Fig. 3.

R65L, N70H, and P95R mutants show loss of their tumor suppressor function in vitro. SKOV3, PEA1 and PEO1 ovarian cell lines were transduced with an empty vector (CTRL), wild-type (OPCML) or mutant (R65L, N70H, and P95R) OPCML and analyzed for protein expression by western blot (a) and protein expression/localization by confocal microscopy (b) with an anti-OPCML antibody. GAPDH or actin were used as loading controls in a. Samples in the right panel in a were run without heat denaturation. Scale bar = 20 μm. Cells were then tested for migration (c) and invasion (d) in transwell assays and for anchorage-independent growth in agarose (e). All the graphs show the mean ± s.d. of three independent experiments. Student t-test compares CTRL and mutants to wild-type OPCML: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. f SKOV3 cells grown as spheroids were serum-starved, treated with the indicated recombinant proteins (rOPCML, rR65L, rN70H, rP95R), embedded into Matrigel and then stimulated with serum. Invasion of the spheroids was quantified after 3 days. Scale bar = 250 μm. The graphs show the mean ± s.e.m. of three independent experiments. Student t-test compares controls and mutant recombinant proteins to wild-type rOPCML: **p < 0.01, ***p < 0.001. g SKOV3 cells expressing the indicated wild-type and mutant OPCML were lysed and analyzed by western blot for the expression of total and phosphorylated levels of EGFR, HER2 and AKT. GAPDH was used as loading control