Fig 2: Effects of arginine supplementation on myogenic differentiation of C2C12 myoblasts. The 80% to 90% confluent C2C12 cells were cultured in differentiation medium containing 0.1, 0.4 (CON), and 1.2 mmol/L arginine for 2 (A) or 4 d (B–C). (A) Relative mRNA expression levels of MyoG and Myomaker normalized to GAPDH mRNA, measured by real-time quantitative PCR in triplicate. (B, C) Protein expression levels of MyoD1 and Myosin were measured by Western blot analysis. Equal loading was monitored with anti-GAPDH antibody. Values are means ± SEM (n = 3). Different letters on bars indicate a significant difference (P < 0.05). CON, control; 0.1 mmol/L, 0.1 mmol/L arginine; 1.2 mmol/L, 1.2 mmol/L arginine.

Fig 3: Characterization of Pik3r1 enhancer-promoter communication. (A) Quantitative local 4C-seq signal for the Pik3r1 viewpoint in skeletal muscle. GR binding sites (GR) are depicted with blue lines, GREs with blue stars, Ctcf binding sites identified by ChIP-seq with cyan arrowheads, indicating convergent motif orientation. H3K4me1 and Myod1 ChIP-seq profiles are presented in green and red, respectively. Enhancer-promoter contact regions are highlighted in orange on the 4C-seq chart. Ctcf-bound promoter and enhancer regions are boxed in cyan. (B, C) Quantitative local 4C-seq signal for the Pik3r1 viewpoint in skeletal muscle of control (upper panels in B and C) mice compared with skeletal muscle GR(i)skm-/- mice (B, lower panel), or with double positive (DP) thymocytes (C, lower panel). Enhancer-promoter contact regions lost upon GR ablation and absent in DP thymocytes are highlighted in red. Contact regions present in both control and GR(i)skm-/- mice are highlighted in light blue. Contact regions present in both control and DP thymocytes are highlighted in light green. Contact regions that are specific for GR(i)skm-/- mice and DP thymocytes are highlighted in dark blue and dark green, respectively. GR binding sites (GBS) are depicted with black lines and GREs with black stars.

Fig 4: NIS blocked arginine-promoted myogenic differentiation of C2C12 myoblasts. C2C12 cells at 80%-90% confluence were cultured in differentiation medium and treated with 1.2 mmol/L arginine in the absence or presence of NIS (10 μmol/L) for 4 (A, B) or 2 d (C, D). (A) Immunostaining of C2C12 myotubes using antibody against myosin. (B) Myotube area is calculated as described in methods. (C, D) Relative mRNA expression levels of MyoD1, MyoG, Cav1.1, RyR1 and Stim1 normalized to GAPDH mRNA, measured by real-time quantitative PCR in triplicate. Values are means ± SEM (n = 3). ∗P < 0.05. ∗∗P < 0.01. Scale bars, 100 μm. CON, control; Arg, 1.2 mmol/L arginine; NIS, 10 μmol/L nisoldipine.

Fig 5: G6PD was down−regulated during skeletal muscle formation. (a) The transcriptional level of G6PD during C2C12 differentiation; data represent mean ± SD; n = 3; * p < 0.05, ** p < 0.01. (b) Western blot analysis for G6PD and MyHC during C2C12 differentiation. (c) The transcriptional level of G6PD in adult mice tissues; n = 3. (d) The protein level of G6PD in adult mice tissues. (e) The transcriptional level of MyHC and Myh3 during embryonic skeletal muscle formation; n = 3. (f) The transcriptional level of MyoD, MyoG, Pax3 and G6PD during embryonic skeletal muscle formation; n = 3. (g) Western blot analysis for MyHC, MyoD, MyoG and G6PD during embryonic skeletal muscle formation. (h) IGV graph shows the binding peak between G6PD promoter and H3K36me3, H3K79me2, H3K27me3 or H3ac. (i,j) Binding of H3K9ac to the G6PD promoter was detected by ChIP assays. DNA isolated from the precipitated complexes is the template for PCR, then PCR products were analyzed on a 1.5% agarose gel (i) and quantified by ImageJ (V1.8.0, National Institutes of Health, Berlin, Germany) (j); * p < 0.05. (k,l) The binding of H3K27ac to the G6PD promoter was detected by ChIP assays. PCR products were analyzed on a 1.5% agarose gel (k), and quantified by ImageJ (l); ** p < 0.01. (m) The percentage of DNA methylation and non-methylation for G6PD promoter was tested by BSP-seq and analyzed by SnapGene; n = 60; * p < 0.05. H3K36me3, histone H3 trimethylation at lysine 36; H3K79me2, histone H3 dimethylation at lysine 79; H3K27me3, histone H3 trimethylation at lysine 27; H3ac, histone H3 acetylation; d0, C2C12 myoblasts cultured in growth medium; myocyte, C2C12 cells cultured in differentiation medium for 80 h; d8, C2C12 cells cultured in differentiation medium for 8 d.