Fig. 6: U87MG cells in which the plexin-A2 gene is knocked-out have abnormal mitotic spindles and display changes in the phosphorylation of AKT and P38.

A The indicated cell types were seeded on fibronectin-coated coverslips and stained with DAPI to visualize cell nuclei (blue) and with antibodies to alpha-tubulin (green) and gamma-tubulin (red). Shown are 3D confocal photographs generated using the Imaris software. Scale bars: 10 μm. B The X–Z axis stack projections of the photographs generated from laser-scanning confocal images taken in 0.3 μm steps were quantified using ZEN 2.3 software. The mitotic spindle angles of the indicated cell types were calculated. Data are represented as mean ± SD. Statistical analysis was done using the one-tailed Mann-Whitney test. *P < 0.05, N = 10 (U87MG), N = 8 (clone 54), N = 11 (T98G ShC), N = 6 (T98G ShA2). C The cDNAs encoding A2InTm, A2ExTM or an empty expression vector (EV) were expressed in clone 54.3 knock-out cells (54.3). The phosphorylation levels of AKT were assayed in the indicated cell types by western blot analysis of cell lysates as described in methods, using an antibody directed against phosphorylated AKT (ser473). Loading was assessed using an antibody directed against total AKT. Shown is a representative western blot. The effect of A2InTm expression on the average phosphorylation levels of AKT was determined (N = 8) (except for A2ExTm expressing cells, N = 1). Below is shown a histogram depicting the ratio between the intensity of phospho-AKT staining and total AKT. Data are represented as mean ± SD. Statistical analysis was done using the one-tailed Mann-Whitney test. ***P < 0.001, D The phosphorylation levels of p38 were assayed in the indicated cell types by western blot analysis of cell lysates as described in methods, using an antibody directed against phosphorylated p38 (Thr180/Tyr182). Loading was assessed using an antibody directed against total p38. Shown is a representative western blot. Below is shown a histogram depicting the average ratio between the intensity of the respective phospho-p38 bands and the total p38 bands. Data are represented as mean ± SD. Statistical analysis was done using the one-tailed Mann-Whitney test. **P < 0.01, N = 6.