Fig. 4: CaM inhibition impaired the invasion and invadopodia formation of SNB19 and LN18 cells.

a The viability of SNB19 and LN18 cells treated by CaM-specific inhibitor W7 at 10μm was measured by cell counting kit-8 assay (Data are presented as means ± SDs of five different absorbance values from every independent experiment). b Expression of endogenous CaM in glioma cells was inhibited by expressing shRNAs targeting human CALM1 and CALM2 but not with the expression of nontargeting control shRNA (shCont). c Using W7 (10 μm) and CaM knockdown both significantly decreased GBM cells migration as detected by wound healing assay (Data are presented as means ± SDs of five different width of the wound from every independent experiment, ****p < 0.0001). d EGF facilitated GBM cells invasion in a dose-dependent manner analyzed by transwell invasion assay (Data are presented as means ± SDs of three different microscopic visions from every independent experiment, ****p < 0.0001). e Using W7 (10 μm) and CaM knockdown both significantly inhibited GBM cells invasion and EGF-induced invasion (**p < 0.01, ****p < 0.0001). f CaM inhibition abolished GBM cells invadopodia formation and focalized proteolysis activity and antagonized EGF-induced invadopodia formation and focalized proteolysis activity (Images were taken from three different microscopic visions from every independent experiment, Invadopodia formation: n = 20, 20, 20, 20, 20, 20 and 20 for Control, EGF, W7, EGF + W7, shcontrol, shCaM, shCaM + EGF in SNB19 and n = 25, 20, 20, 20, 20, 20 and 20 for Control, EGF, W7, EGF + W7, shcontrol, shCaM, shCaM + EGF in LN18; Proteolysis activity: n = 17, 17, 17, 17, 17, 17 and 17 for Control, EGF, W7, EGF + W7, shcontrol, shCaM, shCaM + EGF). Invadopodia quantification and degradation areas were measured by imagej (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)