Fig 2: Effect of anti-PAK siRNAs on JURKAT cell adhesion.Cells were nucleofected with 2 µl esiRNA against PAK1 or PAK2, with the combination of both esiRNAs (PAK1+PAK2) or with non-targeting siRNA (ctrl) and the cell adhesion to fibronectin (ACF- adherent cell fraction) was tested 22 h post nucleofection. The level of PAK1, PAK2 and pPAK2 (Ser141) was assessed at the same time by western-bloting. (A) The experiment with the highest silencing efficacy obtained. The graph of cell adhesivity shows means and standard deviations of sample quadruplicates. (B) Summary of results for all adhesion experiments (N = 7). The adherent cell fraction was expressed as relative to the adhesivity of controls and the resulting values were compared using paired Student’s t-test.

Fig 3: Expression of PAK kinases in the studied cell lines.Cell proteins were separated using gel electrophoresis and the expression levels of PAK1, PAK2, phospho-PAK (Ser 144/Ser141) and phospho-PAK2 (Ser20) were determined using western-blotting. (A) Lanes 1–8 correspond to the individual cell lines: (1) JURL-MK1, (2) MOLM-7, (3) K562, (4) CML-T1, (5) HL60, (6) Karpas-299, (7) Jurkat, (8) HEL. (B) CML-T1 cells were treated with IPA-3 at the indicated concetrations for 2 h, then lysed and subjected to electrophoresis and western-blotting. (C) Relative expression levels of phospho-PAK2 (Ser141) and PAK2 in cell lines treated for 2 h with IPA-3 at different concentrations. The band intensity from western-blots with anti-PAK antibodies was corrected for small differencies in protein loads using actin bands and the resulting values were expressed as relative to controls (without treatment). JURL-MK1:closed circles, MOLM-7:closed triangles, CML-T1:closed squares, K562:open circles, HL60:open squares, JURKAT:open triangles, Karpas-299:open diamonds, HEL:closed diamonds.

Fig 4: Time-course analyses exhibit cardiac dysfunction associated with maladaptive ER stress response following high-fat high-sucrose diet (HFHSD)(A) Representative images and quantification graphs (protein/Gß ratio, AU) from densitometry analysis of PAK2 expression and phosphorylation (pPAK2) and ER stress markers from heart lysates of human subjects. ER stress markers include GRP94, GRP78, phosphorylated IRE1, IRE1, spliced XBP1 (XBP1s), unspliced XBP1 (XBP1u), ATF6, phosphorylated PERK (pPERK), PERK, ATF4, and CHOP.(B–D) Data graphs showing (B) serum insulin, (C) fasted blood glucose, and (D) intraperitoneal glucose tolerance test after 8–24 weeks of feeding in C57BL/6J mice and bar graph displaying AUC.(E) Graphs depicting isovolumetric relaxation time (IVRT) and E/A mitral valve flow from pulse-wave Doppler imaging indicating diastolic function.(F) From M-mode echocardiography: fractional shortening (FS%) and ejection fraction (EF%) indicating systolic function.(G) Relative mRNA expression of Nppb (chow normalized to 1, AU, arbitrary unit).(H) Immunoblots and quantification graphs (protein/Gß ratio, AU) from densitometry analysis of key ER stress transcription factors from heart lysates of C57BL/6J mice. Gß is loading control.Each data point represents one animal or subject. N = 3–4/group in (A), N = 4–5/group in (B–D), N/group, chow = 4, HFHSD = 6 in (E and F), N = 6/group in (G), and N = 3–5/group in (H). All data are presented as mean ± SEM. p values (shown in each panel) versus corresponding controls determined by two-tailed Student's t test in (A); and by ANOVA with Tukey's/Sidak's post hoc tests in (B–H).

Fig 5: A retained ER function is a requisite for anti-inflammatory action of vildagliptin under diabetes(A) Representative immunoblot images and quantification of PAK2 activation and CHOP expression from isolated ARCM in the fact of vildagliptin (Vil) (20 µM) with diabetes-mimicking stress (N = 4).(B) Strategy for Vildagliptin administration (5 mg/kg/day in drinking water) with or without application of AAV9-Pak2 in Pak2cKO mice fed with HFHSD.(C and D) (C) BW (D) intraperitoneal glucose tolerance test.(E and F) (E) IVRT from pulse-wave Doppler analysis and (F) calculated FS from M-mode echocardiography (N = 6 in C–F).(G) Representative immunofluorescence staining (scale bar: 25 µm) and quantification of left: Mac3- (red) and right: CD68- (green)-positive non-cardiomyocytes in heart sections. Cardiomyocytes are counterstained with a-actinin (green) and troponin (red). Nuclei are stained with DAPI (blue) (N = 4/group).(H) Relative mRNA expression (normalized to chow, AU) of pro- and anti-inflammatory markers in Pak2cKO hearts (N = 5–6/group).(I) Immunoprecipitation of acetylated HMGB1 from protein lysate. Immunoblot images and quantification of CHOP and HMGB1 (N = 4/group). Gß is loading control.Inset: key for data points; each data point represents one animal/experiment. All data are presented as mean ± SEM. p values (shown in each panel) versus corresponding controls, determined by ANOVA with Tukey's post hoc tests in (A and C–I).