Figure 3.

RASA5 Functions as a Tumor Suppressor in Carcinoma Cells

(A) Representative colony formation assay by monolayer culture of RASA5-expressing ESCC, NPC, and breast tumor cells. Quantitative analyses of colony numbers shown as values of mean ± SEM of three independent experiments using Student's t test (lower panel), with colonies (>50 cells) in empty vector-transfected cells set as 100; *, p < 0.05.

(B) RASA5 expression was confirmed using both anti-Flag and anti-RASA5 (Ab1, ThermoFisher) by Western blot. The straight arrow represents the specific band of RASA5 detected by anti-Flag antibody. Graphs represent quantification of Western blots with fold change compared with controls (lower panel). Data were presented as mean ± SEM of three independent experiments via Student's t test. ***, p < 0.001.

(C) Characteristic nuclear changes of apoptotic cells after RASA5 expression. Arrows indicate EGFP+ cells.

(D) Cleavage of apoptotic markers, PARP and caspase-3, in carcinoma cells transfected with Flag-tagged RASA5 construct. GAPDH served as a loading control. Graphs represent quantification of Western blots with fold change compared with controls (right). **, p < 0.01; ***, p < 0.001. Data were presented as mean ± SEM of three independent experiments via Student's t test.

(E) Xenograft tumor formation of MB231 carcinoma cells expressing RASA5 or vector only. Cells were injected subcutaneously into nude mice. Representative photographs of H&E staining of xenografts (200x, 400x magnification).

(F) Horizontal bars show mean tumor weight and volume. Statistically significant decrease in tumor weight and growth (**, p < 0.01) upon ectopic RASA5 expression was observed. Data were presented as mean ± SEM of three independent experiments via Student's t test.