Fig 2: 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.

Fig 3: 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 4: 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 5: PI3K regulates Rac-driven macropinocytosis. (A) Immunoblot analysis of phospho-Akt (Thr308) in glucose independent (red) and glucose addicted (blue) cells following 6 h of glucose starvation. (B) Immunoblot analysis of Pak and Akt phosphorylation following treatment with vehicle or 1 µm BKM120 for 1 h. (C) Quantification of FITC-dextran uptake in glucose starved cells treated with either vehicle or 1 µm BKM120 for 1 h prior to incubation with FITC-dextran. Data is normalized to vehicle treated cells. Error bars indicate ± SD of 10 fields scored containing at least 10 cells, and statistics was calculated using Student’s t-test. (D) Immunoblot analysis of Pak signaling in Rac1 KO H1299 control cells or cells stably overexpressing the constitutive active Pak1 mutant T423E following treatment with vehicle or 1 µm BKM120 for 1 h. Densitometry was used to quantify P-Pak band intensities normalized to T-Pak1. (E) Quantification of FITC-dextran uptake in glucose starved Rac1 WT, Rac1 KO, and Rac1 KO/Pak1 (T423E) cells treated with vehicle or 1 µm BKM120 1 h prior to incubation with FITC-dextran. Data is normalized to Rac1 WT cells, error bars indicate ± SD of 10 fields scored containing at least 10 cells. Significance was calculated using ANOVA with Holm-Sidak multiple comparisons between control and BKM120 treated cell lines, ** p < 0.005, *** p < 0.0001.