Fig 1: Aurora-A-mediated PFKFB3 phosphorylation regulates the glycolysis of TC cells.A The interaction between Aurora-A and PFKFB3 was verified by an IP assay in TPC-1 cells. B Interaction between Aurora-A and PFKFB3 is examined by GST pull-down. C Phosphorylation level on PFKFB3 was determined by Anti-PFKFB3 (phospho S461) antibody after in vitro kinase assay. D The expression of p-PFKFB3/PFKFB3 in KTC-1 cells with Aurora-A overexpression and Alisertib-treated 8305 C and CAL-62 cells was analyzed by western blotting. E Comparable level of glycolysis, glycolytic reserve and capacity of 8305 C, TPC-1, and KTC-1 cells measured by Seahorse XF Extracellular Flux analysis. ECAR = extracellular acidification rate. F The ADP/ATP ratio was measured in KTC-1 cells with Aurora-A overexpression or combined with siRNA targeting PFKFB3, in TPC-1 cells with Aurora-A knockdown and in 8305 C cells with Alisertib treatment, respectively. G The expression of p-Aurora-A, Aurora-A, p-ERK, ERK, p-AKT, AKT in KTC-1 cells with Aurora-A overexpression or combined with siRNA targeting PFKFB3. H, I The proliferation, migration and invasion ability in KTC-1 cells with Aurora-A overexpression or combined with siRNA targeting PFKFB3 was analyzed by CCK-8 assay (H) and transwell of migration and invasion assay (I), respectively. *P < 0.05, **P < 0.01, ***P < 0.001, compared with control group. #P < 0.05, ##P < 0.01, ###P < 0.001, compared with Aurora-A upregulation group. J Western blotting analyzed the expression of p-Aurora-A, Aurora-A, p-ERK, ERK, p-AKT, and AKT in 8305 C cells with Alisertib treatment or combined with exogenous ATP (2 mM). K CCK-8 assays showed that Alisertib (10 μmol) significantly inhibited cell proliferation in 8305 C, which can be remarkably restored by exogenous ATP (2 mM) supplementation. *P < 0.05, **P < 0.01, ***P < 0.001, compared with control group. #P < 0.05, ##P < 0.01, ###P < 0.001, compared with the Alisertib-treated group. L Transwell of migration and invasion assay detected the migration and invasion ability of 8305 C cells pretreated with Alisertib for 2 days or combined with exogenous ATP (2 mM). *P < 0.05, **P < 0.01, ***P < 0.001, compared with control group. #P < 0.05, ##P < 0.01, ###P < 0.001, compared with the Alisertib-treated group. The data are presented as the mean ± SD of three independent experiments.