Figure 2

RasGRP3 is expressed in human breast derived ductal adenocarcinoma cell lines and represses cell proliferation of both MCF7 and T-47D derived cells. (A) Q-PCR and Western blot analyses of RasGRP3 expression in multiple breast derived ductal adenocarcinoma cell lines. PC-3 cells were included as a positive control [12]. GAPDH was used as an internal control. The results are representative of three independent experiments. Values represent the mean ± SEM. Expression of RasGRP3 was inhibited using a specific shRNA (shRasGRP3). A non-targeting scrambled shRNA (shSCR) was used as a control. (B) Confirmation of the extent of suppression of RasGRP3 expression was determined by Western blotting. GAPDH was used as an internal control. P indicates the number of subculturing of the RasGRP3 silenced cells. Results are representative of 2 independent experiments. (C) The proliferation of T-47D and MCF7 derived cells was determined using the CyQuant GR cell proliferation assay, with values normalized to the levels of non-transfected control cells. CyQuant assay was conducted every 24 hours. Values represent the mean ± SEM for 2 independent experiments.