Impaired muscle regeneration in satellite cell–specific Igf2 cKO mice. (A) Experimental scheme for satellite cell–specific Igf2 cKO by injection of TMX and muscle regeneration from barium chloride (BaCl₂)–induced injury. (B) Relative expression level of Igf2 in isolated satellite cells comparing control (Cont) and Igf2 cKO cells. (C) H&E staining of TA muscle sections prepared from uninjured and regeneration day 7 mice. TA muscle was injured with BaCl₂ at ZT14 on day 0. Bar, 100 µm. (D) Size distribution of H&E-stained myofibers containing centrally located nuclei at the indicated time points. The minimal Feret’s diameter in each myofiber was measured. n = 3 for each genotype. (E) Average of the minimal Feret’s diameters of myofibers with centrally located nuclei. (F) Immunofluorescence staining of TA muscle with antibodies against eMHC and laminin on day 4. DNA was counterstained with DAPI. Bar, 25 µm. (G) Average of the minimal Feret’s diameters of eMHC⁺ areas on day 4. n = 4 for each genotype. (H) Immunofluorescence staining of TA muscle sections with MyoD antibody and the EdU kit. Mice were injected with EdU on day 2 after injury, and TA muscle was harvested on day 3 for staining. Bar, 25 µm. (I) Frequency of positive cells for EdU uptake and MyoD staining shown in H. n = 3 for each genotype. (J and K) Differentiation index (J) and fusion index (K) of primary myoblasts prepared from control and Igf2 cKO mice, representing biological triplicates with n = 80–110 nuclei in each replicate. Data are presented as mean + SEM. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 with Student’s t test compared with control.