Figure 3. CD157 overexpression protects OV-90 cells from anoikis and enhances motility.

(A) sqRT-PCR (left) and western blot analysis (right) of CD157 in OV-90/CD157 and OV-90/mock cells. GAPDH and β-actin were used as internal controls, respectively. (B) Morphology of colonies formed by OV-90/mock and OV-90/CD157 cells. Representative colonies visualized after crystal violet staining are shown. Scale bar: 200 µM. (C) sqRT-PCR analysis of E-cadherin and N-cadherin in OV-90/mock and OV-90/CD157 cells. Densitometry quantifies the levels of mRNA expression of the indicated molecules relative to GAPDH. (D) Effect of CD157 overexpression on anoikis. After 48, 72. 96 and 192 h of anchorage-independent growth, cells were fixed, stained with propidium iodide and analyzed with a FACSCanto. Anoikis in OV-90/mock and OV-90/CD157 cells was determined by measuring the percent of sub-G1 cells. Results represent the mean ± SEM of three independent experiments. *P<0.05; **P<0.01, two-tailed t test. (E) Anchorage-independent growth of OV-90/CD157 and mock cells was analyzed by soft agar colony formation assay. Graph represents average number of colonies formed from three independent experiments ± SEM after 3 weeks incubation of cells in soft agar. ***P<0.001, two-tailed t test. (F) Effect of CD157 expression on cell migration in a scratch-wound assay in OV-90/CD157 and mock cells. Cells were grown as monolayers, wounded, and photographed at time 0 and at 24 hr. Wound edges are indicated by black dashed lines (scale bar: 200 µM). (G) The ability of cells to close the wound was calculated by measuring 20 randomly chosen distances along the wound edge at time 0 and at 24 hr. Results represent the percentage reduction of the average wound width and are expressed as the mean ± SEM of three independent experiments. *P<0.05; two-tailed t test.