Fig 1: RAC1 (rho family, small GTP binding protein) inhibition mitigated pressure overload‐induced heart failure. A, Representative M‐mode echocardiography, gross appearance of whole hearts (scale bar, 1 mm), heart cross‐sections stained with hematoxylin and eosin (scale bar, 1 mm), histological analysis of heart sections by picrosirius red staining (scale bar, 50 μm) and cell boundaries demarcated with wheat germ agglutinin staining (scale bar, 25 μm) from Pkm2 conditional knockout and Pkm2 f/f mice after transverse aortic constriction surgery cotreated with saline or NSC23766. B, Quantitative analyses of echocardiography showing ejection fraction and fractional shortening, n=9 to 10. C, The ratio of heart weight to body weight, n=9 to 10. D, Statistical results for myocardial interstitial fibrosis analyzed by ImageJ software. n=6 to 7. E, Statistical results for the cell cross‐sectional area, n=9 to 10. F, Western blot and quantification of β‐MHC expression in hearts of Pkm2 conditional knockout and Pkm2f/f mice after transverse aortic constriction surgery cotreated with saline or NSC23766. β‐actin served as loading control. β‐MHC indicates β‐myosin heavy chain; cKO, conditional knockout; EF, ejection fraction; FS, fractional shortening; HE, hematoxylin and eosin; HW/BW, heart weight to body weight; Pkm2, pyruvate kinase M2; PSR, picrosirius red; RAC1, rho family, small GTP binding protein; TAC, transverse aortic constriction; and WGA, wheat germ agglutinin. Values represent as the mean±SEM; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.
Fig 2: eEF2K inhibition increases STAT3 phosphorylation. (A) A549 cells with or without eEF2K depletion were analyzed by immunoblotting analyses with the indicated antibodies. (B) A549 cells with or without eEF2K depletion or overexpression were analyzed by immunoblotting analyses with the indicated antibodies (Ba). The relative phosphorylation levels of the STAT3 to STAT3 were quantified (Bb). (C) eEF2K was immunoprecipitated from lysates of A549 cells. Immunoblotting analyses were performed with the indicated antibodies. (D, E) A549 cell lysates were prepared. Immunoprecipitation and immunoblotting analyses were performed with the indicated antibodies. (F, G) Lysates of A549 cells with or without eEF2K depletion (Fa) or overexpression (Ga) were immunoprecipitated with an anti-PKM2 antibody. Immunoblotting analyses were performed with the indicated antibodies. The relative phosphorylation levels of the STAT3 to PKM2 were quantified (Fb and Gb). **P < 0.01, vs shNC or **P < 0.01, vs Crtl. (H) Lysates of A549 cells with or without eEF2K depletion or overexpression were analyzed by immunoblotting analyses with the indicated antibodies
Fig 3: PKM2 (pyruvate kinase M2) acted as a protein kinase to inhibit RAC1 (rho family, small GTP binding protein) activation.(A through C) Western blot and quantitation of PKM2 and PKM1 (pyruvate kinase M1) expression in neonatal rat cardiomyocytes after Pkm2 knockdown. β‐actin served as loading control, n=4. D, Relative pyruvate production determined by an absorbance assay in NRCMs after Pkm2 knockdown, n=4. E, Immunoprecipitation (IP) assay using anti‐PKM2 antibody in neonatal mouse cardiomyocytes. F, Immunoprecipitation assay using anti‐RAC1 antibody in neonatal mouse cardiomyocytes. G, Representative immunofluorescence images showing the colocalization of PKM2 (green) and RAC1 (red) in adult mouse cardiomyocytes and NRCMs by confocal immunofluorescence analysis (scale bars, 25 μm in adult mouse cardiomyocytes; 10 μm in NRCMs). Line profile analyses showing the distribution and intensity. The Pearson coefficient was measured from the images using ImageJ software (n=21). H, Representative Western blot and Coomassie brilliant blue staining showing the phosphorylation of RAC1 by PKM2 in vitro. Phosphoenolpyruvate was used as the phosphate donor. I, Western blot and quantitation of p‐RAC1 (S71) expression in NRCMs after Pkm2 knockdown. β‐actin served as loading control. J, Western blot and quantitation of RAC1 protein expression in the cycloheximide chase experiment. β‐actin served as loading control. AMCMs indicates adult mouse cardiomyocytes; IB, immunoblot; IgG, immunoglobulin G; IP, Immunoprecipitation; NC, negative control; NRCMs, neonatal rat cardiomyocytes; PKM1, pyruvate kinase M1; PKM2, pyruvate kinase M2; RAC1, rho family, small GTP binding protein; TAC, transverse aortic constriction; and WGA, wheat germ agglutinin. Values represent as the mean±SEM; *P<0.05, **P<0.01,****P<0.0001.
Fig 4: eEF2K depletion promotes tumor growth in mice. a shNC- and sheEF2K-expressing A549 cells were implanted (s.c.) into right flanks of Balb/c nude mice as described in Methods. Tumor issues were dissected until 40 days after injection and were photographed. Statistical analysis of the tumor volumes in each group was performed using one-way analysis of variance (Student’s t-test). n = 6, *p < 0.05, **p < 0.01, vs shNC. b Immunohistochemical (IHC) staining of tumor specimens with an anti-PCNA antibody (left). Quantification of PCNA positive cells from tumor specimens (n = 3) from shNC and sheEF2K treated mice (right). The mean and standard deviation are plotted. c Immunoblotting analyses of tumor specimens were performed with the indicated antibodies. d A model of mechanism underlying eEF2K-regulated metabolism and tumor cell proliferation by phosphorylation of PKM2 and regulation of STAT3 phosphorylation and c-Myc expression. The dotted line in figure represents traditional signaling pathway
Fig 5: Effect of blocking PKM2 on oncogenic role of TRIB2 in cell growth.A, B RTCA station analysis of knocking down or overexpression of TRIB2 on regulating A549 cell migration. Data were expressed as mean ± SD for triplicate experiments, **p < 0.01; Student’s t test. C–F Analysis of xenografts of stable A549 cells expressed of lentivirus si-TRIB2, si-PKM2, or controls (n = 5). Quantitative data of tumor weight (D) and detected tumor volume change (E) of xenografts were analyzed. Protein expression was analyzed by immunoblotting (F). Data were expressed as median (interquartile range), **p < 0.01; Kruskal–Wallis H test. G–I Analysis of xenograft tumors of lv-TRIB2 with lv-si-PKM2 or control treatment (n = 5). Quantitative data of tumor weight (H) and detected tumor volume change (I) of xenografts were analyzed. Protein expression was analyzed by immunoblotting (J). Data were expressed as median (interquartile range), *p < 0.05, **p < 0.01; Kruskal–Wallis H test. K PKM2 and TRIB2 downregulation obviously inhibited A549 cell proliferation. Data are expressed as the mean ± SD of triplicate experiments, **p < 0.01; ANOVA. L Blocking PKM2 attenuated TRIB2-promoting A549 cell proliferation compared with that in control treatment. Data are expressed as the mean ± SD of triplicate experiments, *p < 0.01; ANOVA. M RTCA station analysis of siPKM2 and siTRIB2 on regulating cell migration. Migrative cells were counted on the left. Data were expressed as mean ± SD for triplicate experiments, **p < 0.01; ANOVA. N RTCA station analysis showed that si-PKM2 blocked the TRIB2-promoting cell migration. Data were expressed as mean ± SD for triplicate experiments, *p < 0.05; **p < 0.01; ANOVA.
Supplier Page from Abcam for Recombinant human PKM2 protein (Active)