Fig 1: Direct phosphorylation of PPAR? by PAK4. (A) Recombinant PPAR? was incubated with active PAK4 and [32P]ATP for 30 min at 30°C, and proteins in the mixture were resolved by SDS-PAGE. The band was visualized by autoradiography of 32P-labelled protein. Loading of proteins was confirmed by Coomassie blue staining. (B, C) After transfection of HEK293T cells as indicated, co-IP was performed to determine PAK4 interaction with and phosphorylation of PPAR?. (D) C2C12 myoblasts were PAK4 overexpressed or silenced, and then phosphorylation of PPAR?, ERK, and CDK5 on Day 5 was analysed by western blotting. (E) C2C12 myoblasts were transfected with scrambled siRNA (siCtrl) or siRNA against CDK5 (siCDK5), and then PAK4 phosphorylation of PPAR? was analysed by western blotting. (F) C2C12 myoblasts were pretreated with ERK inhibitor PD98059 (10 µM), and PAK4 phosphorylation of PPAR? was analysed. (G) PTEN-luciferase activities were determined after transfection of HEK293T cells as indicated. (H) C2C12 cells were transfected with wild-type or mutant PPAR? (S273A) along with PAK4, and then PI3K–Akt signalling and myogenic markers on Day 5 were determined. Values are mean ± SD. ** P < 0.01 vs. none; ## P < 0.01 vs. PPAR?; $$ P < 0.01 vs. PAK4 + PPAR?.
Fig 2: Acceleration of skeletal muscle regeneration in PAK4‐silenced mice. The tibialis anterior (TA) muscles of C57BL/6 mice were injected with scrambled siRNA (siCtrl) or siRNA against PAK4 (siPAK4) and then injured by intramuscular injection of NTX as shown in Figure 3A. (A) Western blotting analysis for PAK4 in PAK4 silenced muscles. (B) Immunofluorescence analysis of eMyHC‐positive fibres in TA muscles. (C) H&E and immunofluorescence analyses of sections. Average cross‐sectional area (CSA) of regenerating myofibres and the percentage of myofibres containing two or more centrally located nuclei per field were determined from immunofluorescence sections. (D) Time course analysis of myogenic markers by western blotting. (E) Immunofluorescence staining of Myog‐positive or desmin‐positive myofibres at Day 5. Arrowheads indicate MyoG‐positive myofibres. Values are mean ± SD. ** P < 0.01 vs. D0; ## P < 0.01 vs. siCtrl.
Fig 3: Impairment of skeletal muscle regeneration in PAK4‐overexpressing mice. (A) The tibialis anterior (TA) muscles of C57BL/6 mice were injected with Ad‐LacZ, Ad‐PAK4, or Ad‐PAK4S474A and then injured by intramuscular injection of NTX. (B) Time course analysis of PAK4 and myogenic markers after NTX injection by western blotting. (C) Immunofluorescence analysis of eMyHC‐positive fibres in TA muscles. (D) Immunofluorescence staining of Myog‐positive or desmin‐positive myofibres at Day 5. Arrowheads indicate MyoG‐positive myofibres. (E) Western blot analysis of Myog and eMyHC in injured TA muscles of control and PAK4‐overexpressing mice at 3 or 5 days after injury (n = 3). (F) H&E and immunofluorescence analyses of sections. Average cross‐sectional area (CSA) of regenerating myofibres and the percentage of myofibres containing two or more centrally located nuclei per field were determined from immunofluorescence sections. Values are mean ± SD. * P < 0.05 and ** P < 0.01 vs. Ad‐LacZ; # P < 0.05 and ## P < 0.01 vs. AdPAK4.
Fig 4: Increase of PPAR?-mediated PTEN transcription by PAK4. (A) Genotype-Tissue Expression (GTEx) analysis of human skeletal muscle. Pearson correlation coefficients between PAK4 and PTEN, PAK4 and PPARG, and PPARG and PTEN in human skeletal muscle were calculated. TPM, transcripts per million. (B, C) After transfection of HEK293T cells as indicated, PPRE-luciferase and PTEN-luciferase activities were determined. (D) Maps of human and mouse Pten promoters and ChIP-qPCR assay showing binding of PPAR? to the Pten promoters. (E, F) C2C12 myoblasts were transfected with scrambled siRNA (siCtrl) or siRNA against PPAR? (siPPAR?), and protein levels of PTEN and PI3K-Akt pathway and myotube formation were compared. Values are mean ± SD. ** P < 0.01 vs. none, IgG, or siCtrl; ## P < 0.01 vs. PPAR?, Ad-LacZ, or siCtrl + PAK4; $$ P < 0.01 vs. PAK4 + PPAR? or Ad-PAK4.
Fig 5: Suppression of PI3K-Akt pathway by PAK4. (A, B) Pak4 gene was either overexpressed or silenced in C2C12 cells as indicated, and PI3K-Akt signalling pathway on Day 5 was analysed by western blotting. (C, D) The tibialis anterior (TA) muscle was injected with either PAK4 adenovirus or PAK4 siRNA and PI3K-Akt signalling pathway on 7 days after NTX injection was analysed by western blotting. (E) mRNA levels of Pten were determined in PAK4-overexpressing C2C12 cells and TA muscles. (F, G) C2C12 myoblasts were transfected with siCtrl or siPTEN, and PI3K-Akt signalling and myotube formation were compared. Values are mean ± SD. ** P < 0.01 vs. Ad-LacZ or siCtrl; ## P < 0.01 vs. AdPAK4 or siCtrl + PAK4.
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