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 β-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 ΔT5 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 ΔT5, 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 ΔT5. 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.