Fig 1: ITGB1 is essential for FNDC1-promoted skeletal muscle regeneration in CTX-induced muscle injury in mice.(A) Representative H&E staining of TA muscle from AAV-Scra or AAV-shItgb1 mice at days 5 and 14 post-injury (n = 6 mice). TA muscle from AAV-Scra or AAV-shItgb1 mice were either given Control or mFNDC1 (2.5 mg/kg body weight) every 2 days via intramuscular injection starting with treatment with CTX. Scale bars = 20 μm. (B) Representative immunofluorescence staining of eMYHC+ fibers in TA muscle at day 5 post-injury (n = 6 mice). Staining for eMYHC (in red) marks the newborn myofibrils. Cell membrane was stained with WGA (in green) and nuclei were counterstained with DAPI (in blue). Scale bars = 100 μm. (C, D) Average CSA and frequency distribution of eMYHC+ myofiber minimal Feret’s diameter in TA muscle from Itgb1 knockdown mice treated with Control or mFNDC1 at day 5 post-injury (n = 6 mice). One-way ANOVA, AAV-scra+Control vs. AAV-scra+mFNDC1, P = 1.03 × 10−12; AAV-scra+mFNDC1 vs. AAV-shItgb1+Control, P = 2.6 × 10−14 and AAV-scra+mFNDC1 vs. AAV-shItgb1 + mFNDC1, P = 2.6 × 10−14. (E, F) Representative immunoblotting (E) and quantification (F) of indicated proteins in TA muscle at day 5 post-injury (n = 3 mice). One-way ANOVA, AAV-scra+Control vs. AAV-scra+mFNDC1, P = 4.30 × 10−8; AAV-scra+mFNDC1 vs. AAV-shItgb1+Control, P = 1.04 × 10−8 and AAV-scra+mFNDC1 vs. AAV-shItgb1 + mFNDC1, P = 9.06 × 10−9 for eMYHC. AAV-scra+Control vs. AAV-scra+mFNDC1, P = 2.27 × 10−5; AAV-scra+mFNDC1 vs. AAV-shItgb1+Control, P = 4.64 × 10−7 and AAV-scra+mFNDC1 vs. AAV-shItgb1 + mFNDC1, P = 3.13 × 10−7 for ITGB1; AAV-scra+Control vs. AAV-scra+mFNDC1, P = 1.42 × 10−6; AAV-scra+mFNDC1 vs. AAV-shItgb1+Control, P = 2.13 × 10−8 and AAV-scra+mFNDC1 vs. AAV-shItgb1 + mFNDC1, P = 1.55 × 10−8 for p-FAK/FAK; AAV-scra+Control vs. AAV-scra+mFNDC1, P = 1.58 × 10−5; AAV-scra+mFNDC1 vs. AAV-shItgb1+Control, P = 7.10 × 10−8 and AAV-scra+mFNDC1 vs. AAV-shItgb1 + mFNDC1, P = 6.80 × 10−8 for p-AKT/AKT; AAV-scra+Control vs. AAV-scra+mFNDC1, P = 1.89 × 10−4; AAV-scra+mFNDC1 vs. AAV-shItgb1+Control, P = 8.23 × 10−7 and AAV-scra+mFNDC1 vs. AAV-shItgb1 + mFNDC1, P = 6.54 × 10−7 for p-mTOR/mTOR. Data are represented as mean ± SEM. **p < 0.01. Source data are available online for this figure.
Fig 2: Nuclear LRP6 directs the muscle‐specific splicing of Itgb1D in striated muscle cells. (A) Identification of LRP6 in the nuclei of cells. CM: neonatal rat cardiomyocytes. The α‐tubulin and histone H3 were used as cytoplasmic and nuclear markers, respectively. Right: pooled data. *P < .05 compared with Day 0. (B) Binding of LRP6 protein to small nuclear RNAs (U1/2/4/5/6 snRNAs), as detected by immunoprecipitation from lysates of CM cells (left‐upper and ‐middle) and an in vitro protein‐RNA pull‐down assay with the purified LRP6 proteins and small nuclear RNAs (U1/2/4/5/6 snRNAs) (left‐lower). Right, Coomassie blue staining of the purified LRP6‐flag recombinant proteins. (C) In the nuclei of HeLa cells, LRP6 partially co‐localized with the splice factors U2AF65, PTBP1 and CUG‐BP1. Scale bar, 5 μm. (D) Specific activity of LRP6 on Itgb1 RNA monitored in a cell‐based splice‐reporter minigene assay using the exons that encode the alternative exon D. Top, schematic representation of the Itgb1 splicing reporter. Western blotting examination of LRP6 protein and gel electrophoresis of Itgb1 RNAs were conducted in the neonatal cardiomyocytes (CMs) (Bottom‐left) and the HeLa cells (Bottom‐right) subject to LRP6 knockdown and overexpression. Representative images from five independent experiments with similar results are shown
