Fig. 6

miR-31 prevents hyperactivation of JAK–STAT3 signaling by repressing IL34. a The immunoblots demonstrate the protein levels of IL34, p-STAT3, STAT3, and β-tubulin in proliferating SCs of WT and miR-31-KO mice. b Western blot analysis of MyHC, IL34, p-STAT3, STAT3, and unrelated protein (GAPDH) in 1-day differentiated cultures of WT and miR-31-KO mice. c Single myofibers were isolated from the EDL muscles of WT and miR-31-KO mice. After 42 h of culture, myofibers were fixed and costained for Pax7 and Ki67. Nuclei were identified by labeling with DAPI. Representative merged photomicrographs of cultured myofibers of WT and miR-31-KO mice after staining for Pax7, Ki67, and DAPI. Scale bar: 25 μm. d Quantification of cell doublets containing one Pax7⁺Ki67⁻ cell and one Pax7⁺Ki67⁺ cell among the total dividing SCs on single fibers after culturing for 42 h. *P < 0.05. e The representative immunoblots presented here show IL34, p-STAT3, STAT3 and β-tubulin protein levels in shIL34- and shScramble-treated proliferating SCs of miR-31-KO mice. f Representative merged images of siIL34- and siControl-treated proliferating SCs of miR-31-KO mice after staining for Ki67 and DAPI. Scale bar: 20 μm. g Quantification of the percentage of Ki67⁻ cells in miR-31-KO cultures after siIL34 or siControl treatment. *P < 0.05. h Two days after injecting 1.2% BaCl₂ into the TA muscles of miR-31-KO mice, a lentivirus expressing shRNA against IL34 to downregulate IL34 mRNA of injured miR-31-KO TA muscles or shScramble was delivered to the injured muscles. Samples were collected at day 6 post injury. Representative photomicrograph of H&E-stained TA muscle sections presenting better recovery of injured TA muscles with impaired IL34 function. Scale bar: 50 μm. i Average size of regenerating myofibers at day 6 after injury. **P < 0.001. j The immunoblots presented here show the IL34, p-STAT3, STAT3, and unrelated GAPDH protein levels in regenerating miR-31-KO TA muscle after treatment with IL34 shRNA. k Two days after injecting 1.2% BaCl₂ into the TA muscles of miR-31-KO mice, a STAT3 inhibitor, 5,15 DPP, was delivered to the injured TA muscles. Histology analysis of the regenerating TA muscles of miR-31-KO mice treated with STAT3 inhibitor or vehicle control at 5 days after injury by H&E staining. Scale bar: 50 μm. l Quantification of the myofiber average CSA in regenerating TA muscle 5 days after injury. N = 3 in each group. ***P < 0.001. m WT proliferating SCs grown in differentiation medium were treated with or without 100 ng/ml recombinant IL34 for 24 h. The immunoblots demonstrate the protein levels of p-STAT3, STAT3, and unrelated β-tubulin in cultures after the addition of recombinant IL34. n Two days after injecting 1.2% BaCl₂ into the TA muscles of WT mice, an adenovirus expressing IL34 or mCherry (control) was delivered to the injured muscles of WT mice. Samples were collected at day 5 post injury. H&E-stained TA muscle sections show worse recovery of injured TA muscles with enhanced IL34 function. Scale bar: 50 μm. o Average size of regenerating myofibers at day 5 after injury. N = 3 in each group. ***P < 0.001. p The immunoblots presented here illustrate the levels of Pax7, IL34, p-STAT3, STAT3, and the unrelated protein GAPDH in regenerating WT TA muscles after the overexpression of IL34