Journal: Nature Communications

Article Title: Chromosomally unstable tumor cells specifically require KIF18A for proliferation

doi: 10.1038/s41467-021-21447-2

Figure Lengend Snippet: a Fold change in cell density (cells/mm 2 ) after 96 h in the indicated cell lines following knockdown (KD) of kinesin proteins. Data are normalized to cells treated with control siRNA. Raw cell counts for proliferation assays are available in Source Data. n (wells of cells/independent experiments) = 24/3 (MCF10A, MDA-MB-468, and HCC1806), 64/5 (MDA-MB-231 control and KIF18A KD), 36/3 (MDA-MB-231 KIF18B, KIF4A, KIF22, and MCAK KD). b Representative images of MDA-MB-231 (from five experiments) and MCF10A cells (from three experiments) treated with either control or KIF18A siRNA. Scale bars are 100 microns. c Normalized fold change in cell density (cells/mm 2 ) of MSI and CIN colorectal cancer cell lines after 96 h or until cells in the control populations reached confluence. n (wells of cells/independent experiments) = 88/6 (HCT116); 104/5 (LoVo control KD) and 128/5 (LoVo KIF18A KD); 92/5 (SW480); 44/3 (HT29 control KD) and 48/3 (HT29 KIF18A KD); and 32/3 (LS1034). d Normalized fold change in cell density (cells/mm 2 ) of HeLa Kyoto cells after 96 h or until cells in the control populations reached confluence. n = 40 wells of cells from two independent experiments. Data were analyzed using a one-way ANOVA with post hoc Tukey’s test to compare the control to each of the knockdown conditions ( a ) or with an unpaired, two-tailed t test ( c , d ). All graphs show mean ± SD and individual data points. P values <0.05 from the indicated statistical tests are displayed on plots.

Article Snippet: Human GAPDH (Hs01015428_m1) and human KIF18A (Hs02786624_g1) Taqman probes and primers (Thermo Fisher Scientific) were used for reverse transcription and qRT-PCR.

Techniques: Knockdown, Control, Two Tailed Test

Journal: Nature Communications

Article Title: Chromosomally unstable tumor cells specifically require KIF18A for proliferation

doi: 10.1038/s41467-021-21447-2

Figure Lengend Snippet: a , b Representative images from five and three independent experiments of HT29 ( a ) or HCT116 cells, respectively, ( b ) treated with control or KIF18A siRNAs. DNA (DAPI, blue), microtubules (α-tubulin, white), and centromeres (ACA, red) are labeled. Scale bars are 10 microns. c Percentage of mitotic cells (mitotic index) observed in fixed populations of control or KIF18A siRNA-treated CRC cells. n = three independent biological samples per condition for LS1034, SW480, HCT116, and LoVo; n = 5 for HT29. Data were analyzed via unpaired, two-tailed t test. d Time between nuclear envelope breakdown (NEB) and anaphase onset (AO) in control or KIF18A siRNA-treated cells. Data from three independent experiments are shown for each cell line. n (control KD/KIF18A KD cells) = 185/ 83 (HT29), 182/135 (MDA-MB-231), 203/196 (LS1034), 303/236 (SW480), 332/344 (HCT116), and 338/353 (MCF10A). Data were analyzed via unpaired, two-tailed t test. e Percentage of control or KIF18A siRNA-treated cells that entered mitosis at least 200 min before the end of the movie but did not divide. Data are from the same three experiments as d and were analyzed by a two-sided Chi-square test. n (control KD/KIF18A KD cells) = 191/153 (HT29), 189/195 (MDA-MB-231), 203/202 (LS1034), 310/249 (SW480), 335/351 (HCT116), and 338/367 (MCF10A). f Frames from DIC live-cell imaging of HT29 and MCF10A cells treated with control or KIF18A (KD) siRNAs, showing the progression from NEB to AO. Scale bars are 5 microns, representative of three independent experiments. All graphs show mean ± SD and individual data points. P values <0.05 are displayed.

Article Snippet: Human GAPDH (Hs01015428_m1) and human KIF18A (Hs02786624_g1) Taqman probes and primers (Thermo Fisher Scientific) were used for reverse transcription and qRT-PCR.

Techniques: Control, Labeling, Two Tailed Test, Live Cell Imaging

Journal: Nature Communications

Article Title: Chromosomally unstable tumor cells specifically require KIF18A for proliferation

