Fig 2: miR‐499 directly targets Fnip1 The schematic shows the putative conserved miR‐499 binding site within the 3′UTR of the Fnip1 gene.Luciferase reporters containing the wild‐type Fnip1 3′UTR or Fnip1 3′UTR mutated in the predicted binding site of miR‐499 were used in cotransfection studies in HEK293T cells in the presence or absence of plasmids expressing miR‐499 (n = 3 independent experiments). *P < 0.0001 (Fnip1 3′UTR); *P = 0.023 (Mutated).RT‐qPCR analysis of Fnip1 and Fnip2 mRNA levels in the gastrocnemius muscle of the indicated genotypes (n = 5 mice per group).(Top) Fnip1 protein expression in the gastrocnemius muscle from the indicated mice. (Bottom) Quantification of the Fnip1/tubulin signal ratio (n = 4 mice per group). *P = 0.014.Data information: All values represent the mean ± SEM. P‐value was determined using two‐tailed unpaired Student's t‐test.Source data are available online for this figure.

Fig 3: Restoring the expression of miR‐499 activates the slow‐oxidative muscle fiber program and ameliorates muscular dystrophy in mdx mice Mean expression levels (RT‐qPCR) in gastrocnemius muscle of 8‐week‐old male WT and mdx mice (n = 5 mice per group). *P = 0.009 (Myh7b), *P = 0.0007 (miR‐499), *P = 0.0143 (Myh7), *P < 0.0011 (miR‐208b).Mitochondrial respiration rates were determined from mitochondria isolated from the hindlimb muscle of the indicated mice using pyruvate as substrate. n = 3 separate experiments done with 7–8 biological replicates. *P < 0.01 (ADP).(Left) Representative Western blot analysis of PGC‐1α (Top) and Fnip1 (Bottom) protein expression in the gastrocnemius muscle from the indicated genotypes with α‐tubulin as the loading control. (Right) Quantification of the PGC‐1α/tubulin and Fnip1/tubulin signal ratios normalized (= 1.0) to the WT control. WT, n = 8; mdx, n = 5; mdx/499Tg, n = 7. *P < 0.0001 (versus WT), ‡ P < 0.0001 (versus mdx).Representative Western blot analysis of myoglobin protein expression in the gastrocnemius muscle from the indicated genotypes with α‐tubulin as the loading control (n = 5 mice per group).(Top) Representative MHC1 immunofluorescence (IF) in the soleus of the indicated genotypes. Scale bar: 500 μm. (Bottom) Representative H&E staining of the gastrocnemius muscle of the indicated genotypes. Scale bar: 100 μm.Five‐week‐old male WT, mdx, and mdx/MCK‐miR‐499 mice were euthanized. and serum creatine kinase activity was determined. WT, n = 10; mdx, n = 10; mdx/499Tg, n = 14. *P < 0.0001 (versus WT), ‡ P = 0.0003 (versus mdx).The bars represent the mean running time and distance (± SEM) for 8‐week‐old male mice on a motorized treadmill. WT, n = 10; mdx, n = 13; mdx/499Tg, n = 8. *P = 0.01406 (Running time, versus WT), ‡ P = 0.005756 (Running time, versus mdx). *P = 0.004558 (Running distance, versus WT), ‡ P = 0.001668 (Running distance, versus mdx).Respiratory exchange ratio (RER) during a graded exercise regimen on a motorized treadmill. mdx, n = 8; mdx/499Tg, n = 6. ‡ P < 0.05.Data information: All values represent the mean ± SEM. P‐value in (A, B and H) was determined using two‐tailed unpaired Student's t‐test; P‐value in (C, F and G) was determined using one‐way ANOVA coupled to a Fisher's least‐significant difference (LSD) post hoc test.Source data are available online for this figure.

Fig 4: Inhibition of Fnip1 reactivated AMPK‐PGC‐1α signaling and mitochondrial function in myocytes A(Left) Representative Western blot analysis performed on extracts of myotubes subjected to Fnip1 siRNA or control (Con) siRNA using phospho‐AMPKα (Thr172) and AMPKα antibodies. (Right) Quantification of the p‐AMPKα/AMPKα signal ratios (n = 3 independent experiments). *P = 0.037.B, CResults of RT‐qPCR analysis on WT primary mouse myotubes after transfection with Fnip1 siRNAs or scrambled Con siRNA as indicated. For (C), 48 h post‐siRNA transfection, myotubes were treated for 24 h with DMSO or 10 μm compound C before harvest (n = 3 independent experiments). *P < 0.0001 (Fnip1 in B), *P = 0.0001 (PGC‐1α in B); *P < 0.0001 (versus Con siRNA in C), ‡ P < 0.0001 (versus Fnip1 siRNA in C).D(Top) Representative Western blot analysis performed on extracts of the gastrocnemius muscle isolated from NTG or MCK‐miR‐499 mice using phospho‐AMPKα (Thr172), AMPKα, phospho‐ACC (Ser79), or total ACC antibodies. (Bottom) Quantification of the p‐AMPKα/AMPKα and p‐ACC/ACC signal ratios normalized (= 1.0) to the NTG control (n = 8 mice per group). *P < 0.01.EOxygen consumption rates (OCR) in primary mouse myotubes transfected with Fnip1 siRNA or Con siRNA. n = 7 separate experiments done with 5 biological replicates. *P < 0.05 (Pyruvate), *P < 0.01 (FCCP).FResults of RT‐qPCR analysis for WT primary mouse myotubes subjected to inhibition of both miR‐499 and miR‐208b (anti‐miRs) (n = 4 independent experiments). *P = 0.037 (Ppargc1a), *P = 0.0019 (Fnip1).GOxygen consumption rates (OCR) in primary mouse myotubes transfected with miR‐499/miR‐208b inhibitors alone and together with the presence of Fnip1 siRNA. n = 4 separate experiments done with 5 biological replicates. *P < 0.01 (anti‐miRs versus Control).Data information: All values represent the mean ± SEM. P‐value in (A, B, D–F) was determined using two‐tailed unpaired Student's t‐test; P‐value in (C and G) was determined using one‐way ANOVA coupled to a Fisher's least‐significant difference (LSD) post hoc test.Source data are available online for this figure.