Fig 2: sPIF increases the differentiation of mouse and human myoblasts.C2C12 cells and human myoblasts were cultured in 2% HS and treated with sPIF (300ng/ml) for 10 days. The expression of the different myogenic proteins was determined using western blot analysis (*P < 0.001, control vs sPIF-treated cells; **P < 0.01, MyHC and troponin expression in control vs DMD muscle cells) (a). C2C12 cells were cultured in 2% HS and after 10 days stained with DAPI and anti-myosin heavy chain (MyHC) antibody (FITC). Cell fusion was analyzed using confocal microscopy (b). Human myoblasts derived from three DMD patients (DMD-mus) and two healthy donors (H-mus) were treated with sPIF (300ng/ml) and analyzed for the expression of MyHC and troponin following 10 days in fusion medium using RT-PCR (P < 0.001) (c). The internalization of sPIF-FITC into C2C12 cells was analyzed by live cell imaging using confocal microscopy. The fluorescence of the cells at the initial stage of the experiment and after 72 min is presented (d). The effects of sPIF (300 nM, 2 days) on the expression of Col 1A1, Col 1A2, and TGF-ß in DMD myoblasts were determined using RT-PCR (P < 0.001) (e). The expression of miR-21 was determined in muscle cells obtained from healthy donors and DMD patients (f) and in DMD muscle cells treated with sPIF (*P < 0.001) (g). The effects of miR-21 antagomiR on the expression of Col 1A1 and Col 1A2 in control and sPIF-treated DMD muscle cells were determined after 3 days of treatment (*P < 0.001 for sPIF and miR-21 antagomiR vs control; **P < 0.01 for sPIF+miR-21 antagomiR vs sPIF alone) (h). The results are representative of three similar experiments and are presented as the means ± SE. DAPI, 4',6-diamidino-2-phenylindole; DMD, Duchenne muscular dystrophy; HS, horse serum; RT-PCR, real-time polymerase chain reaction; sPIF, synthetic PIF analog

Fig 3: The regulation of H3K27me3 during embryonic muscle development. (A) GSEA plot of genes involved in the skeletal organ muscle development and calcium signaling pathway on d33 and d90. (B) The gene expression heatmap of HDEGs (left) in the skeletal organ muscle development and calcium signaling pathway. Genome browser tracks (right) showing the H3K27me3 ChIP-seq (orange) and RNA-seq (light blue) data at SLN and MYH7 loci during embryonic muscle development. H3K27me3 peak region was colored in gray. (C) GSEA plot of genes involved in the process of ATP metabolic and lipid metabolic on d33 and d90. (D) The gene expression heatmap of HDEGs (left) in the process of ATP metabolic and lipid metabolic. Genome browser tracks (right) showing the H3K27me3 ChIP-seq (orange) and RNA-seq (light blue) data at AK1 and SLC25A4 loci during embryonic muscle development. H3K27me3 peak region was colored in gray.

Fig 4: SLI increased MyHC expression in the GA muscle, decreased MAFbx and Mstn expression, and regulated JNK and ERK to inhibit FOXO entry into the nucleus. (a) Using tubulin as an internal control, the expression of procaspase3 and active caspase3 was detected by Western blotting; the relative expression level of protein was detected by ImageJ software. Tubulin was used as internal controls and corrected to the NC group. Data are shown as mean ± SD, n = 3/group; **p < 0.01, ***p < 0.001 versus group LLC + DDP. (b) Using tubulin as an internal control, the expression of the MyHC, MAFbx, Mstn, JNK/FoxO, and ERK/FoxO signaling pathways was detected by Western blotting; the expression of nuclear FoxO3a using Lamin A as an internal control. (c) The relative expression level of protein was detected by ImageJ software. Tubulin and Lamin A were used as internal controls and corrected to the NC group. Data are shown as mean ± SD, n = 3/group; *p < 0.05, **p < 0.01, ***p < 0.001 versus group LLC + DDP. (d) The qRT-PCR method to detect the expression of MyoG, Mstn, Fbxo32, and Ub mRNA in the GA muscle; RPS18 was used as an internal control. Data are shown as mean ± SD, n = 5/group; **p < 0.01, ***p < 0.001 versus group LLC + DDP.

Fig 5: lncMGPF regulates HuR-mediated mRNA stability through its core binding region and increases HuR accumulation in the cytoplasm. (A) RNA pulldown assays of full-length (FL) and truncated fragments of lncMGPF show that FL and F3 (1176–1795) specifically interact with HuR in C2C12 myoblasts. ß-actin and GAPDH were used as negative controls. (B, C) RNA pulldown assays of truncated fragments of lncMGPF show that F7 (1630–1795) is the core HuR binding region for the interaction of lncMGPF with HuR (B) and deletion of the classical binding motifs (1764–1779) does not affect this interaction (C) in C2C12 myoblasts. ß-actin and GAPDH were used as negative controls. (D, E) qRT-PCR of RNA stability assays in differentiated C2C12 myoblasts shows that lncMGPF enhances the stabilities of MyoD (D) and MyoG (E) mRNAs via the specific HuR binding region. (F) RNA pulldown assays of truncated fragments of lncMGPF show that the deletion of any binding motif alone (F7?1764–1779; F7?1730–1743; F7?1747–1763) does not affect the binding of lncMGPF and HuR, while no binding of lncMGPF and HuR is observed after deletion of three binding motifs simultaneously (F7?1730–1779) in C2C12 myoblasts. (G, I) Western blotting analyses of nuclear and cytoplasmic protein fractionations show that lncMGPF overexpression increases cytoplasmic HuR (G), whereas lncMGPF knockdown reduces HuR accumulation in the cytoplasm (I) in C2C12 myoblasts. a-Tubulin was used as a cytoplasmic protein marker and H3 was used as a nuclear protein marker. (H, J) Representative images of HuR immunofluorescence staining assays confirm that lncMGPF overexpression (H) and knockdown (J) increase and decrease HuR accumulation in the cytoplasm in C2C12 myoblasts, respectively. Magnification, 63×. (K) Western blotting of differentiated C2C12 myoblasts shows that lncMGPF knockdown significantly reduces and HuR-CP1 protein levels. (L) Western blotting of differentiated C2C12 myoblasts shows that lncMGPF overexpression significantly increases MyHC and HuR-CP1 protein levels. The relative mRNA levels were normalized to ß-actin. The data are presented as mean ± SD of three independent experiments; * P < 0.05, ** P < 0.01.