Fig 2: Inhibition of ULK1 increases the cancer cells sensitivity to taxol. (A) Immunofluorescence images of cells with different chromosome segregation defects. Cells were synchronized by thymidine (2 mM) double block and released into fresh medium for 9 h. ‘No chrom. segregation defects’, ‘Mild chrom. segregation defects’ or ‘Severe chrom. segregation defects’ indicate cells with 0, 1–5, or more than 5 chromosomes segregation defects in anaphase, respectively. (B) HCT116 WT cells, ULK1-KO cells, rWT cells and rK46I cells were treated with thymidine for 24 h and then released into medium with or without 5 nM taxol for 9 h before staining for DNA and quantification. (C) HCT116 WT cells were treated with or without 2 µM SBI throughout the course of the experiment. Cells were treated with thymidine for 24 h and then released into fresh medium or into medium containing 5 nM taxol for 9 h before staining for DNA and quantification. (D) A set of RNAi-resistant rescue forms of MAD1 plasmids (MAD1-rWT or rS546A) were stably transfected into HCT116 MAD1-KD cells. Cells were treated with thymidine for 24 h and then released into fresh medium or into medium containing 5 nM taxol for 8.5 h before staining for DNA and quantification. (E) HCT116 WT cells, ULK1-KO cells, ULK1-rWT cells and ULK1-rK46I cells were treated with taxol (5 nM). After 24, 48, 72 and 96 h, the cells were harvested and counted to generate a growth curve. (F) HCT116 WT cells were treated with or without 2 µM SBI throughout the course of the experiment. Cells were treated with taxol (5 nM). After 24, 48, 72 and 96 h, the cells were harvested and counted to generate a growth curve. (G) A set of RNAi-resistant rescue forms of MAD1 plasmids (MAD1-rWT or rS546A) were stably transfected into HCT116 MAD1-KD cells. Cells were treated with taxol (5 nM) for different times, then harvested and counted to generate a growth curve. (H) HCT116 WT cells, ULK1-KO cells, ULK1-rWT cells and ULK1-rK46I cells were treated with or without taxol (5 nM). After 2 weeks, the cloning formation was analyzed with Crystal Violet staining. (I) Quantification of the colony numbers in (H). Asterisks indicate significance (the Tukey test; ***: P < 0.001). (J) Colony formations of indicated treatment in (G). (K) Quantification of the colony numbers in (J).

Fig 3: Mad1-Ser546 phosphorylation is involved in Mad1 kinetochore location. (A) GFP-MAD1-WT, GFP-MAD1-S546A or GFP-MAD1-S546D was transfected into stable MAD1-RNAi (#1) HCT116 cells. Cells were synchronized by sequential thymidine-RO3306 treatment and released into fresh medium for 40 min or thymidine-nocodazole treatment for 10 h; scale bars: 5 µM. (B) Quantification of the kinetochore GFP/CENP-B ratio in (A). Asterisks indicate significance (the Dunnett test; ***: P < 0.001). (C) Flag-MAD1-WT or Flag-MAD1-S546A was transfected into HCT116 MAD1-KD cells. Cells were synchronized by thymidine (2 mM) double block and released into fresh medium for 8.5 h, or into medium containing nocodazole (330 nM) for 10 h; scale bars: 5 µM. (D) Quantification of the kinetochore S546ph/CENP-B ratio in (C). (E) HCT116 WT cells were transfected with MAD1 siRNA (siMAD1). At 48 h after transfection, MAD1-rWT or MAD1-rS546A was transfected into HCT116 cells. Cells were synchronized by sequential thymidine-RO3306 treatment and released into fresh medium prior to CDC20 immunoprecipitation. (F) Time-lapse analysis of the duration of mitosis. MAD1-KD cell lines expressing RFP-H2B stably were transfected with GFP-MAD1-WT, GFP-MAD1-S546A or GFP-MAD1-S546D. Indicated cells were treated with thymidine for 24 h and released into fresh medium for 6 h prior to treatment with 200 nM nocodazole. n indicates number of cells, m is the median duration of mitosis. Asterisks indicate significance (the Tukey test; ***: P < 0.001). (G) Time-lapse analysis of the duration of mitosis. HCT116 WT cells and ULK1-KO cells expressing RFP-H2B stably were transfected with GFP-MAD1-S546D. Indicated cells were treated with thymidine for 24 h and released into fresh medium for 6 h prior to treatment with 200 nM nocodazole. n indicates number of cells, m is the median duration of mitosis. Asterisks indicate significance (the Tukey test; ***: P < 0.001).

Fig 4: Mad1 is a substrate of ULK1. (A) Identification of S546 phosphorylation on His-MAD1 incubated in vitro with ULK1 by using mass spectrometry analysis. (B) Schematic of the phosphorylation sites Ser546 in human and other species MAD1. (C) Flag-ULK1 or Flag-ULK1-K46I proteins were immunopurified from transfected HEK293 cells. Then in vitro phosphorylation assays were performed. (D) Flag-ULK1 protein was immunopurified from transfected HEK293 cells. In vitro phosphorylation assays were then performed in the presence of His-MAD1 or His-MAD1-S546A proteins. (E) An empty plasmid and an expression plasmid for Flag-ULK1 or Flag-ULK1-K46I were co-transfected with GFP-MAD1 or GFP-MAD1-S546A into HCT116 cells for 48 h. Proteins were then extracted for western blotting. (F) HCT116 WT cells and ULK1-KO cells were synchronized by sequential thymidine-RO3306 treatment and released into fresh medium. Proteins were then extracted for western blotting. (G) HCT116 WT cells and ULK1-KO cells were synchronized by thymidine (2 mM) double block and released into fresh medium for 8.5 h; scale bars: 5 µM. (H) Quantification of the kinetochore S546ph/CENP-B ratio in (G). Asterisks indicate significance (the Dunnett test; ***: P < 0.001).

Fig 5: GSK3ß inhibition induced aberrant spindle polarity and altered expression of TPR and dynein in CRC cellsHCT116 cells were immunostained with anti-a-tubulin (spindle apparatus marker; red) and anti-GSK3ß (green) antibodies, counterstained with DAPI (chromatin; blue) and examined by confocal microscopy. (A) Representative images of HCT116 cells in the process of mitosis in control group and groups treated with 25 µM of AR-A014418 or SB-216763. (B) Comparison of images of mitotic HCT116 cells transfected with control or GSK3ß-specific siRNA for 72 hours. (A, B) Scale bar = 5 µm. (C) Expression of GSK3ß, TPR, MAD1, p150, and dynein in HCT116 (left panels) and SW480 (right panels) cells treated with 25 µM AR-A014418 or SB-216763 for indicated periods. (D) Comparison of immunoblotting indicating levels of expression of GSK3ß, TPR, and its associated proteins (MAD1 and dynein) in HCT116 (left panels) and SW480 (right panels) cells transfected with control (siCTL) or GSK3ß-specific siRNA (siGSK3ß) for 72 hours.