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: Diagram depicting the current model for the regulation of NIS functional residency at the PM by the SRC/RAC1/PIP5K/PAK/EZRIN/NIS pathway. In brief, NIS localization and function at the PM depends on its binding to SRC kinase, the activity of which, triggered by yet-unknown factors, leads to the recruitment and activation of the small GTPase RAC1. RAC1 signals not through p38 nor through JNK (two kinases downstream of RAC1 known to participate in the transcriptional regulation of NIS [59,60]), but through PAK1 and PIP5K. The combined activity of these kinases promotes ARP2/3-mediated actin polymerization, and the recruitment and binding of the actin anchoring protein EZRIN to NIS, promoting its residency and function at the PM of normal and TC cells. The figure was created with BioRender.com, accessed on 26 July 2021.

Fig 4: 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 5: LPS-induced endotoxemia leads to myocardial inflammation, increased myocardial PGE 2 levels, and MEF2 activationMEF2-lacZ reporter mice (BALB/c background, 6–12 weeks old) were treated with 7 mg/kg lipopolysaccharide from Escherichia coli (O111:B4) or saline intraperitoneally and were sacrificed after 24 h. mRNA levels of different inflammatory cytokines, as indicated. The graphs show relative mRNA levels, fold increase compared to saline-treated controls, normalized to 18s content.PGE2 was quantified after mechanical homogenization of deeply frozen hearts using nano-liquid chromatography-tandem mass spectrometry.Histologic and macroscopic stainings of ß-galactosidase activity in MEF2-lacZ reporter mice show MEF2 activation (blue cells and precipitates, respectively) in the myocardium upon LPS treatment. Saline-treated littermates served as control. Scale bar of histological stainings is 100 µm. Quantification of whole-heart stainings is shown in the right panel (pixel intensity in blue channel normalized to total intensity).Induction of PKD, HDAC5, and PAK2 in myocardial inflammations. Immunoblots were performed on extracts of hearts of saline- and LPS-treated mice. Representative blots are shown in the left panel, and the quantification is in the right panel.Data information: Values are mean ± s.e.m. The exact n values are shown in the bottom of the bar graphs. *P < 0.05, Welch's two-tailed unpaired t-test was used for statistical analysis, n.s. = not significant. In (C), representative images of myocardial sections are shown from three independent experiments and representative whole hearts of six independent experiments. P-values: IL6, P = 0.0007 (A); TNFa, P < 0.0001 (A); PGE2, P = 0.0016 (B); blue pixel intensity, P = 0.0083 (C); phospho-PKD-Ser-744/Ser-748, P = 0.0049 (D); phospho-PKD-Ser-916, P = 0.2684 (D); phospho-HDAC5-Ser-498 (D), P = 0.0019; phospho-HDAC5-Ser-259, P = 0.0003 (D); phospho-PAK2-Ser-192/Ser-197, P = 0.1962 (D); phospho-PAK2-Thr-402, P = 0.0446 (D). The exact n and P-values can also be found in the Source Data Excel file for Fig 7.Source data are available online for this figure.