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. 2017 Nov 17;8(65):109120–109134. doi: 10.18632/oncotarget.22621

Figure 1. Nuclear PKM2 interacts with H2AX upon DNA damage.

Figure 1

(A) Cytosolic and nuclear extracts were prepared from MCF7 cells treated with or without etoposide, followed by western blot analysis for the indicated proteins. LaminB was a nuclear marker, and β-actin acted as a cytosol marker. (B) Heat map of gene ontology (GO) for DNA damage signaling pathway based upon the identified PKM2-interacting proteins from LC-MS/MS. GO analysis (DAVID 6.7) was applied to the specific PKM2-associated complex under etoposide treatment and untreatment for molecular function and biological process enrichment [27]. The colors in the map represent the quantitative value (normalized total spectra) according to the Scaffold_4.3.3. Color code ranges from 0 to 10. (C) Co-IP analysis of the interaction between endogenous PKM2 and H2AX or γ-H2AX using antibodies against PKM2 in MCF7 cells treated with or without etoposide for 1 hour. IP using rabbit IgG was a negative control. Input, 10% whole cell lysate. (D) GST pull-down analysis of H2AX with PKM2 using purified PKM2 and GST-tagged H2AX fusion protein. (E) Co-IP analysis of the interaction between endogenous PKM2 and γ-H2AX using antibodies against γ-H2AXin MCF7 cells treated with etoposide. (F) Immunofluorescent staining of endogenous PKM2 and γ-H2AX in MCF7 cells treated with or without etoposide for 1 hour. The bottom panel showed the amplified images for cells lined with white line. All these experiments were repeated at least for three times with the same results.