doi: 10.1038/s41467-021-21447-2

Figure Lengend Snippet: a Representative images from five independent experiments of MDA-MB-231 cells treated with either control (top) or KIF18A (bottom) siRNAs. Pericentriolar material (γ-tubulin, red) is numbered to show poles with and without centrioles (centrin-1, green). Microtubules (α-tubulin) are labeled in white. b Percent of mitotic cells from each indicated cell line with multipolar spindles following treatment with either control or KIF18A siRNA. n = 9 (control KD) and 5 (KIF18A KD) independent experimental replicates for MDA-MB-231. n = 3 (HT29, HCC1806, SW480), 4 (HeLa), and 5 (MCF10A) independent experimental replicates. Data were analyzed via unpaired, two-tailed t test. c Plot of multipolar spindle percentage as a function of fold change (FC) in cell number for the indicated cell lines following KIF18A KD. R-squared value is 0.79 using a linear regression model. d Representative images from three independent experiments of an MDA-MB-231 cell with a third pole (γ-tubulin, magenta) lacking centrin-1 (green). e Percent of multipolar MDA-MB-231 cells in mitosis with fragmented pericentriolar material (PCM), as indicated by the presence of γ-tubulin puncta lacking centrin-1 puncta. n = 18 (control KD) and 58 (KIF18A KD) multipolar cells from three independent experiments. Data were analyzed via a two-sided Chi-square test. f Intercentriolar distance measurements (in microns) for MDA-MB-231 cells in each indicated category. n = 81 (control KD bipolar), 38 (control KD multipolar), 94 (KIF18A KD bipolar), and 91 (KIF18A KD multipolar) centriole pairs from three independent experiments. Data were analyzed via one-way ANOVA with post hoc Tukey’s test for multiple comparisons. g Representative still frames from three independent experiments of a live MDA-MB-231 KIF18A KD cell labeled with siR-tubulin. Arrows indicate pole splitting and separation. h , i Percent of live, siR-tubulin-labeled MDA-MB-231 cells that ( h ) enter mitosis with more than two spindle poles or ( i ) split and separate spindle poles during mitosis. n = 126 (control KD) and 172 (KIF18A KD) cells from three independent experiments. Data were analyzed by a two-sided Chi-square test. All graphs show mean ± SD and individual data points. P values <0.05 are displayed. All scale bars are 10 microns.

Article Snippet: Human GAPDH (Hs01015428_m1) and human KIF18A (Hs02786624_g1) Taqman probes and primers (Thermo Fisher Scientific) were used for reverse transcription and qRT-PCR.

Techniques: Control, Labeling, Two Tailed Test

Journal: Nature Communications

Article Title: Chromosomally unstable tumor cells specifically require KIF18A for proliferation

doi: 10.1038/s41467-021-21447-2

Figure Lengend Snippet: a , b Percent of fixed MDA-MB-231 cells ( a ) in mitosis or ( b ) with multipolar spindles after the indicated siRNA knockdown (KD). n = 3 independent biological samples for all conditions in a , and n = 3 (MAD2 KD) or 4 (control, KIF18A, and KIF18A & MAD2 KD) in b . Data were analyzed via one-way ANOVA with post hoc Tukey’s test for multiple comparisons ( a ) or two-sided Chi-square test ( b ). P values <0.05 are displayed. c , d Percent of live, siR-tubulin-labeled MDA-MD-231 cells that ( c ) split poles during mitosis or ( d ) entered mitosis with more than two spindle poles. n (number of cells/number of independent experiments) = 126/3 (control), 172/3 (KIF18A), 127/2 (MAD2), and 117/2 (KIF18A and MAD2). All bars represent mean, and error bars represent SD. Individual data points are shown. e Stacked histogram showing relative frequencies of the duration of time between NEB and pole splitting for siR-tubulin-labeled MDA-MB-231 cells following KIF18A KD or KIF18A and MAD2 KD. f Western blots of MAD2, KIF18A, and GAPDH following treatment with either a double dose of control siRNA or a combination of KIF18A and MAD2 siRNA.

Article Snippet: Human GAPDH (Hs01015428_m1) and human KIF18A (Hs02786624_g1) Taqman probes and primers (Thermo Fisher Scientific) were used for reverse transcription and qRT-PCR.

Techniques: Knockdown, Control, Labeling, Western Blot

Journal: Nature Communications

Article Title: Chromosomally unstable tumor cells specifically require KIF18A for proliferation

doi: 10.1038/s41467-021-21447-2

Figure Lengend Snippet: a Percent of MDA-MB-231 cells with multipolar spindles in control or KIF18A KD cells treated with either DMSO, 20 nM Paclitaxel (Pac), or 5 μM Nocodazole (Noc) for 3 h. n = 151 (control KD + DMSO), 263, (KIF18A KD + DMSO), 189 (control KD + pac), 218 (KIF18A KD + pac), 155 (control KD + noc), and 158 (KIF18A KD + noc) cells from three independent experiments. Data were analyzed via a two-sided Chi-square test. b Percentage of mitotic cells with multipolar spindles in fixed MDA-MB-231 or MCF10A after the indicated treatments. n = 9 (control KD), 5 (KIF18A KD), 3 (DMSO), and 5 (paclitaxel) independent experimental replicates for MDA-MB-231 cells. n = 7 (control KD), 5 (KIF18A KD), 3 (DMSO), and 6 (paclitaxel) independent experimental replicates for MCF10A cells. Data were analyzed via a two-sided Chi-square test. c Percentage of fixed MDA-MB-231 and MCF10A cells in mitosis following the indicated treatments. n = 4 independent biological samples for all conditions. Data were analyzed via one-way ANOVA with post hoc Tukey’s test for multiple comparisons. All graphs show mean ± SD with individual data points indicated. P values <0.05 from the indicated statistical tests are displayed.

Article Snippet: Human GAPDH (Hs01015428_m1) and human KIF18A (Hs02786624_g1) Taqman probes and primers (Thermo Fisher Scientific) were used for reverse transcription and qRT-PCR.

