Fig 1.
Mitotic defects caused by OTSSP167.
(A) Cytokinesis failure. Nocodazole arrested mitotic MCF7-mRFP-H2A cells were washed and released either into DMSO or 100 nM OTSSP167 (OT)-containing medium. Initiation of cleavage furrow (for DMSO treated cells, n = 19) or cell flattening (for OT treated cells, n = 27) together with chromosome decondensation marked exit from mitosis. OT treated cells exited from mitosis without division. (B) Mitotic checkpoint defects. Cells arrested in nocodazole were further exposed to either DMSO or OTSSP167 (n>63). Cell flattening and chromosome decondensation marked exit from mitosis.
Fig 2.
OTSSP167 causes loss of MAD2 from the MCC.
(A) Outline of cell synchronization protocol. Following single thymidine arrest (STA) for 24 h (numbers above arrows indicate time in hours), HeLa cells were released into nocodazole (Noc) or taxol for 12 h and then treated with DMSO or OTSSP167 (100nM) ("+") plus MG132 (to prevent mitotic exit). After 2 h, cells were processed for lysates and immunoprecipitation (IP). (B&C) Nocodazole (B) or Taxol (C) arrested cells were treated with DMSO (“-“) or OTSSP167 (“+”) and the lysates were subjected for nonimmune rabbit IgG or BUBR1 IP and Western blot. The MCC and APC/C components were probed together with MELK. Molecular weight markers (in kDa) were labeled.
Fig 3.
MELK knockdown does not compromise MCC assembly.
HeLa cells were transfected with MELK shRNA for 24 hrs. Transfected cells were selected by puromycin while synchronized as described in Fig 2A. The nocodazole (A) and taxol (B) arrested cell lysates were subjected to IP and then probed for proteins as indicated.
Fig 4.
OTSSP167 inhibits Aurora B and MELK.
(A) In vitro kinase assays with ~40 nM of recombinant 6×His-Aurora B/INCENP(822–918) and 1 μg each of histone H3.3 and myelin basic protein (MBP). OTSSP167 (OT) was added at different concentrations (nM) with DMSO as a control (lane “-“). The samples were separated by SDS-PAGE, blotted and then processed for autoradiography (“32P”). The same blot was then stained shortly with Coomassie brilliant blue (CBB) to show equal loading of substrates, and probed by immunoblotting (“IB”) for Aurora B. The numbers on the right indicate molecular weight markers (kDa). (B) Similar as in (A), except that ~40 nM of recombinant GST-MELK(1–340) was used. (C) HeLa cells arrested with nocodazole and MG132 were further treated with DMSO (“ctrl”), ZM447439 (ZM, 2.5 μM final concentration) or OTSSP167 (OT, 100 nM final concentration) and the lysates were used for in vitro Aurora B IP kinase assays. Alternatively the IPs from DMSO treated lysates were used in kinase assays in the presence of ZM or OT. The kinase reactions were applied to SDS-PAGE followed by Western blot. The membranes were stained for recombinant histone H3.3 by Coomassie staining before blocking. The phosphorylated H3.3 was probed with anti-phospho-H3S10 antibody. (D) Immunofluorescence of HeLa cells arrested with taxol and MG132 and then further treated with DMSO or OTSSP167 (OT). Anti-phospho-H3S10 antibody was probed to detect Aurora B activity on chromosomes. Anti-CENP-I is a marker for centromeres. DAPI stains DNA. Bar = 10 μm.
Fig 5.
OTSSP167 mislocalizes Aurora B in mitotic cells.
Taxol treated HeLa cells were further exposed to DMSO or 100 nM OTSSP167 for 2 hrs, and then fixed to probe Aurora B (A) or Borealin (B) together with CENP-A and DAPI. Maximum projections of z-stacks are shown. Bar = 10 μm. For quantification of intensities, only kinetochore pairs that fit within one z-plane were used. Relative intensities of Aurora B and Borealin were normalized to CENP-A signals and shown to the right.
Fig 6.
OTSSP167 inhibits Haspin and BUB1 kinases.
(A-D) Taxol arrested and DMSO or OT treated HeLa cells were fixed for immunofluorescence and probed for DAPI, CENP-A and phospho-H3T3 (A), phospho-H2AT120 (B), Sgo1 (C) or BUB1 (D). Bar = 10 μm. For quantification, only kinetochore pairs that fit within one z-plane were used. Relative intensities of proteins were normalized to CENP-A signals and shown to the right. (E) HeLa cells arrested in mitosis by nocodazole and MG132 were further exposed to DMSO (“ctrl”), 5-ITU (ITU1 and ITU2 are two batches) or OTSSP167 (OT), and the whole cell lysates prepared in 1×SDS sample buffer were separated for Western blot. The membrane was stained by Coomassie blue first before being destained and probed for MAD2 or phospho-H3T3. (F) HeLa cells arrested with nocodazole and MG132 were further treated with DMSO (“ctrl”), or OTSSP167 (OT) and the lysates were used for in vitro BUB1 IP kinase assays. In one lane OT was also added directly to the reactions containing the IP from DMSO treated lysates. The kinase reactions were applied to SDS-PAGE followed by Western blot. The membrane was stained for recombinant histone H2A before blocking. The phosphorylated H2A was probed with anti-phospho-H2AT120 antibody.
Fig 7.
OTSSP167 causes GFP-MELK localization to cell cortex in prometaphase cells.
Single-plane images of GFP-MELK transfected HeLa cells were shown in all panels. Bar = 10μm. (A) A metaphase and an anaphase cell in the same field were shown in the absence of drugs. Note the cortex association of GFP-MELK only appears obviously during anaphase. (B) Shown are three representative transfected prometaphase cells after treatment with OTSSP167 for 2 hrs. (C) GFP-MELK transfected prometaphase cells do not show cortex association after exposure to hesperadin (Aurora B inhibitor), reversine (MPS1 inhibitor) or Plk1 inhibitor III.