Figure 7. BI-D1870 prolongs the SAC signal.

(A) BI-D1870 inhibits the association of CDC20 with APC/C complex. HL60 cells were synchronized at metaphase using a thymidine/nocodazole double block. One hour before release, cells were treated with BI-D1870 (5 μM). Cells were released and cultured in media with or without BI-D1870 (5 μM) for 0.5 hours. Total lysates were immunoprecipitated using an anti-CDC20 or anti-MAD2 antibody. IP products and total lysates were analyzed by immunoblotting for APC2, MAD2, phospho-Serine/Threonine, CDC20 and β-Tubulin as a loading control. Relative amounts of bound MAD2 or Ser/Thr-phosphorylated MAD2 were quantified by densitometric measurements. (B–D) Inactivation of SAC or MAD2 knockdown releases cells from BI-D1870-induced metaphase arrest. (B) Mps1-IN-1 treatment completely released cells from metaphase arrest. HL60 cells were cultured with BI-D1870 (5 μM) or DMSO vehicle control for 12 h. Mps1 kinase inhibitor Mps1-IN-1 (10 μM) was added 90 min before collecting cells to assess the effect of SAC inhibition on BI-D1870-induced metaphase arrest. Cells were fixed and stained with DAPI and antibodies against Cyclin A, Cyclin B, and p-H3. Bivariate flowcytometric profiles show cell populations at each cell cycle stage. (C) MAD2 knockdown alleviates the RSK inhibitor BI-D1870-induced metaphase arrest. HL60 cells were transduced with pLKO.1 lentiviral vector expressing MAD2 shRNA or luciferase shRNA and then selected with puromycin. Suppressed expression of MAD2 was confirmed at the protein level by immunoblotting and in mRNA levels by qRT-PCR. (D) MAD2 knockdown cells were treated with BI-D1870 (5 μM) for 12 hours, and then cells were fixed and analyzed for cell cycle distribution. Fixed cells were stained with DAPI and antibodies against Cyclin A, Cyclin B, and p-H3. The mitotic cell population was determined by DNA content and protein levels of p-H3, Cyclin A, and Cyclin B (top). Flow cytometric profiles represent one out of three experiments with similar results.