Journal: Molecular Cancer Research

Article Title: Coregulation of NDC80 Complex Subunits Determines the Fidelity of the Spindle-Assembly Checkpoint and Mitosis

doi: 10.1158/1541-7786.MCR-23-0828

Figure Lengend Snippet: Destruction of SPC24 leads to paradoxical activation and impairment of SAC function. A, Loss of SPC24 abolishes mitotic progression in synchronized cells. mAID SPC24 KO cells were synchronized using double thymidine block and released into either drug-free or DI-containing medium. The cells were harvested at the indicated time points and analyzed with immunoblotting and flow cytometry. B, SPC24-depleted cells undergo mitotic arrest followed by mitotic slippage. mAID SPC24 KO cells were either left untreated or treated with DI (containing 2 μg/mL of Dox and 100 μg/mL of IAA). Individual cells were tracked using live-cell imaging for 24 hours. Key: interphase (gray); mitosis (red); cell death (truncated bars); and defective mitotic exit (blue; including mitotic slippage and defective sister chromatid separation followed by cytokinesis failure). The percentages of cells with defective mitotic exit in the population exiting mitosis before and after t = 12 hours of DI treatment are indicated. Box-and-whisker plots show the elapsed time between mitotic entry and exit (or cell death for cells that died during mitosis). Cells exhibiting defective mitotic exit are highlighted in blue. The duration of mitosis in cells treated with PTX was also analyzed. C, Silencing of SPC24 results in mitotic and postmitotic cell death. mAID SPC24 KO cells were treated and imaged as described in B . Representative images of an untreated cell undergoing normal mitosis (top), a DI-treated cell undergoing prolonged mitotic arrest and subsequent cell death (middle), and a DI-treated cell undergoing aberrant sister chromatid separation followed by cytokinesis failure and cell death (bottom) are shown. Time: h:min. Scale bar, 10 μm. D, Depletion of SPC24 induces mitotic block or mitotic slippage depending on the duration of DI treatment. mAID SPC24 KO cells were synchronized using double thymidine block. The cells were left untreated or treated with DI at the time of second thymidine release ( t = 0) or three hours after release ( t = 3 hours). Another population was treated with DI at 16 hours before the second thymidine release ( t = −16 hours). At four hours after release from thymidine, the cells were subjected to live-cell imaging analysis. The raw data are shown in Supplementary Fig. S1C. Box-and-whisker plots show the elapsed time between mitotic entry and exit (or cell death for cells that died during mitosis). Cells exhibiting defective mitotic exit are highlighted in blue. E, Silencing of SPC24 leads to transient enrichment of MAD2 at kinetochores. mAID SPC24 KO cells were treated with DI for either 8 hours or 24 hours. The cells were also incubated with NOC for 16 hours to activate the SAC. The cells were then processed for immunostaining for MAD2 and CREST to quantify the MAD2/CREST signals ( n = 30 cells). ****, P < 0.0001; ns P > 0.05. F, Silencing of SPC24 results in transient activation of SAC. mAID SPC24 KO cells were treated with DI for either 8 hours or 24 hours. The cells were also incubated with NOC to activate the SAC (due to toxicity, cells treated with DI for 24 hours were incubated with NOC for the last 16 hours). Lysates were prepared and analyzed with immunoblotting. MAD2–CDC20 complex was examined by immunoprecipitation followed by immunoblotting. Lysates from asynchronously growing cells immunoprecipitated using normal rabbit serum (NRS) served as a negative control.

Article Snippet: The following antibodies were obtained from the indicated sources: β-actin (A5316; Sigma-Aldrich), APC4 (ab72149; Abcam), APC11 (14090; Cell Signaling Technology), phospho-AURKA Thr288 /AURKB Thr232 /AURKC Thr198 (2914; Cell Signaling Technology), CDC20 (sc-5296; Santa Cruz Biotechnology), CDC27 (610455; BD Biosciences), CREST (90C-CS1058; Fitzgerald Industries), cyclin B1 (sc-245; Santa Cruz Biotechnology), cyclin E (HE12, sc-247; Santa Cruz Biotechnology), phosphorylated histone H3 Ser10 (sc-8656R; Santa Cruz Biotechnology), FLAG (F3165; Sigma-Aldrich), KNL1 (ab70537; Abcam); MAD2 (17D10, sc-47747; Santa Cruz Biotechnology), MAD2 (raised against bacterially expressed GST-MAD2; ref. ), NDC80 (sc-81283; Santa Cruz Biotechnology), NUF2 (sc-271251; Santa Cruz Biotechnology), cleaved PARP1 (552597; BD Biosciences), pericentrin (ab220784; Abcam), PTTG1 (DCS-280, sc-56207; Santa Cruz Biotechnology), SPC24 (A16601; ABClonal), SPC25 (HPA047144; Atlas Antibodies), acetylated α-tubulin (sc-23950; Santa Cruz Biotechnology), and α-tubulin (Alexa-Fluor-488–conjugated, 5063; Cell Signaling Technology).

Techniques: Activation Assay, Blocking Assay, Western Blot, Flow Cytometry, Live Cell Imaging, Whisker Assay, Incubation, Immunostaining, Immunoprecipitation, Negative Control

Journal: Cell Chemical Biology

Article Title: Modulating Protein-Protein Interactions of the Mitotic Polo-like Kinases to Target Mutant KRAS

doi: 10.1016/j.chembiol.2017.07.009

Figure Lengend Snippet:

Article Snippet: Cells were washed in PBS containing 0.1% Tween-20 then incubated with anti-alpha tubulin antibody (Santa Cruz sc-32293) and anti-CREST antibody (Europa 90C-CS1058).

Techniques: Recombinant