Fig. 7

Targeting SHCBP1 in combination with low-dose DNA-damaging drugs triggers mitotic catastrophe in tumour cells due to G2–M checkpoint abrogation. A, B A549, NCI-H1299 and HeLa cells were transfected with control or SHCBP1 siRNA for 24 h, and then treated with low-dose etoposide (A549 cells, 1 μM; NCI-H1299 cells, 5 μM; HeLa cells, 3 μM) or vehicle for 24 h, respectively. Representative immunofluorescence images of PHH3 (red) and α-tubulin (green) co-staining (A) and statistics of PHH3-positive cell counts (B) are shown. Scale bars, 200 μm. At least three fields of view were randomly selected with at least 300 cells per field for cell counting. Data are expressed as mean ± SD and analyzed by unpaired Student’s t test; ns = not significant; ***, P < 0.001. C NCI-H1299, HeLa and A549 cells were transfected with control or SHCBP1 siRNA for 24 h, followed by treatment with either low-dose etoposide (ETOP, same as in A and B) or cisplatin (CDDP) or corresponding vehicle (DMSO) for 24 h or 40 h; CDDP: NCI-H1299 (10 μM), HeLa (5 μM), A549 (5 μM). Flow cytometry plots and histograms of cells stained with PHH3 (Ser10)-FITC antibody and propidium iodide (PI) are shown. Mitotic cells that are positive for PHH3(Ser10) were circled out by small boxes. Black arrows indicate the sub G1-phase cells. D Western blot analysis of SHCBP1, PHH3(Ser10), γH2AX and P53 in NCI-H1299, HeLa and A549 cells, cells were transfected with control or SHCBP1 siRNA for 24 h, followed by 24 h treatment with various chemotherapeutic drugs, respectively; docetaxel (DTX), paclitaxel (PTX), etoposide (ETOP) and cisplatin (CDDP). GAPDH was used as internal reference. E NCI-H1299 and HeLa cells transfected with control or SHCBP1 siRNA were synchronized in G2 phase by allowing progression for indicated time after double thymidine release and then pulsed with 15 μM etoposide for 1.5 h, then washed out into fresh media with or without nocodazole (NOCO) for 13 h. Western blot analysis of the SHCBP1, p-cdc2(Tyr15), PHH3, and γH2AX was performed in these cells with GAPDH as internal reference. F Western blotting was performed for SHCBP1, WEE1, p-cdc2 (Tyr15) and total cdc2 in A549, NCI-H1299 and HeLa cells which were transfected with control or SHCBP1 siRNA for 24 h, followed by incubation with vehicle solution, low-dose ETOP or CDDP (concentrations are the same as previously described) for 24 h, respectively. G HeLa and NCI-H1299 cells transfected with control or SHCBP1 siRNA for 24 h were subjected to low-dose ETOP (3 μM for HeLa cells and 5 μM for H1299 cells) or corresponding vehicle for another 24 h. Representative immunofluorescence images of α-tubulin (red) and γH2AX (green) co-staining in these cells are shown (scale bars, 100 μm). The right panel of NCI-H1299 cells displays the magnified views highlighted in the left panel by the white boxes, and the white arrows indicate the cells undergoing mitotic catastrophe with micronuclei formation. The white-arrowheads in the HeLa cells highlight cells with higher γH2AX level after SHCBP1 knockdown. H NCI-H1299 and HeLa cells received the same treatment as in (G), and stained with Liu stain for quick checking (left panel). The area marked by the box is magnified, and the black arrows indicate cells undergoing mitotic catastrophe with micronuclei formation; Scale bars, 50 μm. Histogram (right panel) shows the percentage of micronucleated cells over total cells. Data are expressed as mean ± SD and analyzed by unpaired Student’s t test; ****, P < 0.0001. I NCI-H1299 and HeLa cells received the same treatment as in (G). Representative immunofluorescence images of PHH3 (red) and α-tubulin (green) co-staining in cells using single-photon confocal microscope. White arrows indicate cells with multipolar spindles. White-arrowheads indicate chromosomal fragmentation. Scale bars, 20 μm. J A549, HeLa, and NCI-H1299 cells transfected with control or SHCBP1 siRNA for 24 h were subjected to low-dose ETOP for 36 h or synchronized through TdR release, followed by exposure to low-dose ETOP for 36 h. Representative flow cytometry plots show co-staining with 7-AAD and APC-labeled Annexin V for identification of early (Annexin V + /7-AAD-) and late (Annexin V + /7-AAD +) apoptosis