Fig. 3

A DAPI and anti-ANILLIN staining in control (?Dox) and BRAF^V600E-expressing (+Dox) anaphase RPE-1 cells. Images are maximum intensity projections of z-stacks. Scale bar = 7.5?µM. B Mean ANILLIN fluorescence intensity at the equator of control and BRAF^V600E-expressing anaphase RPE-1 cells. N?=?79 cells for ?Dox and N?=?53 for +Dox. Unpaired Student?s t test. Error bars represent mean?±?SEM. C DAPI and anti-RHOA staining in -BRAF^V600E (?Dox) cells, BRAF^V600E-expressing (+Dox) cells, and BRAF^V600E-expressing (+Dox) RPE-1 cells treated with MEKi or ERKi. Drugs were added coincident with Dox administration. Images are maximum intensity projections of z-stacks (0.20?µM). Scale bar = 7.5?µM. D Mean RHOA fluorescence intensity at the equator of control RPE-1 cells, BRAF^V600E-expressing RPE-1 cells, and BRAF^V600E-expressing RPE-1 cells treated with MEKi or ERKi. N?=?41 cells for ?Dox, N?=?68 for +Dox, N?=?19 for +Dox +MEKi, and N?=?19 for +Dox +ERKi. Brown-Forsythe and Welch one-way ANOVA with Dunnett?s multiple comparisons test. Error bars represent mean?±?SEM. E Western blot analysis of immunoprecipitated RHOA-GTP from control (?Dox) and BRAF^V600E-expressing (+Dox) RPE-1 cell lysates at different time points post thymidine release. Total RHOA protein and alpha tubulin were used as a controls. A representative of three independent biological replicates is shown. F Western blot quantification of immunoprecipitated RHOA-GTP levels from control (?Dox) and BRAF^V600E-expressing (+Dox) RPE-1 cell lysates at different time points post thymidine release. Samples were normalized to the ?Dox condition. N?=?3 independent experiments. Unpaired Student?s t test. Error bars represent mean?±?SEM. G Fold change (FC) in G1 tetraploid RPE-1 FUCCI cells following addition of RHOA activators. LPA (1?µM) and S1P (1?µM) were added coincident with Dox administration. Fold changes in G1 tetraploids relative to the control (+Dox no drug) are shown. N?=?3 independent experiments. Unpaired Student?s t test. Error bars represent mean?±?SEM. H G1 tetraploid generation following expression of HA-tagged-BRAF^V600E in RHOA^Q61L-inducible cells. Experimental design (top): RPE-1 FUCCI cells with Dox inducible RHOA^Q61L were transiently transfected with an HA-tagged-BRAF^V600E-expressing construct and selected accordingly. G1 tetraploids were quantified (bottom left and middle) by gating HA-positive, Cdt-1:mCherry-positive cells with increased Hoechst incorporation. Fold change in G1 tetraploids (bottom right), normalized to control (?Dox) cells. N?=?3 independent experiments. Unpaired Student?s t test. Error bars represent mean?±?SEM.