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. 2020 Oct 26;11(10):919. doi: 10.1038/s41419-020-03104-6

Fig. 6. Threonine 360 of SKA3 protein is essential for PLK1 binding and subsequent biological functions.

Fig. 6

A Schematic diagram of wild-type SKA3 protein (SKA3-WT), SKA3 N-terminal domain deletion (SKA3-ΔN), and SKA3 C-terminal domain deletion (SKA3-ΔC). Numbers indicate amino acid positions. B LSCC cells were cotransfected with HA-tagged PLK1-expression plasmid and FLAG-tagged SKA3-WT-, SKA3-ΔN-, and SKA3-ΔC-expressing plasmids for 48 h; CoIP was performed using a FLAG antibody, and PLK1 was detected by western blotting. C LSCC cells were transfected with SKA3-WT, SKA3-ΔN, and SKA3-ΔC-expression plasmid or an empty vector for 48 h, and the expression levels of PLK1 and phosphorylated AKT (Ser473 and Thr308) were determined by western blotting. D FD-LSC-1 and TU-177 cells were transfected with SKA3-WT-, SKA3-ΔN-, and SKA3-ΔC-expression plasmid, or an empty vector for 48 h, and glycolysis was measured. E FD-LSC-1 and TU-177 cells stably expressing the Flag-tagged wild-type SKA3 or domain deletion, or the empty vector, were seeded in 96-well plates. Cell proliferation was measured using a high-content screening system for 5 days. Top: fold changes in cell proliferation relative to day 1; bottom: representative cell images. Scale bar, 200 μm. F FD-LSC-1 and TU-177 cells were cotransfected with HA-tagged PLK1-expression and the SKA3-WT plasmid (Flag-SKA3-WT) or a phosphorylation-site mutant (T360A) expression plasmid (Flag-SKA3–T360A) for 48 h. The cell lysates were immunoprecipitated with an anti-Flag antibody, and the precipitates were analyzed using western blot analysis with the indicated antibodies. G FD-LSC-1 and TU-177 cells were transfected with the Flag-SKA3-WT or Flag-SKA3–T360A-expression plasmid for 48 h. The levels of PLK1 and phosphorylated AKT (Ser473 and Thr308) were determined using western blot analysis. H FD-LSC-1 and TU-177 cells were transfected with the Flag-SKA3-WT or Flag-SKA3–T360A-expression plasmid, or the empty vector for 48 h, and then glycolytic capacity was measured. I FD-LSC-1 and TU-177 cells stably expressing the wild-type SKA3 or SKA3–T360A were seeded in 96-well plates. Cell proliferation was measured using a high-content screening system for 5 days. Top: fold changes in cell proliferation relative to day 1; bottom: representative cell images. Scale bar, 200 μm. The data are expressed as means ± SD of three independent experiments. *P < 0.05.