Fig. 2

A Generation of BRAF^V600E-expressing RPE-1 FUCCI cell lines with a lentiviral-based doxycycline-inducible vector. B Western blot showing inducible expression of BRAF^V600E (+Dox) in RPE-1 FUCCI cells using a BRAF and BRAF^V600E-specific antibody. Expression of the Tet repressor protein is shown. TUBULIN was used as the loading control. A representative of three independent biological replicates is shown. C Flow cytometry plots of control (−Dox) and BRAF^V600E-expressing (+Dox) RPE-1 FUCCI cells. Tetraploid cells accumulating in G1 were quantified based on Cdt1-mCherry positivity and Hoechst incorporation. Percentages of G1 tetraploid cells in control and BRAF^V600E-expressing cultures are indicated. D Fold change (FC) in G1 tetraploids normalized to the control (−Dox) are shown for BRAF^V600E, BRAF^WT and BRAF^K483W (kinase-dead) -expressing RPE-1 FUCCI cell lines. N = 5 independent experiments for BRAF^V600E, N = 3 for BRAF^WT and N = 3 for BRAF^K483W. One-way ANOVA with Tukey’s multiple comparisons test. Error bars represent mean ± SEM. E Merged phase contrast and GFP photomicrographs of H2B-GFP expressing control (−Dox) and BRAF^V600E-expressing (+Dox) RPE-1 cells that have recently undergone mitosis. White dotted lines indicate 2 cells with 1 nucleus each that have separated following a successful cytokinesis (−Dox) and 1 cell with 2 nuclei that has failed cytokinesis (+Dox). F Percentages of cells exhibiting cytokinesis failure in H2B-GFP RPE-1 control (−Dox) and BRAF^V600E-expressing (+Dox) cells. N = 4 independent experiments examining -Dox = 934 and +Dox = 568 cells. Unpaired Student’s t test. Error bars represent mean ± SEM.