Techniques: Control

Journal: Nature Communications

Article Title: Chromosomally unstable tumor cells specifically require KIF18A for proliferation

doi: 10.1038/s41467-021-21447-2

Figure Lengend Snippet: a Percent of monopolar MDA-MB-231 cells with three or more γ-tubulin puncta in control or KIF18A KD cells treated with both monastrol (20 μM) and either DMSO or 20 nM Paclitaxel (Pac). n (number of monopolar mitotic cells/number of independent experiments) = 112/4 (control KD + DMSO), 102/4 (KIF18A KD + DMSO), 132/3 (control KD + Pac), and 149/3 (KIF18A KD + Pac). The plot shows mean ± SD with individual data points indicated. Data were analyzed via a two-sided Chi-square test, and P values <0.05 are displayed. b Representative images (from three independent experiments) of MDA-MB-231 cells treated with 20 μM monastrol and either DMSO or 20 nM Paclitaxel. DNA (DAPI, blue), microtubules (α-tubulin, red), and centrosomes (γ-tubulin, green) are labeled. Scale bar is 5 microns.

Article Snippet: Human GAPDH (Hs01015428_m1) and human KIF18A (Hs02786624_g1) Taqman probes and primers (Thermo Fisher Scientific) were used for reverse transcription and qRT-PCR.

Techniques: Control, Labeling

Journal: Nature Communications

Article Title: Chromosomally unstable tumor cells specifically require KIF18A for proliferation

doi: 10.1038/s41467-021-21447-2

Figure Lengend Snippet: a Representative images (from three independent experiments) of MDA-MB-231 cell density 96 h after the start of high-contrast bright-field imaging. Cells were treated with either control or KIF18A siRNAs in combination with DMSO or 500 nM UMK57. Scale bar is 100 microns. b Fold change in cell density after 96 h in MDA-MB-231 cells treated with the specified siRNAs and either 500 nM UMK57 or DMSO. n = 76 (control KD + DMSO), 52 (control KD + UMK57), 76 (KIF18A KD + DMSO), and 68 (KIF18A KD + UMK57) wells of cells from three independent experiments. Data were analyzed via one-way ANOVA with post hoc Tukey’s test for multiple comparisons. c , d Percent of total mitotic cells ( c ) and mitotic cells with multipolar spindles ( d ) in fixed populations after the indicated treatments. n = 3 independent experimental replicates per condition. Data were analyzed via one-way ANOVA with post hoc Tukey’s test for multiple comparisons. e , f Percent of live, siR-tubulin-labeled MDA-MB-231 cells that ( e ) split poles or ( f ) entered mitosis with more than two spindle poles after the indicated treatments. n = 126 (control KD + DMSO), 158 (control KD + UMK57), 172 (KIF18A KD + DMSO), and 152 (KIF18A KD + UMK57) mitotic cells from three independent experiments. Data were analyzed via a two-sided Chi-square test. All graphs show mean ± SD and individual data points. P values <0.05 are displayed.

Article Snippet: Human GAPDH (Hs01015428_m1) and human KIF18A (Hs02786624_g1) Taqman probes and primers (Thermo Fisher Scientific) were used for reverse transcription and qRT-PCR.

Techniques: Imaging, Control, Labeling

Journal: Nature Communications

Article Title: Chromosomally unstable tumor cells specifically require KIF18A for proliferation

doi: 10.1038/s41467-021-21447-2

Figure Lengend Snippet: Schematic summarizing the differential responses of CIN and near-diploid cells to KIF18A knockdown (KD). Increased microtubule polymerization rates in CIN cells may sensitize them to further deregulation of microtubule dynamics caused by loss of KIF18A function, leading to force imbalances that disrupt the integrity of the spindle. See “Discussion” for additional detail.

Article Snippet: Human GAPDH (Hs01015428_m1) and human KIF18A (Hs02786624_g1) Taqman probes and primers (Thermo Fisher Scientific) were used for reverse transcription and qRT-PCR.

Techniques: Knockdown

Journal: Journal of Virology

Article Title: HPV16 entry requires dynein for minus-end transport and utilizes kinesin Kif11 for plus-end transport along microtubules during mitosis

doi: 10.1128/jvi.00937-24

Figure Lengend Snippet: Kif11 inhibitor reduces transport of EdU-labeled PSVs to mitotic chromosomes. HaCaT cells were infected with WT EdU-labeled PsVs for 24 h and then treated with 1.5 µM Kif11 inhibitor (Eg5 inh+) or left untreated (Ctrl) for 10 min prior to the cells being stained and subjected to the Click-iT reaction as previously described in Materials and Methods. ( A ) AF555 dye was used to stain EdU-labeled pseudogenomes (red), mouse anti-α-tubulin antibody was used to stain for α-tubulin (white), and rabbit anti-Kif11 antibody was used to stain for Kif11 (green). Left-hand panel of Ctrl row of ( A ) is of an infected HaCaT cell in prophase, and the right-hand panel of Ctrl row of ( A ) is of an infected HaCaT cell in metaphase. Since Eg5 inh+ treatment locks cells into a monoastral phenotype , both the left-hand and right-panels of the Eg5 inh+ row of ( A ) are of pre-monoastral cells. Two smaller close-up images, rotated at a 45° angle on the x-, y-, and z-axes, are included on the right-side of the main image(s) to better show close association, or lack thereof in Eg5 inh+, between EdU-labeled HPV genome and Kif11. ( B ) AF555 dye was again used to stain EdU-labeled pseudogenomes in cells undergoing mitosis at prophase or metaphase (red), and mitotic chromosomes were visualized using Hoechst (blue). Images represent single medial slices. ( C ) Quantification of EdU-labeled HPV16 PsV in whole cells +/−Eg5 inh+ treatment; at least 20 cells per group, control and Eg5 inh+ were counted, with 69 total cells counted (group numbers of 21, 26, and 22). Localization of EdU puncta (red) was analyzed with IMARIS using spots/surface analysis. The number of pseudogenomes in whole cells was established based on alpha tubulin and EdU-labeled HPV genome signals; determining genome and the localization on mitotic chromosomes was based on Hoechst staining. Lines in graph represent mean with SEM, and statistical significance was assessed by Student t -test, N = 3, ns: P > 0.05. ( D ) Quantification of EdU-labeled HPV16 PsV (red) co-localized with mitotic chromatin (blue) upon Kif11 inhibitor treatment using IMARIS. At least 20 cells per group, control and Eg5 inh+ were counted, with 66 total cells counted (group numbers of 21, 25, and 20). Lines in graph represent mean with SEM, and statistical significance was assessed by Mann Whitney test, N = 3, ***: P < 0.001. ( E ) Uninfected HaCaT cells were treated with 1.5 µM Eg5 inhibitor for 10 min and then stained for Kif11 and Kif18a as previously described in Materials and Methods, except cells were also subjected to the Click-iT reaction, but without the AF555 dye to stain for kinesins under denaturing conditions. Either Kif11 or Kif18A was stained using anti-rabbit Kif11 or anti-rabbit Kif18A antibodies, respectively (red). Mouse anti-α-tubulin antibody was used to stain α-tubulin (green), and cell nuclei were visualized using Hoechst (blue). Images represent single medial slices.

