Fig. 1: Silencing plexin-A2 expression in U87MG and A172 cells inhibits their proliferation and the development of subcutaneous tumors.

A Western blot showing plexin-A2 expression levels in U87MG cells expressing a non-specific shRNA (ShC) or a shRNA targeting plexin-A2 (ShPlexA2#1). Cell lysates were probed with antibodies directed against plexin-A2 or vinculin. B U87MG or U87MG cells silenced for plexin-A2 expression using two different shRNAs were seeded in triplicate (2 × 10⁴ cells/well) in 24 well dishes. Cells were counted in a coulter counter after 3 days. Shown is the percentage of attached cells on day 3 compared with the number of cells attached on day 0 which was taken as 100%. Data are represented as mean ± SD. Statistical analysis was done using the one tailed Mann-Whitney test. *P < 0.05, N = 3. C Western blot showing plexin-A2 expression levels in A172 cells expressing a non-specific shRNA (ShC) or a shRNA targeting plexin-A2 (ShPlexA2#1). Cell lysates were probed with antibodies directed against plexin-A2 or vinculin. D A172 cells were infected with lentiviruses encoding a control shRNA (ShC) or a shRNA targeting plexinA2 (ShPlexA2#1). Each group of cells was seeded in quadruplicate in 96 well dishes (3 × 10³ cells/well). Cell proliferation was measured using the WST-1 proliferation assay as described in materials and methods, and the values presented were calculated as described under (B). Data are represented as mean ± SD. Statistical analysis was done using the one tailed Mann-Whitney test. *P < 0.05, N = 3. E U87MG expressing a nonspecific shRNA (ShC) or U87MG cells silenced for plexin-A2 expression using ShPlexA2#1 were injected subcutaneously into NOD/SCID mice (3 × 10⁶ cells/mouse). After 30 days the experiment was terminated, tumors were excised, weighed and photographed. Each group contained 5 mice. Shown are pictures of the mice (upper panel) and of the excised tumors (lower panel). F U87MG expressing non-specific shRNA (ShC) or U87MG cells silenced for plexin-A2 expression using ShPlexA2#1 were injected subcutaneously into Athymic/Nude mice (2 × 10⁶ cells/mouse). Each group contained 7 mice. Tumor development was measured twice a week using calipers. Data are represented as mean ± SD. G Shown is the mean weight of 19 tumors excised from three independent experiments done as in (F). Data are represented as mean ± SD. Statistical analysis was done using the one tailed Mann-Whitney test. *P < 0.05.