Per1/Per2–Igf2 axis and myoblast differentiation. (A and B) Differentiation index (A) and fusion index (B) during differentiation of Igf2 KD cells. (C) Relative expression levels of five muscle genes during differentiation of Igf2 KD cells. The value obtained with day 0 control (Cont) KD cells was defined as 1.0. (D) Temporal profile of the frequency of EdU⁺ nuclei in Igf2 KD cells during differentiation. (E) Western blotting of Clock, Bmal1, Per1, and Per2 after retrovirus-mediated transduction of these genes in C2C12 cells. Empty vector (Emp Vec) was used as a control. Cells transduced with Igf2 shRNA clone 1 (Igf2 KD1) and those with control shRNA were compared. Histone H2B was used as a loading control. (F) EdU uptake and immunofluorescence staining of undifferentiated and differentiating primary myoblasts with antibodies against MHC and MyoD. DNA was counterstained with DAPI. Bar, 100 µm. (G) Differentiation index (top) and fusion index (bottom) of control and Per KO C2C12 cells cultured with exogenous Igf2 at different concentrations for 3 and 5 d. Igf2 was not added to the control cells. (H) Relative expression levels of Ckm (top) and Myog (bottom) of control and Per KO C2C12 cells treated with exogenous Igf2 for 3 and 5 d. The expression level of the control cells before differentiation was defined as 1.0. Data are presented as mean + or ± SEM of biological triplicates. Each replicate includes n = 1,000–1,500 nuclei in A and B. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 with Student’s t test compared with control. NS, P ≥ 0.05. Asterisks were only added to the values with 1 ng/ml Igf2 in G and H.