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. 2016 Aug 3;7:12431. doi: 10.1038/ncomms12431

Figure 5. PKM2 dephosphorylation is required for β-catenin transactivation and c-Myc-dependent expression of glycolytic genes and Cdc25A.

Figure 5

Immunoprecipitation and immunoblotting analyses were performed with the indicated antibodies. Data are representative of at least three independent experiments. (a) U87/EGFR cells with depleted Cdc25A and reconstituted expression of rCdc25A WT or rCdc25A Y59F were transfected with the TOP-FLASH plasmid and treated with or without EGF (100 ng ml−1) for 10 h. Data represent the mean±s.d. of three independent experiments. (b,c) U87/EGFR cells with depleted Cdc25A and reconstituted expression of rCdc25A WT or rCdc25A Y59F were infected with a lentivirus expressing Flag-PKM2 and then treated with or without EGF (100 ng ml−1) for 6 h. ChIP analyses with anti-Flag antibody (b) and anti-acetyl-H3-K9 antibody (c) and PCR with primers for MYC promoters were performed. Data represent the mean±s.d. of three independent experiments. (d) U87/EGFR cells with depleted Cdc25A and reconstituted expression of rCdc25A WT or rCdc25A Y59F were treated with or without EGF (100 ng ml−1) for 24 h. (e) U87/EGFR cells were treated with EGF (100 ng ml−1) for 24 h. (f) U87/EGFR cells were infected with or without a lentivirus expressing Flag-Cdc25A Y59F and treated with or without EGF (100 ng ml−1) for 6 h. Nuclear fractions were extracted for detection of nuclear PKM2 phosphorylation levels. Total lysates of the cells treated with EGF for 24 h were prepared for examination of c-Myc and Cdc25A expression. (g) Endogenous PKM2-depleted U87/EGFR cells were reconstituted with the expression of rPKM2 WT or rPKM2 S37D. (h) U87/EGFR cells with depleted PKM2 and reconstituted expression of rPKM2 WT or rPKM2 S37D mutant were transfected with the TOP-FLASH plasmid and treated with or without EGF (100 ng ml−1) for 10 h. Data represent the mean±s.d. of three independent experiments. (i) U87/EGFR cells with depleted PKM2 and reconstituted expression of rPKM2 WT or rPKM2 S37D were treated with or without EGF (100 ng ml−1) for 24 h.