Fig. 3

MAPK and AKT signaling pathways are required for cancer cell invasion and survival mediated by TMEM52B suppression. (A) Phase-contrast images of SW480sub control cells and TMEM52B-knockdown cells. Arrowheads indicate representative membrane ruffling or protrusive spot. Number of cells displaying lamellipodia or protrusive spots (more than 3) were counted. Cell spreading area per cell (at least 200 cells) was calculated using ImageJ software. Values represent mean ± SD. *P < 0.05. Scale bar, 25 μm. (B) Cells were transfected with siRNA or shRNA specific to TMEM52B for 48 h prior to lysis for immunoblot analysis or RT-PCR analysis. Transfected cells were incubated for 48 h under suspension culture conditions prior to lysis for immunoblot analysis of cleaved caspase-3 and PARP. (C) Cells were transfected with shRNA specific to TMEM52B for 24 h and then treated with pharmacological inhibitors: PD098059 (5 μM SW480sub and 20 μM HCT-15); SP600125, 15 μM; wortmannin, 10 μM or 25 μM, or 0.1% DMSO as a vehicle control for 24 h before whole-cell lysates were prepared for immunoblotting. (D, E) Cells were transfected with shRNA specific to TMEM52B for 48 h. Invasion (D) and cell survival (E) assays were performed as described in Fig. 2A and B in the presence of pharmacological inhibitors: PD098059 (5 μM SW480sub and 20 μM HCT-15); SP600125, 15 μM; wortmannin, 10 μM, or DMSO. Cell invasion was determined by calculating the cell-stained area relative to the total area using ImageJ software. All determinations were performed in three independent experiments. Values represent mean ± SD. *P < 0.05 compared with shControl + DMSO; ^§P < 0.05 compared with shTMEM52B + DMSO