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. Author manuscript; available in PMC: 2020 Jan 14.
Published in final edited form as: Nat Cell Biol. 2019 Jun 17;21(7):845–855. doi: 10.1038/s41556-019-0337-y

Figure 7. G-actin regulates Rac activity in melanoma cells.

Figure 7

(a,b) B16F10 conditional lines expressing control or siRNA-resistant Flag-ArhGAP12 derivatives were transfected with control or ArhGAP12 siRNA. (a) Invadopodia formation assessed as in Figure 6b. 4 fields per well have been imaged and averaged. Data shown represent n=24 independent wells pooled from three independent experiments. At least 22,437 cells were imaged per condition. Data are means ±SEM, two-tailed Mann-Whitney test. (b) Experimental metastasis assay, displayed as in Figure 6d. Representative results of three experiments are shown, n=5 mice, two-tailed Mann-Whitney test. (c) HGF-induced Rac GTP loading imaged using the RaichuEV-Rac biosensor. siRNA-resistant Flag-ArhGAP12 WT or R582A were transiently re-expressed in serum-starved ArhGAP12-depleted B16F10 cells. Images were acquired from control (n=96), +ArhGAP12 WT (n=58) and +ArhGAP12 R582A (n=37) cells. (i) Basal Rac GTP loading, measured by FRET/CFP ratio over 10 min before stimulation. Data are means ±SEM, two-tailed Mann-Whitney test. Note the lower expression level of ArhGAP12 R582A. (ii) Kinetics of Rac GTP loading following HGF stimulation, normalised taking the basal activity in control cells as 1.0. Data are means ±SEM. (d) Immunoblot analysis of GST-PAK Rac pulldown assays using lysates of B16F10 cells cotransfected with Flag-ArhGAP12 derivatives and Myc-Rac. Representative immunoblot of three independent experiments. (e) B16F10 cells, maintained in 0.3% FCS, following treatment with Cytochalasin D (CD) or Latrunculin B (LatB) for 30 min before Rac.GTP pulldown assay. Data are means ±SEM n=3, two-tailed unpaired t-test. (f) Cells transfected with control or ArhGAP12 siRNA were maintained in 10% FCS and treated with CD for 30 min before Rac.GTP pulldown assays. Data are means ±SEM, n=5 (control), n=11 (siArhGAP12) independent experiments, two-tailed unpaired t-test. (g) Cells as in (f) were treated with LatB for 30 min before Rac.GTP pulldown assay. Representative immunoblot of three independent experiments. (h) Wildtype and ArhGAP12-knockout MEFs were treated with LatB (30 min), and PDGF (5 min) before Rac.GTP pulldown assay. Data are means ±SEM, n=5 (WT), n=3 (KO) independent experiments, two-tailed unpaired t-test. (i) Global and local regulation by RPEL rhoGAP proteins. See Supplementary Figures 5 and 6 for related data. Source data for a-c, e, f and h are shown in Supplementary Table 1. Unprocessed blots are shown in Supplementary Figure 8.