Article Snippet: Primary antibodies used for the immunofluorescence studies were as follows: mouse monoclonal antibody (mAb) anti-α-tubulin (Cell Signaling; #3873S), mouse mAb AlexaFluor (AF) 488-conjugated anti-α-tubulin (Cell Signaling; #8058), mouse mAb anti-γ-tubulin for the detection of the MTOC (Sigma-Aldrich; #T6557), rabbit mAb anti-dynein intermediate chain 1 (Abcam; #ab171964); all rabbit mAbs for detection of kinesins such as Kif11 (Cell Signaling; #14404S), Kif18a (Novus Biologicals; #NBP1-85126), and Kif25 (Novus Biologicals; #NBP1-92055).

Techniques: Labeling, Infection, Staining, Control, MANN-WHITNEY

Journal: Journal of Virology

Article Title: HPV16 entry requires dynein for minus-end transport and utilizes kinesin Kif11 for plus-end transport along microtubules during mitosis

doi: 10.1128/jvi.00937-24

Figure Lengend Snippet: Kinesin motor proteins are in proximity to HPV16 L2 protein during infection. ( A ) HEK293TT cells were transfected with plasmids encoding HA-tagged HPV16 L2 and FLAG-tagged Kif11 to perform a co-immunoprecipitation (CoIP) assay using anti-HA beads; pCMV6 plasmid was used for the CoIP empty vector controls. CoIPs were performed with (right-hand panel) or without (left-hand panel) transfected cells treated with Kif11 inhibitor (1.5 µM; “Eg5i”) 1 or 2 h prior to cell harvesting for CoIP. Tagged HPV16 L2 and Kif11 proteins were probed for via Western blot using anti-HA and anti-FLAG antibodies, respectively, in both whole-cell lysates (“Lysate”) and anti-HA bead elutions (“elute”); anti α-tubulin Western blots were used as a loading control for the lysates. The enclosed immunoblots are representative of at least two independent experiments ( N = 2). ( B ) Mock-infected (“Mock”) and PsV16-infected (“Infected”) HaCaT cells were processed using the proximity ligation assay (PLA) protocol 21 hpi. PLAs were performed using an antibody cocktail to target the HPV16 L2 protein and an individually selected kinesin, either Kif11, Kif18a, or Kif25. The HPV16L2 antibodies used are the same throughout all PLAs, while the specific kinesin antibody used for the indicated PLA is identified in the left-hand margin of the images. PLA puncta were visualized in the representative images as red dots. Confocal images were acquired as z-stacks and processed as described in Materials and Methods. The white arrows highlight mitotic cells in their representative images for the PLAs. ( C ) The average PLA puncta numbers per interphase cells were counted for at least 200 cells per group in one experiment and are presented as median with 95% CI in the corresponding graphs for the PLAs in the middle panels adjacent to their representative images. The average PLA puncta numbers per mock-infected versus PSV-infected interphase cell were (median) 0.43 vs 6.8 (Kif11), 0.83 vs 5.8 (Kif18A), and 1.8 vs 4.1 (Kif25). Differences between mock and infected cells were analyzed using Mann–Whitney test, ( N = 3); **** P < 0.0001. ( D ) The average PLA puncta numbers were counted per at least 20 mitotic cells for each condition and are presented as median with 95% CI. Mock-infected vs PSV-infected PLA puncta numbers per mitotic cell were (median) 0.0 vs 6.0 (Kif11), 0.0 vs 5.0 (Kif18A), and 1.0 vs 5.5 (Kif25). Differences between mock and infected cells were analyzed using Mann–Whitney test, **** P < 0.0001.

Article Snippet: Primary antibodies used for the immunofluorescence studies were as follows: mouse monoclonal antibody (mAb) anti-α-tubulin (Cell Signaling; #3873S), mouse mAb AlexaFluor (AF) 488-conjugated anti-α-tubulin (Cell Signaling; #8058), mouse mAb anti-γ-tubulin for the detection of the MTOC (Sigma-Aldrich; #T6557), rabbit mAb anti-dynein intermediate chain 1 (Abcam; #ab171964); all rabbit mAbs for detection of kinesins such as Kif11 (Cell Signaling; #14404S), Kif18a (Novus Biologicals; #NBP1-85126), and Kif25 (Novus Biologicals; #NBP1-92055).

