Fig. 4: AGPG affects PFKFB3 stability by preventing its ubiquitination.

a In vitro-synthesized AGPG was incubated with protein lysates from KYSE30 cells transfected with vectors expressing FLAG-tagged FL or truncation mutants of PFKFB3. RNA pull-down and western blotting assays were then performed. Truncation mutants included FL, N-terminal (N) and C-terminal (C) constructs. b RIP assays were performed using anti-FLAG antibodies in cells transfected with vectors expressing FLAG-tagged FL or truncation mutants of PFKFB3. c AGPG knockdown reduced PFKFB3 expression in ESCC cells. d PFKFB3 downregulation by AGPG CRISPR KO was rescued by AGPG FL but not by AGPG ΔT5. e PFKFB3 downregulation by AGPG knockdown was abolished by MG-132 (10 μm, 12 h). f Western blotting detection of PFKFB3 levels in KYSE150 cells transfected with shCtrl or shAGPG followed by treatment with CHX (100 µg per ml) for the indicated times. g IP assays showed that AGPG knockdown increased PFKFB3 ubiquitination levels. FLAG-tagged PFKFB3 was expressed in cells, which were then subjected to IP assays. h Active APC/C could be immunoprecipitated from cells using monoclonal Cdc27 antibody. CoIP assays showed that AGPG CRISPR KO significantly increased the interaction between PFKFB3 and Cdc27. i IP assays showed that AGPG knockdown did not increase ubiquitination of the PFKFB3 K302A mutant. j Cells were infected with FLAG-tagged PFKFB3 WT or K302A and treated with CHX (100 µg per ml) for the indicated time. FLAG levels were detected by western blotting. k PFKFB3 K302A overexpression significantly reversed the decreased ECAR and cell proliferation caused by AGPG CRISPR KO, whereas PFKFB3 WT could only partially rescue these effects in KYSE150 cells. l PFKFB3 K302A overexpression significantly reversed the decreased glycolysis caused by AGPG CRISPR KO, whereas PFKFB3 WT could only partially rescue this effect. m PFKFB3 K302A overexpression abolished the G1/S arrest caused by AGPG CRISPR KO, whereas PFKFB3 WT could only partially rescue this effect. Data in b, k–m are representative of three independent experiments and presented as mean±S.D., n = 3 biologically independent samples, the P value was determined by one-way ANOVA with Tukey’s multiple comparisons test. No adjustments were made for multiple comparisons.