Fig 2: Effect of blocking Src-mediated EPS8 phosphorylation on EPS8 downstream targets.a HN4 cells were stably transfected with an EPS8 expression plasmid encoding the EPS8-FFFF mutant. Total cell lysates were prepared, resolved by SDS-PAGE and western blotted with the indicated antibodies. b–d Relative expression of FOXM1, AURKA and AURKB, normalised to GAPDH expression, is shown. Data are representative of independent biological experiments performed at least three times. **p < 0.01. e HN4 cells were incubated in the presence of dasatinib (400 nM) or an equivalent volume of DMSO. Twenty-four hours later, total cell lysates were prepared, resolved by SDS-PAGE and western blotted with the indicated antibodies. f–h. Relative expression of total and Y416-phosphorylated Src, FOXM1, AURKA and AURKB, normalised to GAPDH expression, is shown. Data are representative of independent biological experiments performed at least three times. **p < 0.01.

Fig 3: Contribution of individual Src phosphorylation sites in EPS8 on EPS8 downstream target expression.a HN4 cells stably transfected with EPS8 expression plasmids encoding FFFF, 485F, 525F, 602F and 774F mutants were used to prepare total cell lysates and then western blotted with the indicated antibodies. b, c Graphs indicate relative expression of FOXM1 and AURKA, in controls and mutant-expressing cells, normalised to GAPDH expression. Data shown are combined from three independent biological experiments. *p < 0.05, **p < 0.01, ***p < 0.001. d HN4 cells were stably transfected with an EPS8 expression plasmid or the FFF-EPS8 mutant. Total cell lysates were prepared, resolved by SDS-PAGE and western blotted with the indicated antibodies. e–g Graphs indicate relative expression of FOXM1, AURKA and AURKB, normalised to GAPDH expression. Data are representative of independent biological experiments performed on at least three separate occasions. *p < 0.05, **p < 0.01, ***p = 0.001.

Fig 4: Overexpression of SIRT6 leads to up-regulation of various APC/C substrates and defective mitosis. (a) 293 T and HeLa cells stably expressing empty vehicle (EV), SIRT6-FLAG (SIRT6), SIRT6-FLAG, and CDH1-HA simultaneously (ST6 + CDH1) were treated with thymidine block and subjected to western blotting. Levels of TPX2, cyclin B1, AURKA, AURKB, securin, FLAG, and CDH1 were detected using the corresponding antibodies. Actin was used as a loading control. Short exposures of 293 T CDH1 are shown in the original image section of the supplementary file. (b) Proportions of cells with three centrosomes or more were counted in control and SIRT6 overexpressing 293 T and HeLa cells (n = 100). (c) Proportions of cells with two or more nuclears were counted in control and SIRT6 overexpressing 293 T and HeLa cells (n = 100). (d) Mechanistic illustration of the current study.

Fig 5: RecQL4 directly interacts with AURKB.a Endogenous RecQL4 was detected in anti-Flag pull-down against Flag-AURKB. Flag-GFP immnoprecipitate was used as control. b Endogenous AURKB was detected in anti-Flag-RecQL4 immnoprecipitate determined by Western blotting (lane 2 of Flag IP), and only N-terminus of RecQL4 (Flag-RecQL4-NT, lane 3 of Flag IP) showed an interaction with AURKB. Flag-GFP immunoprecipitate was used for negative control. c Direct interaction between RecQL4 and AURKB was shown by in vitro GST pull-down assay