Techniques: Infection, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Plasmid Preparation, Cell Harvesting, Western Blot, Control, Proximity Ligation Assay, MANN-WHITNEY

Journal: Journal of cell science

Article Title: Kinesin KIF18A is a novel PUM-regulated target promoting mitotic progression and survival of a human male germ cell line.

doi: 10.1242/jcs.240986

Figure Lengend Snippet: Fig. 2 The effect of PUM1 and PUM2 on the expression of KIF18A LUC-3’UTR construct

Article Snippet: TCam-2 cells were transfected with 40 nM siRNA mixture of 3 different KIF18A sequences (Santa Cruz Biotechnology, sc-96629), PUM1 (Santa Cruz Biotechnology, sc-62912), PUM2 (Santa Cruz Biotechnology, sc-44773) and control scrambled siRNA (Santa Cruz Biotechnology, sc-37007) (sequences in Table S1).

Techniques: Expressing, Construct

Journal: Journal of cell science

Article Title: Kinesin KIF18A is a novel PUM-regulated target promoting mitotic progression and survival of a human male germ cell line.

doi: 10.1242/jcs.240986

Figure Lengend Snippet: Fig. 3 Effect of KIF18A, PUM1 and PUM2 knockdown on cells proliferation. A - The MTS

Article Snippet: TCam-2 cells were transfected with 40 nM siRNA mixture of 3 different KIF18A sequences (Santa Cruz Biotechnology, sc-96629), PUM1 (Santa Cruz Biotechnology, sc-62912), PUM2 (Santa Cruz Biotechnology, sc-44773) and control scrambled siRNA (Santa Cruz Biotechnology, sc-37007) (sequences in Table S1).

Techniques: Knockdown

Journal: Journal of cell science

Article Title: Kinesin KIF18A is a novel PUM-regulated target promoting mitotic progression and survival of a human male germ cell line.

doi: 10.1242/jcs.240986

Figure Lengend Snippet: Fig. 4 The effect of KIF18A siRNA silencing on apoptosis, as measured using imaging flow

Article Snippet: TCam-2 cells were transfected with 40 nM siRNA mixture of 3 different KIF18A sequences (Santa Cruz Biotechnology, sc-96629), PUM1 (Santa Cruz Biotechnology, sc-62912), PUM2 (Santa Cruz Biotechnology, sc-44773) and control scrambled siRNA (Santa Cruz Biotechnology, sc-37007) (sequences in Table S1).

Techniques: Imaging

Journal: Journal of cell science

Article Title: Kinesin KIF18A is a novel PUM-regulated target promoting mitotic progression and survival of a human male germ cell line.

doi: 10.1242/jcs.240986

Figure Lengend Snippet: Fig. 5 The effect of KIF18A siRNA silencing on the cell cycle, as measured by flow

Article Snippet: TCam-2 cells were transfected with 40 nM siRNA mixture of 3 different KIF18A sequences (Santa Cruz Biotechnology, sc-96629), PUM1 (Santa Cruz Biotechnology, sc-62912), PUM2 (Santa Cruz Biotechnology, sc-44773) and control scrambled siRNA (Santa Cruz Biotechnology, sc-37007) (sequences in Table S1).

Techniques:

Journal: Communications Biology

Article Title: Identification of PIM1 substrates reveals a role for NDRG1 phosphorylation in prostate cancer cellular migration and invasion

doi: 10.1038/s42003-020-01528-6

Figure Lengend Snippet: A – D Validation of endogenous PIM1 substrates. LNCaP-AS PIM1 thiophosphorylates PUM1 A , CHMP7 B , NDRG1 C , and KIF18A D . Endogenous proteins were immunoprecipitated from LNCaP-WT PIM1 and LNCaP-AS PIM1 and analyzed by western blot for the presence of thiophosphorylation (ThioP), or immunoprecipitated PUM1, CHMP7, NDRG1, and KIF18A. E – H Phosphorylation site validation using WT and phosphorylation site mutant substrates. Substrates (Myc-PUM1, FLAG-CHMP7, FLAG-NDRG1, and GFP-6HIS-KIF18A; either wild-type (WT) or the indicated PIM1 phosphorylation site mutation) were expressed in 293 T cells with AS PIM1 and thiophosphorylation labeling completed. Substrates were immunoprecipitated using Myc, FLAG, or GFP-magnetic beads, and Western blot performed to detect thiophosphorylated, or the immunoprecipitated substrates. Western blots are representative of two independent experiments.

Article Snippet: The antibodies used were as follows: PIM1 (sc-13513; Santa Cruz Biotechnology), NDRG1 (HPA006881, Sigma), NDRG1 pS330 (ab124713, Abcam), AR (sc-7305, Santa Cruz Biotechnology), HSP90 (610418, BD Biosciences), FLAG (F3165, Sigma), α-Tubulin (ab7291, Abcam), CHMP7 (HPA036119, Sigma), Thiophosphate ester antibody [51–8] (ab92570, Abcam), KIF18A (C-19, Santa Cruz Biotechnology), PUM1 (ab92545, Abcam), NaK + ATPase (EP1845Y, Abcam), SP1 (PA5-29165, Thermofisher), Histone H3 (ab1791, Abcam).

