Fig. 4

SHCBP1 knockdown slows tumour cell cycle but promotes premature mitotic entry by compromising the WEE1-mediated G2–M checkpoint. A Cell cycle analysis of A549 cells transfected with control or SHCBP1 siRNA at indicated time points after double thymidine (TdR) release. (i), flow cytometry plots of cells released at various time points and normally growing asynchronous (asyn) cells analyzed by using ModFit LT 5.0 and FlowJo software. Cells were stained with or without PHH3 (Ser10)-FITC antibody followed by propidium iodide (PI) staining. PHH3 (Ser10) positive cells indicating cells in M phase have been marked by small boxes on the dot plot. (ii) the line plot shows the change trend of cell proportion in each cell cycle phase (cell proportion of G1, S, G2 and M phase to the whole cell cycle, and the M phase to the G2 + M [4N] phase) over the released time. Data are expressed as mean ± SD (n = 3 independent experiments) and analyzed by the two-way ANOVA test (ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). B Click-EdU pulse chase flow cytometry analysis of A549 cells. Cells transfected with siCtrl or siSHCBP1 were pulsed with 10 µM EdU for 45 min, and then changed to medium containing 20 µM thymidine to chase and analyze cells at 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 h, cells were harvested at indicated time point for click-EdU reaction and PI staining and then subjected to flow cytometry analysis. Fractions of cells in the EdU-negative G1-phase compartment (i), EdU-negative G2/M phase compartment (ii), fraction of EdU positive cells that have divided (iii), and fraction of EdU positive cells at chase 0 h (iv) were analyzed. Data are expressed as mean ± SD (n = 3 independent experiments) and analyzed by the two-way ANOVA test (i-iii) and unpaired Student’s t test (iv), respectively (ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). C, D A549 (C) and NCI-H1299 (D) cells transfected with control or SHCBP1 siRNA were subjected to 0.1 μM docetaxel (DTX) treatment for 0 h, 3 h, 5 h, 7 h, 10 h, 12 h and 24 h for cell cycle analysis, respectively. The legends of (i) and (ii) are similar to those in A. E, F NCI-H1299 and HeLa cells transfected with control or SHCBP1 siRNA were synchronized to late G2 phase after the indicated times of release from double thymine (TdR), followed by treatment with nocodazole (NOCO) or vehicle for 0 h, 3 h and 6 h. Representative western blot images of SHCBP1, phos-cdc2(Tyr15), phos-cdc25c(Thr48) and PHH3 (Ser10) with GAPDH as internal reference at each time point (E) and the corresponding density analysis (F) are shown. Data are expressed as mean ± SD, P values were determined by unpaired Student’s t test (ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). G Representative western blot images of WEE1, p-cdc2(Tyr15) and total cdc2 in A549, NCI-H1299 and HeLa cells analyzed after SHCBP1 knockdown. H WEE1 mRNA expression of A549, NCI-H1299 and HeLa cells relative to GAPDH mRNA expression in cells transfected with control or SHCBP1 siRNA are shown. Relative WEE1 mRNA expression of siSHCBP1 group was calculated as a fold-change versus the siCtrl group. Data are expressed as mean ± SD (n = 3 independent experiments) and analyzed by the unpaired Student’s t test (***, P < 0.001; ****, P < 0.0001)