Fig. 6

RhoH affects cell shape in PC3 cells. Analysis of cell shape. PC3 cells were transfected with the indicated siRNAs targeting RhoH or control siRNA. After 48 h, cells were seeded on Matrigel-coated glass coverslips. 72 h after transfection, cells were fixed and stained for F-actin (Alexa Fluor 546 phalloidin), tubulin (fluorescein isothiocyanate (FITC)-labelled anti-tubulin antibody) and DNA (4′,6-diamidino-2-phenylindole (DAPI)). a Representative images of cells. Scale bar 10 μm. Arrows indicate examples of binucleated cells. b–d PC3 cell area, shape factor and elongation factor were measured using ImageJ from F-actin-stained images. Scatter dot plots show mean +/− SEM; at least 60 cells per condition were analysed from three independent experiments, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, Student’s t test. e Quantification of the number of extended protrusions during cell migration. Panels show representative images of siControl- or siRhoH-transfected cells. Arrows indicate protrusions on migrating cells. Graph shows the average number of protrusions per cell relative to control ***p ≤ 0.001. f T47D cells were transfected with the indicated siRNAs. After 72 h, cells were fixed and stained for F-actin (Alexa Fluor 546 phalloidin) and DNA (DAPI). Cell area was measured using ImageJ from F-actin-stained images. Scatter dot plots show mean +/− SEM; n = 3, **p ≤ 0.01, ***p ≤ 0.001, Student’s t test. Relative RhoH mRNA levels in T47D cells were determined by qPCR using GAPDH as control. Mean +/− SEM, n = 3, ***p ≤ 0.001, Student’s t test. g RhoH depletion leads to an increase in binucleated cells. Cells were fixed 72 h after siRNA transfection and stained for F-actin (Alexa Fluor 546 phalloidin) and DNA (DAPI). The % of cells with two or more nuclei were scored; at least 250 cells per condition were analysed from five independent experiments. Graphs show mean +/− SEM, n = 5, *p ≤ 0.05, ***p ≤ 0.001, Student’s t test. h RhoH depletion reduces cell number. PC3 cell numbers were counted 96 h after siRNA transfection. Graphs show mean +/− SEM, n = 3, *p ≤ 0.05, **p ≤ 0.01, Student’s t test