Techniques: Biomarker Discovery, Immunoprecipitation, Western Blot, Phospho-proteomics, Mutagenesis, Labeling, Magnetic Beads

Journal: Nature

Article Title: Whole genome doubling confers unique genetic vulnerabilities on tumor cells

doi: 10.1038/s41586-020-03133-3

Figure Lengend Snippet: (a) Workflow used to identify gene essentiality in WGD + cancer cells from Project Achilles data (see ). (b) Top hits from PSL analysis (text color indicates genes associated with indicated pathways). (c) Gene expression fold changes in WGD + tumors relative to WGD – tumors plotted against combined FDR values across all tumor types with select PSL genes highlighted. (d) Population doublings after 8 days of AZ3146 treatment (n = 3 independent experiments; two-way ANOVA with interaction; graph shows mean +/− SEM; interaction p-values = 0.0085, 0.0020, 0.0156, respectively). (e) Relative viability of indicated cell lines 7 days after treatment with indicated siRNA (n = 3 independent experiments; graph shows mean +/− SEM). (f) Mean LC 50 for 5 WGD – and 5 WGD + breast cancer cell lines for indicated drug treatments (n = 3 independent experiments; nonlinear regression; graphs show mean LC 50 +/− 95% CI). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: We infected cells with a SMARTvector Inducible Lentiviral shRNA (Horizon) targeting KIF18A and selected cells with puromycin (Santa Cruz Biotechnology) at 2 μg/mL.

Techniques: Gene Expression

Journal: Nature

Article Title: Whole genome doubling confers unique genetic vulnerabilities on tumor cells

doi: 10.1038/s41586-020-03133-3

Figure Lengend Snippet: (a) Mitotic duration of indicated cells following indicated treatments (n = 200 cells; Student’s unpaired t-test – two-sided; graph shows mean +/− SEM; p-values = <0.0001, <0.0001, 0.0265, respectively). (b) The fraction of mitoses that generate micronuclei following indicated treatments (n = 200 cells; Student’s unpaired t-test – two-sided; p-values = <0.0001, <0.0001, <0.0001, <0.0001, 0.0002, <0.0001, respectively). (c) Relative viability of 2N and 4N HCT116 cells 7 days after treatment with indicated siRNA at indicated concentrations with Western blot showing protein knockdown 48 hours after treatment with siRNA (n = 3 independent experiments; graph shows mean +/− SEM at each dose; for gel source see ). (d) Relative viability of 2N and 4N MCF10A cells 7 days after treatment with indicated siRNA at 50 pM concentration (n =3 independent experiments; Student’s unpaired t-test – one-sided; graph shows mean +/− SEM; p-values = <0.0001, <0.0001). (e) Relative viability of 2N and 4N RPE cells 5 days after treatment with indicated siRNA at 50 pM concentration (n = 3 independent experiments; Student’s unpaired t-test – one-sided; graph shows mean +/− SEM; p-values = 0.090, 0.0007, respectively). (f) Representative Western blot showing knockdown of indicated proteins 48 hours after treatment with indicated siRNA (n = 3 independent experiments; for gel source see ). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: We infected cells with a SMARTvector Inducible Lentiviral shRNA (Horizon) targeting KIF18A and selected cells with puromycin (Santa Cruz Biotechnology) at 2 μg/mL.

Techniques: Western Blot, Knockdown, Concentration Assay

Journal: Nature

Article Title: Whole genome doubling confers unique genetic vulnerabilities on tumor cells

doi: 10.1038/s41586-020-03133-3

Figure Lengend Snippet: (a-b) Dose-response to indicated treatment after 7 days in indicated cell lines with accompanying LC 50 (n = 3 independent experiments; nonlinear regression with variable slope; graphs show mean relative viability +/− SEM at each dose and mean LC 50 +/− 95% CI). (c-e) Dose-response curves for 5 WGD – and 5 WGD + breast cancer cell lines 7 days after indicated drug treatment at the indicated concentrations (n = 3 independent experiments; nonlinear regression with variable slope; graph shows mean +/− SEM at each dose). (f) Representative Western blot showing knockdown of indicated proteins in breast cancer cell lines 48 hours after treatment with indicated siRNA (n = 3 independent experiments; for gel source see ). (g) Relative viability decrease in WGD + and WGD – breast cancer cell lines 7 days after treatment with indicated siRNA (Wilcoxon rank-sum test – two-sided; graph shows mean +/− SEM; p-values = <0.0001, 0.0027, respectively) * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: We infected cells with a SMARTvector Inducible Lentiviral shRNA (Horizon) targeting KIF18A and selected cells with puromycin (Santa Cruz Biotechnology) at 2 μg/mL.

