Fig. 4: SKA3 binds PLK1, thus promoting glycolysis via AKT activation.

A, B CoIP-western blot analysis of TU-177 cells. The lysates were incubated with anti-SKA3 (A) and anti-PLK1 (B) antibodies, and the fractionated immunoprecipitates were probed on western blots using antibodies against SKA3 and PLK1. C FD-LSC-1 and TU-177 cells were cotransfected with a Flag-tagged SKA3-overexpression plasmid and a HA-tagged PLK1-overexpression plasmid for 48 h. Immunofluorescence staining was performed using anti-FLAG and anti-HA antibodies, and cell nuclei were stained using DAPI (blue). Subcellular localization of SKA3 (green) and PLK1 (red) was observed using a laser confocal microscope. D Western blot analysis of the expression of PLK1, phosphorylated PTEN, and phosphorylated AKT (Ser473 and Thr308) in SKA3-knockdown or SKA3-overexpressing FD-LSC-1 and TU-177 cells, respectively. E FD-LSC-1 and TU-177 cells were transfected with the SKA3-overexpression plasmid for 36 h and then were treated with the PLK1 inhibitor BI2536 (30 nM) or an equal volume of DMSO for 12 h. AKT and phosphorylated AKT (Ser473 and Thr308) levels were determined by western blot analysis. F Knockdown of PLK1 and overexpression of SKA3 in FD-LSC-1 and TU-177 cells. The levels of total and phosphorylated AKT (Ser473 and Thr308) were determined by western blot analysis. G Effect of SKA3 knockdown and PLK1 overexpression on the levels of phosphorylated AKT (Ser473 and Thr308) determined by western blot analysis. H FD-LSC-1 and TU-177 cells were transfected with the SKA3-overexpression plasmid for 36 h, and then treated with the AKT inhibitor MK-2206 (20 µM) for 12 h. Expression of phosphorylated AKT (Ser473 and Thr308), HK2, PFKFB3, and PDK1 was determined by western blot analysis. I LSCC cells were cotransfected with siRNAs targeting PLK1 and an SKA3-overexpression plasmid for 48 h, at which time glycolysis was measured using the Seahorse energy metabolism instrument. J LSCC cells were transfected with the SKA3-overexpression plasmid for 36 h, and then treated with the PLK1 inhibitor BI2536 (30 nM) or AKT inhibitor MK-2206 (20 µM) for 12 h. Glycolysis was measured using a Seahorse energy metabolism instrument. The data are expressed as means ± SD of three independent experiments in (I) and (J). *P < 0.05; **P < 0.001.