Fig. 5: U87MG cells in which the plexin-A2 gene was knocked out acquire some properties of senescent cells.

A The indicated cell types were seeded on fibronectin-coated coverslips and stained with DAPI to visualize cell nuclei (blue) and with fluorescent phalloidin (green) to visualize actin fibers. Shown are merged confocal photographs generated using ZEN 2.3 software. B-C The indicated cell types were assayed at pH-6 for the expression of the senescence marker SA-β galactosidase as described in materials and methods. Shown are representative photographs. D Equal numbers of U87MG cells (N = 9 independent experiments), plexin-A2 knock out U87MG cells (clone 54.3 (N = 7), clone 35 (N = 3)) or clone 54.3 cells in which full-length plexin-A2 (PlexinA2) (N = 5) or A2InTm (N = 5) were re-expressed, were collected and lysed in 100 µL of 0.1 M phosphate buffer (pH 6.0). β-galactosidase activity was then determined at pH 6.0 as described. Data are represented as mean ± SD. Statistical analysis was done using the one-tailed Mann-Whitney test. *P < 0.05, **P < 0.01, ns: non-specific. E Cytofluorimetric analysis of propidium iodide-stained triplicates of U87MG cells, U87MG clone 35 cells and clone 54.3 cells infected with empty lentiviruses (EV) or A2InTm. The peaks represent cells in G0/G1 (G1), Cells in S (S), cells in G2/M (G2), and aneuploid cells (3 N,4 N). F The percentage of cells in the different cell cycle phases is shown for each of the cell types shown in (E). Data are represented as mean ± SD. N = 3.