Techniques: Western Blot, Knockdown

Journal: Nature

Article Title: Whole genome doubling confers unique genetic vulnerabilities on tumor cells

doi: 10.1038/s41586-020-03133-3

Figure Lengend Snippet: (a) Dose-response to MG132 treatment after 7 days in indicated cell lines with accompanying LC 50 (n = 3 independent experiments; nonlinear regression with variable slope; graphs show mean relative viability +/− SEM at each dose and mean LC 50 +/− 95% CI). (b) Progression free survival and overall survival in patients with upper tertile tumor expression of KIF18A in the TCGA (Cox proportional-hazards regression; graph shows hazard ratios +/− 95% CI). (c) Representative Western blot showing KIF18A levels following transfection with the indicated siRNAs in the indicated cell lines (n = 3 independent experiments; for gel source see ). (d) Anaphase phenotypes following depletion of KIF18A (n = 20 cells per condition; stars indicate p-value for two-sided Fisher’s exact test comparing the fraction of anaphases with lagging chromosomes; p-values = <0.0001, 0.0033, 0.0187, respectively). (e) Representative confocal images showing phases of mitosis in indicated cell lines 48 hours after transfection with indicated siRNA (representative images from 2 independent experiments; scale bar 10 μm). (f) Representative still images from 2N and 4N MCF10A cells progressing through mitosis after transfection with the indicated siRNAs. H2B-GFP labeled chromosomes are shown in white. Arrows in enlarged images show oscillating chromosomes during metaphase and the generation of a micronucleus (hrs: min; scale bar 10 μm) * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: We infected cells with a SMARTvector Inducible Lentiviral shRNA (Horizon) targeting KIF18A and selected cells with puromycin (Santa Cruz Biotechnology) at 2 μg/mL.

Techniques: Expressing, Western Blot, Transfection, Labeling

Journal: Nature

Article Title: Whole genome doubling confers unique genetic vulnerabilities on tumor cells

doi: 10.1038/s41586-020-03133-3

Figure Lengend Snippet: (a) Relative viability of indicated cell lines 8 days after transfection with the indicated siRNAs (n = 3 independent experiments; each condition normalized to respective control; Student’s unpaired t-test – two-sided; graph shows mean +/− SEM; p-values = 0.0019, 0.0109, 0.0017, respectively). (b) Mitotic duration and fate after treatment with indicated siRNA (n = 200 cells per condition; black stars indicate p-value for two-sided Student’s t-test comparing mean mitotic duration; blue stars indicate p-value for two-sided Fisher’s exact test comparing the fraction of mitoses that give rise to micronuclei; dotted line represents mean mitotic duration). (c) Measurement of spindle length (centrosome-to-centrosome) after transfection with indicated siRNA (n = 20 cells per condition; two-way ANOVA with interaction; graph shows mean +/− SEM; scale bar 10 μm; interaction p-values = 0.0001, 0.0011, 0.0032, respectively). (d) Image demonstrating measurement of chromosome oscillations immediately prior to anaphase by assessing the widest oscillating chromosomes in each poleward direction and the cross-sectional area of all the chromosomes (scale bar 10 μm). (e) Widest oscillating chromosome in each poleward direction immediately prior to anaphase (n = 20 cells per condition from 2 independent experiments; two-way ANOVA with interaction; interaction p-values = 0.0025, <0.0001, <0.0001, respectively). (f) Two-dimensional cross-sectional area of the entire body of chromosomes immediately prior to anaphase (n = 20 cells per condition; Student’s unpaired t-test – two-sided; graph shows mean +/− SEM; p-values = 0.0012, <0.0001, 0.0525, 0.0318, <0.0001, 0.0318, 0.0432, respectively). (g) Representative confocal images showing phases of mitosis in indicated cell lines 48 hours after transfection with indicated siRNA (scale bar 10 μm). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: We infected cells with a SMARTvector Inducible Lentiviral shRNA (Horizon) targeting KIF18A and selected cells with puromycin (Santa Cruz Biotechnology) at 2 μg/mL.

Techniques: Transfection, Control

Journal: Nature

Article Title: Whole genome doubling confers unique genetic vulnerabilities on tumor cells

doi: 10.1038/s41586-020-03133-3

Figure Lengend Snippet: (a) Representative image of a 4N MCF10A cell 4 days after transfection with siKIF18A and stained for cGAS. Graph shows the fraction of micronuclei in 2N and 4N MCF10A cells with indicated treatment that stained positive for cGAS (n = 200 micronuclei per condition; Fisher’s exact test – two-sided; scale bar 10 μm; p-values = <0.0001, 0.0069, respectively). (b-c) Representative confocal images of indicated cell lines 48 hours after transfection with indicated siRNA. Arrows highlight MAD1 positive kinetochores in misaligned chromosomes (scale bar 10 μm; representative images from 2 independent experiments). (d) Representative Western blot of indicated protein levels after treatment with indicated siRNA and accompanying graphs showing relative protein levels normalized to loading control (n = 3 independent experiments; Student’s unpaired t-test – one-sided; graph shows mean +/− SEM; p-values = 0.0337, 0.0030, 0.0674, 0.0421, 0.0067, 0.0227, respectively; for gel source see ). (e) Cell fates of indicated cell lines tracked for 3 days beginning 18 hours after transfection with indicated siRNA (n = 40 cells per condition; two-sided Fisher’s exact test comparing fraction of cells arresting/delaying in interphase relative to control group; p values = 0.0016, <0.0001, <0.0001, respectively). (f) Relative viability of indicated cell lines 4 days after transfection with the indicated siRNA (n = 3 independent experiments; Student’s unpaired t-test – two-sided; graph shows mean +/− SEM; p-values = 0.0132, 0.0310, 0.8808, 0.8615, respectively). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: We infected cells with a SMARTvector Inducible Lentiviral shRNA (Horizon) targeting KIF18A and selected cells with puromycin (Santa Cruz Biotechnology) at 2 μg/mL.

