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. Author manuscript; available in PMC: 2013 Aug 17.
Published in final edited form as: Cell. 2012 Aug 17;150(4):685–696. doi: 10.1016/j.cell.2012.07.018

Figure 4. PKM2-Dependent H3-T11 Phosphorylation Promotes EGF-Induced Expression of Cyclin D1 and c-Myc.

Figure 4

Please also see supplemental Figure S6.

Immunoprecipitation, immunoblotting, and ChIP analyses were performed with the indicated antibodies.

(A) U87/EGFR cells with or without depletion of endogenous PKM2 and reconstituted expression of WT rPKM2 or rPKM2 K367M were treated with or without EGF (100 ng/ml) for 10 h.

(B) 293T cells with or without expressing Flag-tagged WT H3 or H3-T11A were treated with EGF (100 ng/ml) for 6 h.

(C, D, E) U87/EGFR cells with depleted endogenous histone H3 and reconstituted expression of WT rH3 or rH3-T11A were treated with or without EGF (100 ng/ml) for 10 h. ChIP analyses were performed with an anti-PKM2 (C) or an anti-acetyl-H3K9 antibody (D). (E) Quantitative real time polymerase chain reaction (PCR) was performed with specific primers for CCND1 (left panel) or MYC mRNA (right panel). Data represent the mean ± SD of three independent experiments.

(F) U87/EGFR cells with depleted endogenous histone H3 and reconstituted expression of WT rH3, rH3-T11A, or rH3-K9R were treated with or without EGF (100 ng/ml) for 6 h for detection of H3 acetylation or 24 h for examination of cyclin D1 and c-Myc expression.

(G) U87/EGFR cells with endogenous PKM2 depletion and reconstituted expression of WT rPKM2 or rPKM2 K367M were treated with or without EGF (100 ng/ml) for 24 h.