Figure 5. PKM2-Dependent H3-T11 Phosphorylation Is Required for Cell Cycle Progression, Cell Proliferation, and Tumor Development.
Please also see supplemental Figure S7.
(A) WT rH3 or rH3-T11A expression was reconstituted in U87/EGFRvIII cells with depleted endogenous H3. Immunoblotting analyses were performed with the indicated antibodies.
(B) U87/EGFRvIII cells with depleted PKM2 or endogenous H3 and reconstituted expression of WT rH3 or rH3-T11A were stained with propidium iodide and analyzed for DNA staining profiles by flow cytometry. Data represent the mean ± SD of three independent experiments.
(C) A total number of 2 × 104 U87/EGFRvIII cells with depleted PKM2 or endogenous H3 and reconstituted expression of WT rH3 or rH3-T11A were plated and counted 7 days after seeding in DMEM with 2% bovine calf serum. Data represent the mean ± SD of three independent experiments.
(D, E, F, G) A total of 5 × 105 endogenous histone H3–depleted U87/EGFRvIII (D, E) or GSC11 (F, G) cells with reconstituted expression of WT rH3 or rH3-T11A were intracranially injected into athymic nude mice for each group. The mice were sacrificed and examined for tumor growth. H&E-stained coronal brain sections show representative tumor xenografts. Tumor volumes were measured by using length (a) and width (b) and calculated using the equation: V = ab2/2. Data represent the means ± SD of seven mice. (E) Immunoblotting analysis with anti–phospho-H3-T11 antibody was performed on lysates of the tumor tissue derived from the mice injected with U87/EGFvIII cells with reconstituted expression of WT histone H3 and the counterpart tissue derived form the mice injected with U87/EGFvIII cells with reconstituted expression of histone H3 T11A mutant. (F) WT rH3 or rH3-T11A expression was reconstituted in GSC11 cells with depleted endogenous H3. Immunoblotting analyses were performed with the indicated antibodies.