Fig. 4: Increased citrate levels enhance PFKM-dependent histone H3 phosphorylation.

a HEK293T cells were transfected with Flag-PFKM. The in vitro kinase assay was performed by incubating immunoprecipitated Flag-PFKM proteins with commercial recombinant histone H3 in the presence of increasing doses of citrate. b HEK293T cells were transfected with empty vector or Flag-histone H3. A co-IP assay was performed in the presence or absence of citrate (0.5 mM). c U87 cells were transfected Flag-PFKM and treated with different doses of citrate. A co-IP assay was performed. d U87 cells were treated with different doses of citrate. Histone H3S10ph levels were examined by immunoblotting analysis. e The U87 cells with CS depletion were supplemented with citrate (2.5 mM) for 24 h. Histone H3S10ph levels were examined by immunoblotting analysis. f The recombinant Sumo-His-PFKM treated with or without citrate was analyzed by gel filtration chromatography, followed by immunoblotting analysis. g PFKM-knockout HEK293T cells were transfected with Flag-rPFKM WT or F639L. The in vitro kinase assay was performed by incubating immunoprecipitated Flag-rPFKM WT or F639L with commercial recombinant histone H3. h PFKM-knockout HEK293T cells were transfected with HA-rPFKM WT or F639L and empty vector or Flag-histone H3. A co-IP assay was performed. i PFKM-knockout HeLa cells were transfected with HA-rPFKM WT or K617A, immunoprecipitation experiments were performed. j PFKM-knockout HeLa cells were rescued with rPFKM WT or K617A. Histone H3S10ph levels were detected. (a–j) Immunoprecipitation and immunoblotting experiments were performed with the indicated antibodies. Data are representative of three independent experiments. Source data are provided as a Source Data file.