Fig. 7: Point mutations in the FARP2 binding site and in the Fyn phosphorylation sites of the plexin-A2 intracellular domain inhibit restoration of cell proliferation induced by A2InTm and inhibit the A2InTm mediated rescue of AKT phosphorylation.

A U87MG cells, U87MG clone 54.3 cells infected with either control lentiviruses (EV) or with lentiviruses encoding A2InTm or A2InTm variants containing point mutations in the FARP2 binding site (54.3 + A2InTm FARP2 mut), in the Fyn phosphorylation sites (54.3 + A2InTm FYN mut), in the Rho binding domain (RBD) (54.3 + A2InTm RBD mut) or in the juxtamembrane cytosolic dimerization interface (54.3 + A2InTm Cytosolic mut) of plexin-A2 were seeded in 24 well dishes (1 × 10⁴ cells/well). Triplicate wells were counted every two days using a coulter-counter. Data are represented as mean ± SD. Shown is a representative experiment out of five similar experiments. B Shown are the average population doubling times of the clone 54.3 derived indicated cell types. Results were derived from N = 5 independent experiments. Data are represented as mean ± SD. Statistical analysis was done using the one-tailed Mann-Whitney test. *P < 0.05. C Single-cell suspensions of clone 54.3+EV, clone 54.3 + A2InTm, clone 54.3 + A2InTm FARP2 mut or clone 54.3 + A2InTm FYN mut cells, were seeded in soft agar (1 × 10³ cells/well) in triplicates. After 17 days colonies of cells were stained with crystal violet. Shown are photographs of representative wells. D, E The average number of colonies per well with an area between 5000 and 10,000 µm² or 10,000 and 15,000 µm² was determined. Data are represented as mean ± SD. Statistical analysis was done using the one-tailed Mann-Whitney test. *P < 0.05, N = 3. F U87MG cells, clone 54.3+EV cells, clone 35 cells, clone 54.3 + A2InTm cells, clone 54.3 + A2InTm FARP2 mut cells and clone 54.3 + A2InTm FYN mut cells 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. Red arrows point at multinucleated cells. G The percentage of multinucleated cells of the various cell types described in (F) was determined by counting 23 microscopic fields per each cell type. H An empty lentiviral expression vector (EV) or lentiviruses expressing cDNAs encoding A2InTm or A2InTm variant containing point mutations in the FARP2 binding site (A2InTm FARP2 mut) 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. Below is shown a histogram depicting the ratio between the intensity of the respective phospho-AKT bands and the total AKT bands. I An empty lentiviral expression vector (EV) or lentiviruses expressing cDNAs encoding A2InTm or A2InTm variant containing point mutations in the Fyn phosphorylation sites (A2InTm FYN mut) were expressed in clone 54.3 knock-out cells (54.3). The levels of AKT phosphorylation were determined as described under (H). Below is shown a histogram depicting the ratio between the intensity of the respective phospho-AKT bands and the total AKT bands.