antibodies for indicated proteins; α-Tubulin was used as loading control. Representative of three independent experiments. e Representative pictures of colonies stained with crystal violet at 14 days after irradiation (RT) of ERMS cells transfected with EPH-A2siRNA, EPH-B2siRNA, CTRsiRNA or treated with 3.5 μM GLPG1790 (upper panel). Colony formation efficiency was calculated by crystal violet absorbance. Results represent the mean values ± SD of three independent experiments. Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001 vs. CTRsiRNA arbitrarily set at 1 (lower panel)