Techniques: Transfection, Staining, Western Blot, Control

Journal: Nature

Article Title: Whole genome doubling confers unique genetic vulnerabilities on tumor cells

doi: 10.1038/s41586-020-03133-3

Figure Lengend Snippet: (a) Western blot showing endogenous KIF18A levels in indicated cell lines with graph showing respective protein levels normalized to GAPDH loading control (representative blot from 3 independent experiments; for gel source see ). (b) Representative Western blot showing KIF18A levels 48 hours after transfection with indicated siRNA (n = 3 independent experiments; for gel source see ). (c) Relative viability decrease in WGD + and WGD – breast cancer cell lines 7 days after treatment with indicated siRNA (n = 3 independent experiments; Wilcoxon rank-sum test – two-sided; graph shows mean +/− SEM; p-value < 0.0001). (d) Relative viability 7 days after induction of Cas9 in cells with sgRNA targeting KIF18A with Western blot showing protein depletion 72 hours after induction (n = 3 independent experiments; graph shows mean +/− SEM; Student’s unpaired t-test – two-sided; p-values = 0.0007, < 0.0001, respectively; for gel source see ). (e) Relative viability 7 days after induction of shRNA targeting KIF18A with Western blot showing protein depletion 120 hours after induction (n = 3 independent experiments; graph shows mean +/− SEM; Student’s unpaired t-test – one-sided; p-value < 0.0001; for gel source see ). (f) Widest oscillating chromosome in each poleward direction immediately prior to anaphase (n = 20 cells per condition; Student’s unpaired t-test – two-sided; p-values = 0.0022, 0.1781, 0.1487, 0.0136, 0.0820, <0.0001, <0.0001, <0.0001, <0.0001, 0.4132, respectively). (g) Two-dimensional cross-sectional area of the entire body of chromosomes immediately prior to anaphase (n = 20 cells per condition; Student’s unpaired t-test – two-sided; p-values = 0.1178, 0.7545, 0.1440, 0.0034, 0.9989, 0.0005, 0.0033, 0.0012, 0.0110, 0.9089, respectively; graph shows mean +/− SEM). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: We infected cells with a SMARTvector Inducible Lentiviral shRNA (Horizon) targeting KIF18A and selected cells with puromycin (Santa Cruz Biotechnology) at 2 μg/mL.

Techniques: Western Blot, Control, Transfection, shRNA

Journal: Nature

Article Title: Whole genome doubling confers unique genetic vulnerabilities on tumor cells

doi: 10.1038/s41586-020-03133-3

Figure Lengend Snippet: (a) Relative viability of cell lines 8 days after transfection with the indicated siRNAs (n = 3 independent experiments; graph shows mean +/− SEM). (b) Mitotic duration and fate following transfection with indicated siRNA (n = 80 cells per condition across 2 independent experiments; dotted line represents mean mitotic duration; black stars indicate p-values for two-sided Student’s unpaired t-test comparing mean mitotic duration; blue stars indicate p-values for two-sided Fisher’s exact test comparing fraction of mitoses that give rise to micronuclei; red stars indicate p-values for two-sided Fisher’s exact test comparing fraction of cell that die in mitosis). (c) Depletion of KIF18A impairs WGD + cell viability through two distinct mechanisms: A) Widely oscillating chromosomes fail to properly attach to microtubules, thus activating the spindle assembly checkpoint and leading to prolonged mitosis and death. B) Larger spindles and wider oscillations increase the distance some chromosomes must traverse in anaphase leading to lagging chromosomes, micronuclei formation, and cellular arrest. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: We infected cells with a SMARTvector Inducible Lentiviral shRNA (Horizon) targeting KIF18A and selected cells with puromycin (Santa Cruz Biotechnology) at 2 μg/mL.

Techniques: Transfection

Journal: Nature

Article Title: Whole genome doubling confers unique genetic vulnerabilities on tumor cells

doi: 10.1038/s41586-020-03133-3

Figure Lengend Snippet: (a) Measurement of spindle length (centrosome-to-centrosome) after transfection with indicated siRNA (n = 20 cells per condition; Student’s unpaired t-test – two-sided; graph shows mean +/− SEM). (b) Anaphase phenotypes following depletion of KIF18A (n = 20 cells per condition; stars indicate p-value for two-sided Fisher’s exact test comparing the fraction of anaphases with lagging chromosomes). (c) The fraction of cells in each cell line that undergo indicated fates after completing a mitosis deficient of KIF18A that resulted in micronuclei formation (n = 25 cells per condition). (d) The fraction of cells in each cell line that experience mitotic death in their first and second mitoses following KIF18A depletion (n = 25 cells per condition). (e) KIF18A essentiality scores for WGD – and WGD + cell lines segregated into “highly aneuploid” (AS > 10) and “non-highly aneuploid” categories based on aneuploidy score (AS) (see ) (dotted lines show mean; Wilcoxon rank-sum test – two-sided; p-values = 0.02583, 0.3682, respectively). (f) Aneuploidy scores and WGD status for 998 cancer cell lines in the CCLE. (g) Relative viability of indicated cell lines 7 days after transfection with the indicated siRNAs (n = 3 independent experiments; each condition normalized to respective control; one-way ANOVA with Dunnett’s post hoc test; graph shows mean +/− SEM; p-values = 0.1676, > 0.9999, 0.0040, 0.2698, 0.0007, respectively). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: We infected cells with a SMARTvector Inducible Lentiviral shRNA (Horizon) targeting KIF18A and selected cells with puromycin (Santa Cruz Biotechnology) at 2 μg/mL.

Techniques: Transfection, Control