Fig 3.
Effect of shRNA mediated BRAF suppression on MA cell cycle distributions and expression of cell cycle regulatory proteins. A. MA cell lines with (left) or without (right) BRAFV600E mutation were infected with lentivirus expressing empty control (PLKO.1) or BRAF shRNA (6289), then treated with puromycin to select infected cells. Pooled puromycin resistant cells were subjected to flow cytometry to determine cell cycle distributions. For all cell lines with BRAFV600E, suppression of BRAF expression resulted in increased proportion of G1 phase cells. For 3 of th 4 cell lines with wild-type BRAF, shRNA suppression had little effect on the G1 fraction of cells. B. Lysates from the same transduced cells as in “A” were subjected to Western Blot analysis to assess the effects of BRAF knockdown on the expression of proteins with known roles in regulating G1 phase cell cycle transit: CYCLIN D1 and CYCLIN D3, enzymatic subunits CDK4 and CDK6, and cyclin dependent kinase inhibitor p27Kip1. The left lane in each blot results from control shRNA and the right lane result is from use of the BRAF shRNA lentivirus. Comparison of results for any two cell lines shows that BRAF suppression effects are variable for the five G1 regulatory proteins examined, but are consistent in demonstrating that BRAF suppression results in decreased expression of one or more of the proteins that promote G1 transit, and/or increases the expression of the negative G1 transit regulator p27Kip1.