Fig. 3

Knock-out of AMPK caused severe differentiation defect in C2C12 cells. a The strategy for knocking-out (KO) AMPKa1 (Prkaa1) and a2 (Prkaa2) in C2C12 cells. For knocking-out AMPKa1 (Prkaa1), CRISPR/Cas9 knockout plasmid and homology-directed repair (HDR) plasmid from Santa Cruz were used. For knocking-out AMPKa2 (Prkaa2), a sgRNA targeting exon 4 of AMPKa2 (Prkaa2) [19] was used. Complete KO of AMPKa1 and a2 was confirmed by sequencing. The location of the two-base pair (TT) insertion in exon 2 of Prkaa1 and deletions in exon 4 of Prkaa2 alleles are indicated (purple dashed lines). b Knockout of AMPK in C2C12 cells was confirmed by Western blot analysis. GAPDH was used as a control. Shown are three independent clones of AMPK-KO. c, d Differentiation defects of AMPK-KO C2C12 cells. C2C12 cells were subjected to differentiation in 2% horse serum. MHC (Myosin heavy chain 1, MYH1) was detected by Western blot analysis (c) and immunofluorescence staining (d) during differentiation. Proteins examined are indicated. DAPI: 4′,6-diamidino-2-phenylindole. e Knockout of AMPK does not significantly change the expression of Tet1, Tet2 and Tet3 at mRNA levels in C2C12 cells. Expression data were retrieved from RNA sequencing data. TPM: transcripts per million. f Gene ontology analysis of downregulated genes in AMPK-KO C2C12 cells compared to wild-type cells at myoblast (differentiation d0) and myotube (differentiation d8) stages. g The expression of Pax7 and myogenic regulatory factors (MRFs) was examined by RT-qPCR analysis. mRNA levels are presented relative to the levels in wild-type myoblasts (differentiation day 0) and were normalized to those of Gapdh. Data are presented as mean ± SD from three independent experiments performed in triplicates. *p < 0.05; **p < 0.01