Figure 2. RAPGEF1 and UCK1 positively regulate melanocyte proliferation.
A. Crystal violet assays following RAPGEF1 knockdown in immortalized melanocytes. Equal numbers of cells either infected with two RAPGEF1 shRNAs or vector control were seeded after infection and drug selection. Cells were fixed and stained with crystal violet at multiple timepoints after seeding. Crystal violet was then solubilized from stained cells and measured by OD560. OD560 values were normalized to the reading at D1, and the mean of six biological replicates and SEM were plotted. P-value was calculated from two-way ANOVA; B. Quantitative RT-PCR for RAPGEF1 in immortalized melanocytes following infection with shRNAs targeting RAPGEF1 relative to empty vector control. RNA was collected and assayed at D5 after infection. C. FACS analysis of immortalized melanocytes infected with either empty vector or RAPGEF1 shRNA at D5 after infection. BrdU and 7-AAD staining were used to characterize the cell cycle status of different cells. The images shown here are from a representative experiment from three biological replicates. D. Crystal violet assays following UCK1 knockdown in immortalized melanocytes. Equal numbers of cells either infected with two UCK1 shRNAs or vector control were seeded after infection and drug selection. Cells were fixed and stained with crystal violet at multiple timepoints after seeding. Crystal violet was then solubilized from stained cells and measured by OD560. OD560 values were normalized to the reading at D1, and the mean of six biological replicates and SEM were plotted. P-value was calculated from two-way ANOVA. E. Quantitative RT-PCR for UCK1 in immortalized melanocytes following infection with shRNAs targeting UCK1 relative to empty vector control. RNA was collected and assayed at D7 after infection F. FACS analysis of immortalized melanocytes infected with either empty vector or UCK1 shRNA at D7 after infection. BrdU and 7-AAD staining were used to characterize the cell cycle status of different cells. The images shown here are from a representative experiment from three biological replicates. G. Immortalized melanocytes infected with a dox-inducible vector (pCW57.1) to express RAPGEF1 were seeded after infection and drug selection. Dox-treated cells were treated with 200ng/mL dox at seeding (D0) and cells were fixed and stained with crystal violet at multiple timepoints after seeding. Crystal violet was then solubilized from stained cells and measured by OD560. OD560 values were normalized to the reading of D1 and the mean of 12 biological replicates and SEM were plotted. P-value was calculated using two-way ANOVA. H. Western blot for RAPGEF1 in dox-treated immortalized melanocytes. Whole cell extract was collected 48 hours post-dox treatment at D7 after infection.