Fig. 10

PAK2 mediates the effects of RhoH on cell shape. a, b RhoH co-immunoprecipitates with PAKs. COS7 cells were transfected with constructs encoding GFP-RhoH and myc epitope-tagged PAKs (a) or HA-RhoH, GFP-Rac1, Myc-PAK2 or GFP alone (b). After 24 h, lysates were incubated with GFP-Trap beads (GFP IP) followed by western blotting with the indicated antibodies. Results shown are representative of three independent experiments. c PAK2 depletion reduces cell migration. PC3 cells were transfected with the indicated siRNAs and Oris™ migration assay carried out as in Fig. 2. Migration is shown as a percentage of siControl +/− SEM, n = 3; *p ≤ 0.5, ***p ≤ 0.001, Student’s t test. Representative immunoblot from cell lysates probed with the indicated antibodies. GAPDH was used as loading control. d–f PAK2 depletion increases cell area and decreases migration. PC3 cells were transfected with the indicated siRNAs. d After 72 h, cells were fixed and stained for F-actin (Alexa Fluor 546 phalloidin) and DNA (DAPI). Cell area was measured from F-actin-stained images. Scatter dot plots show mean +/− SEM; n = 3 (> 125 cells/condition), ***p ≤ 0.001, Student’s t test. e Representative images used for d; scale bar 10 μm. f Cells were seeded 48 h after transfection in RPMI containing 0.1% FCS and monitored by time-lapse microscopy for 24 h, images taken every 5 min. At least 50 cells were tracked from two independent experiments. Boxes of box and whisker plots show median, 25th and 75th percentiles; whiskers show minimum and maximum values; *p ≤ 0.05, **p ≤ 0.01, Student’s t test. g RhoH does not alter PAK activity. PC3 cells were transfected with the indicated siRNAs, cultured for 24 h in medium containing 0.1% FCS then stimulated with 20 ng/ml HGF for 10 min. Cell lysates were probed with the indicated antibodies. Anti-phospho-PAK recognises PAK1, PAK2 and PAK3. GAPDH was used as loading control. Graph shows densitometric quantification of western blots. n = 3; ns non-significant, Student’s t test