Fig 3: Roles of splicing repressors in the splicing switching of Itgb1 isoforms. (A) Western blotting examination of hnRNP A1 and PTB proteins during myodifferentiation of C2C12 myoblasts. DM, differentiation medium. Bottom: pooled data. *P < .05 compared with day 0. (B) Schematic of hnRNP A1 and PTB‐binding sites in the flanking introns of alternative exon D in the splicing reporter of Itgb1. (C) Effects of hnRNP A1 on the protein expression of ITGB1D in myocytes. (D) Effects of LRP6 on the protein expression of hnRNP A1 and PTBP1. The proteins were extracted from neonatal cardiomyocytes subject to hnRNP A1‐ or Lrp6‐siRNAs for 48 h. Right: pooled data. *P < .05 compared with si‐Ctrl. (E‐F) Effects of the splice repressor PTBP1 on the splicing of Itgb1D in the presence and absence of LRP6 in culture neonatal cardiomyocytes. Western blotting examination of LRP6, PTBP1 and ITGB1D proteins (E). E‐right: pooled data. *P < .05 compared with si‐Ctrl. (F‐top) Gel electrophoresis analysis of Itgb1 RNAs; (F‐bottom) gel electrophoresis analysis of the specific activity of PTBP1 on Itgb1 pre‐mRNA using the minigene. (G) Model of the LRP6‐mediated splicing of Itgb1D in striated muscle cells. Representative blots from five independent experiments with similar results are shown
Fig 4: Interaction of LRP6 with exon splicing enhancer determines the muscle‐specific splicing of Itgb1D. (A) Quantitative PCR detection of precursor Itgb1 and mature Itgb1D mRNAs retrieved by LRP6‐specific antibody compared with immunoglobulin G (IgG) in the RIP assay within NRCMs cells transfected with vectors expressing pre‐Itgb1 and Itgb1D mRNAs, respectively. (B) Identification of the LRP6 modules responsible for Itgb1D RNA binding. Gel electrophoresis (Top) and qPCR analysis (bottom) of Itgb1D RNAs immunoprecipitated by the LRP6 domains fused to the Flag tag. (C) In vivo selection of exonic splicing enhancer sequences. Top, schematic representation of serial deletions of the 81‐bp Exon D in the splicing reporter minigene construct; bottom, gel electrophoresis analysis of Itgb1 RNAs extracted from neonatal cardiomyocytes expressing NLS‐ICD and minigene mutants. Mut: mutation. Wt: wild type. (D) A point mutation‐dependent strategy utilized to visualize the cis‐regulatory element. Upper, diagram showing the point mutation; middle, gel electrophoresis analysis of Itgb1 RNAs extracted from neonatal cardiomyocytes expressing NLS‐ICD and minigene mutants. M: mutation. Lower, analysis of the identity of ESE in AED between mouse and human. (E) Identification of the amino acid residues responsible for LRP6 binding to the exonic splicing enhancer. Top, Diagram showing the point mutation within LRP6 C‐terminus. Bottom‐left, western blotting verification of the expression of wild and mutated NLS‐ICD (NLS‐GFP‐ICDWT and NLS‐GFPICDMut) proteins in cultured neonatal cardiomyocytes. Bottom‐right, Gel electrophoresis analysis of Itgb1 RNAs. Representative blots from five independent experiments with similar results are shown. Mut, mutation. WT, wild type
Fig 5: Integrin α5β1 is the receptor for FNDC1 in myoblast.(A) The top 10 proteins enriched with FNDC1 by cross-linking/Co-IP/MS analysis. (B) Co-IP of FNDC1 and Integrin β1 (ITGB1) in C2C12 cells. (C, D) Immunoprecipitation of FNDC1 and Integrin αv (ITGAV) or Integrin α5 (ITGA5) in C2C12 cells. (E, F) Pull-down assays. 100 nM His-tagged FNDC1 (truncated recombinant FNDC1 protein) was incubated with the indicated Flag-tagged integrin (5 nM) and then affinity adsorbed through a nickel column. Immunoblotting was performed to analyze protein interactions between integrins (α5 or αv) and mFNDC1 (E) or to analyze protein interaction between integrin α5β1 and mFNDC1 (F). (G) Surface plasmon resonance analysis of the FNDC1-Intergrin α5β1 interaction. Numbers above curves indicate the analyte concentration tested. All experiments were performed in triplicate with a mobile phase of HBS-EP. (H) Representative immunofluorescence staining of MYHC (in green) in C2C12 cells treated with Control or mFNDC1 4 days post-differentiation in the presence or absence of K43c. Scale bars = 300 μm. (I) Quantification of fusion index (a MYHC+ cell with at least three nucleus) (n = 6 independent experiments). One-way ANOVA, Control vs. mFNDC1, P = 8.87 × 10−11; Control vs. K43c, P = 1.15 × 10−10; Control vs. mFNDC1 + K43c, P = 6.10 × 10−10; mFNDC1 vs. K43c, P = 2.30 × 10−14; mFNDC1 vs. mFNDC1 + K43c, P = 2.30 × 10−14. Data are represented as mean ± SEM. **p < 0.01. Source data are available online for this figure.
Supplier Page from Abcam for Anti-Integrin beta 1d antibody [2B1]