Fig. 4: Re-expression of a truncated plexin-A2 containing the intracellular and trans-membrane domains (A2InTm) of plexin-A2 restores cell proliferation of plexin-A2 knock-out cells.

A Plexin-A2 knock-out clone 54.3 cells were infected with an empty lentiviral expression vector (EV) or a lentiviral vector directing expression of a V5 tagged full length plexin-A2 (PlexA2) or a truncated plexin-A2 containing the intracellular and trans-membrane domains of plexin-A2 (A2InTm). Western blots prepared from cell lysates were probed with antibodies directed against V5 and vinculin. B Representative growth curves of U87MG cells and of U87MG KO clone 54.3 cells infected with lentiviruses encoding plexin-A2 (PlexinA2), A2InTm or an empty expression vector (EV). Cells were seeded in quadruplicate in 24 well dishes (1 × 10⁴ cells/well). Cells were counted every two days using a coulter-counter. Data are represented as mean ± SD. C The average population doubling time of U87MG cells (N = 11) was compared with the average population doubling time of clone 54.3 + EV (N = 11), clone 54.3 + PlexinA2 (N = 7) and clone 54.3 + A2InTm (N = 8). Data are represented as mean ± SD. Statistical analysis was done using the one tailed Mann-Whitney test. ***P < 0.001 D Clone 54.3 cells infected with an empty lentiviral expression vector (EV) or with a lentiviral vector directing expression of plexin-A2 were seeded in soft agar in 24 well dishes (1 × 10³ cells/well) in triplicates. Colonies were stained with crystal violet after 13 days. Shown are photographs of representative wells. E, F The average number of colonies per well with an area between 5000 and 10,000 µm² or 10,000 and 15,000 µm² were determined. Data are represented as mean ± SD. Statistical analysis was done using the one-tailed Mann-Whitney test. *P < 0.05, N = 3. G Lentiviruses were used to express control (ShC) or a plexin-A4 targeting shRNA (ShPlexA4) in KO clone 54.3 cells expressing A2InTm. The expression of plexin-A4 was then examined using qRT-PCR. Data are represented as mean ± SD, N = 2. H Representative growth curve of U87MG cells and of clone 54.3 cells expressing an empty expression vector (EV) or expressing A2InTm and clone 54.3 cells expressing A2InTm as well as a non-specific shRNA (ShC) or Clone 54.3 cells expressing A2InTm and an shRNA targeting plexin-A4 (ShPlexinA4). The various cells were seeded in quadruplicate in 96 well dishes (3 × 10³ cells/well). The proliferation of the cells was determined every two days using the WST-1 assay as previously described. Data are represented as mean ± SD. The experiment was repeated three times with similar results.