Fig 1: Clinical relevance of the p53-AGPG-PFKFB3 axis in ESCC.a Representative IHC staining for Ki67, PFKFB3, CDK1, p27, and p53 in ESCC patients (SYSUCC, n = 104) with low or high AGPG expression. Scale bar, 100 µm. b Percentage of specimens with low or high Ki67, PFKFB3, CDK1, p27, and p53 expression in the low or high AGPG expression groups (SYSUCC, n = 104, Chi-square test, two-sided). c, d Representative IHC images and statistical analysis of PFKFB3 expression in ESCC and matched normal tissues (SYSUCC). Scale bar, 100 µm. Data are representative of three independent experiments and presented as mean±S.D., n = 20 cases, the P value was determined by a two-tailed unpaired Student’s t test. e Kaplan–Meier analysis of the overall survival of ESCC patients (SYSUCC) with low (n = 52) or high (n = 52) PFKFB3 expression (log-rank test, two-sided). f Kaplan–Meier analysis of overall survival of ESCC patients (SYSUCC) with low (low expression of both AGPG and PFKFB3, n = 42), high (high expression of both AGPG and PFKFB3, n = 41) or intermediate (n = 21) AGPG/PFKFB3 expression (log-rank test, two-sided). g Graphical abstract showing that the lncRNA AGPG regulates glucose metabolism remodeling by affecting PFKFB3 stability.
Fig 2: 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 ?T5. 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.
Fig 3: AGPG directly associates with PFKFB3.a, b PFKFB3 in cell lysates a or purified His-tagged recombinant PFKFB3 b was pulled down by biotin-labeled AGPG but not by AGPG antisense RNA. S, sense. AS, antisense. c RIP assays indicated that AGPG precipitated with PFKFB3 in whole-cell lysates. The RNA levels of AGPG and ß-actin were measured by qPCR analysis. d AGPG-binding proteins were detected by MTRAP and western blotting analysis. PFKFB3 bound to AGPG was captured by anti-FLAG antibody affinity agarose beads; IP complexes were separated and identified by specific antibodies. e Immunofluorescence analysis showed that AGPG and PFKFB3 colocalized not only in the nucleus but also in the cytoplasm. Scale bar: 5 µm. f qPCR detection of AGPG expression and western blotting detection of PFKFB3 expression in human ESCC cells. PFKFB3 expression was positively correlated with AGPG expression. (Pearson’s correlation analysis, n = 10). g In vitro-synthesized FL and truncation mutants of AGPG were incubated with protein lysates from KYSE150 and KYSE30 cells or with purified His-tagged recombinant PFKFB3. RNA pull-down and western blotting assays were then performed. h CLIP-qPCR showed that the T5 fragment of AGPG was the region responsible for PFKFB3 binding. i RNA pull-down assays showed that AGPG ?T5 could not interact with PFKFB3. j AGPG CRISPR KO cell lines were generated using the CRISPR/Cas9 genome-editing system. Overexpression of AGPG FL, but not of AGPG ?T5, was sufficient to reverse the decreased ECAR and cell proliferation caused by AGPG CRISPR KO. k Western blotting showed that CDK1 downregulation and p27 upregulation by AGPG CRISPR KO were abolished by AGPG FL but not by AGPG ?T5. l HomeR was used to perform the motif analysis on the binding peaks obtained by the Piranha and CIMS analyses. Both methods suggested that CCAGCCA might be responsible for PFKFB3 binding. Data in c, f, h, j are representative of three independent experiments and presented as mean±S.D., n = 3 biologically independent samples, the P value was determined by a two-tailed unpaired Student’s t test.
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