Figure 2.

Inhibition of endogenous RasGRP3 expression retarded cell proliferation in multiple human melanoma cell lines. (A) Individual cell lines were transiently transfected with RasGRP3 siRNA pool and Control siRNA pool 1 at 80 nM final concentration. After 96 hours, cell proliferation was determined by using the CyQuant NF cell proliferation assay, with values normalized to the levels of untreated cells. Values represent the mean ± SEM for four independent experiments. Confirmation of the extent of suppression of RasGRP3 was determined by immunoblotting. Results are representative of 3 experiments. (B and C)The Tet-on stable cell lines created from the M14 and SK-MEL-5 cell lines were seeded at a density of 10⁴ cells/ml in complete growth medium. Cells were treated with/without 1 μg/ml tetracycline for 120 hours and proliferation was determined by using the CyQuant NF cell proliferation assay, with values normalized to the levels of parental M14 or SK-MEL-5 cells. Values represent the mean ± SEM of four independent experiments. Endogenous RasGRP3 expression was determined by RT-PCR (M14) or immunoblot (SK-MEL-5) 120 hours after induction. Results are representative of three independent experiments.