Fig 2: Pak activity is required for regulation of centrosome separation downstream of Tiam1.(a,b) MDCK II cells with dox-inducible overexpression of WT, L106F (kinase active) or T402A (kinase inactive) Pak2-CFP or tet-ON construct only (Control) were transfected with non-targeting control (NT) or Tiam1 siRNA and dox added 1 day later for 1 day. (a) Cells were lysed and analysed by immunoblotting with the indicated antibodies. Arrow indicates expression of exogenous Pak2-CFP constructs; *endogenous Pak2. (b) Cells were stained for IF with antibodies against GFP, ?-tubulin and DAPI, and intercentrosomal distance of cells in prophase expressing the different Pak2-CFP constructs (as determined by presence of positive staining with GFP antibody, see Supplementary Fig. 6b) was measured (>50 cells over three independent experiments). (c) MDCK II cells were treated for 6 h with 2 µM FRAX597 and either lysed and analysed by immunoblotting for Pak activity (P*Pak) or fixed and stained for IF with antibodies against a-tubulin, ?-tubulin and DAPI then intercentrosomal distance in prophase measured (>50 cells over three independent experiments). All graphs show mean+s.e.m. Unpaired two-sided t-test: *P<0.05, **P<0.001.

Fig 3: PAK1 overexpression exerts anti-atrophic effects in two models of in vitro C2C12 atrophy. (A) The circulating levels of IL6 in the plasma of C26-bearing mice is drastically increased with respect to PBS-injected mice (n = 3–6, Mann Whitney's test, *P = 0.02). (B) Upon 5 h treatment with 10 or 100 ng/mL murine IL6, C2C12 myoblasts transiently expressing Stat3 4X-FLuc reporter plasmids induce FLuc (n = 3, one-way anova followed by Dunnett's test, **P = 0.01, ****P = 0.0001). (C) IL6-induced Stat3-FLuc signal is reduced in myoblasts expressing GFP-PAK1 with respect to those expressing GFP. Myoblasts treated for 5 h with IL6 (100 ng/mL) were previously transfected with Stat3 4X-FLuc plasmid, Renilla luciferase plasmids, and GFP or PAK1-expressing plasmids. The results of three independent experiments are shown. Mean is reported (n = 14, unpaired t-test, ***P = 0.0002). (D) Rates of long-lived protein degradation were measured in myotubes transfected on the third day of differentiation with plasmids for pDSRed2-PAK1 or empty vector and differentiated for one more day, when they were exposed for 24 h to 10 ng/mL IL6 (n = 4, unpaired t-test, *P = 0.05). (E) The mRNA content of Pak1 is reduced in atrophying myotubes exposed for 24 h to 10 µM dexamethasone (n = 6, Mann Whitney's test, **P = 0.002, ****P = 0.0001). (F) Myotubes transfected on the third day of differentiation with plasmids for GFP-PAK1 or only GFP were exposed for 24 h on the fourth day of differentiation to vehicle or 1 µM dexamethasone or 10 µM IPA-3. In these conditions, the mRNA levels of MuRF1 were determined by quantitative polymerase chain reaction (n = 4, unpaired t-test, *P = 0.05). (G) Myoblasts were transfected, as indicated earlier, for 24 h, total protein was then extracted. Immunoblot analysis reveals the protein content of p-FOXO3, FOXO3, atrogin-1, and vinculin that is used as loading control. (H) Quantification of the ratio between p-FOXO3 over total FOXO3 is shown (n = 6–8, unpaired t-test, *P = 0.05). SEM is indicated in all figures. IL6, interleukin-6; FLuc, firefly luciferase; PAK1, p21 protein-activated kinase 1; TBP, Tata-Binding Protein.

Fig 4: Tiam1 and Pak modulate responses to Eg5 inhibitors.(a–e) MDCK II cells were transiently transfected with two siRNAs against Pak2 or non-targeting control (NT) as indicated, and 2 days later treated with 25 µM monastrol for 16 h. (a) Cells were lysed and analysed for knockdown by immunoblotting with the indicated antibodies. (b) Cells were fixed and stained for FACS analysis with propidium iodide and MPM2 antibody to detect % of mitotic cells (n?3, 10,000 cells/rep). (c–e) Cells were fixed and then stained by IF with antibodies against a-tubulin, ?-tubulin and DAPI. (c) Example mitotic spindles: a-tubulin=green, ?-tubulin=red and DAPI=blue, scale bars, 5 µm. (d) Quantification of the proportion of bipolar spindles (n?3). (e) Quantification of the proportion of mitotic cells in anaphase and telophase (n?3). (b–e) All graphs show mean+s.e.m. Unpaired two-sided t-test: **P<0.01. (f) MDCK II cells expressing histone-2B-GFP (H2B-GFP) and a-tubulin-RFP were transiently transfected with siRNAs against Tiam1, Pak1, Pak2 or non-targeting control (NT) as indicated for 2 days then treated with 25 µm monastrol before being imaged using time-lapse confocal microscopy as described in Methods. Graph shows % of cells which reached metaphase as a function of time after the start of prophase (first frame with evidence of condensed chromosomes). Small vertical lines indicate cells at the end of imaging time. Gehan-Breslow-Wilcoxon test: **P<0.001 (compared with NT control).

Fig 5: IPA-3 treatment reduces myogenin expression throughout Mabs and C2C12 myogenic differentiation process. (A) Immunoblot analysis revealing p-Pak1 (S144) and total Pak1 in C2C12 protein extracts following 2 h treatment with various concentrations of the inhibitor IPA-3 or vehicle (dimethyl sulfoxide). Pak1 and p-Pak1 signals have been developed on separate filters and vinculin serves as a loading control. (B) Immunofluorescence microphotographs of Mabs labelled with an antibody against myogenin (green). (C, D) Bar plot representing the percentage of myogenin positive cells for Mabs and C2C12, respectively. (E) The graph illustrates the percentage difference of myogenin positive cells during Mabs myogenic differentiation program. (F) Graph showing the different percentage of myogenin positive cells during C2C12 differentiation. The values represented are means of at least three independent experiments + SEM. Statistical significance has been evaluated using the unpaired t-test, *P = 0.05, **P = 0.01. Scale bar: 100 µm. PAK1, p21 protein-activated kinase 1; Mabs, mesoangioblasts.