Journal: The EMBO Journal

Article Title: Weakened APC/C activity at mitotic exit drives cancer vulnerability to KIF18A inhibition

doi: 10.1038/s44318-024-00031-6

Figure Lengend Snippet: ( A ) Linear correlation between 5-day KIF18Ai toxicity and mitotic duration in cells without errors from live-cell wide-field time-lapse microscopy of H2B/α-Tubulin fluorescently tagged cell lines from Fig. . ( B ) Metaphase-to- anaphase duration from live-cell wide-field time-lapse microscopy of H2B/α-Tubulin fluorescently tagged cell lines from Fig. . Error bars represent mean ± SD. N ≥ 50 cells per condition. Full sample size information is listed in Dataset . ( C ) Linear correlation between metaphase-to-anaphase duration in ( B ) and mitotic duration in KIF18Ai from live-cell wide-field time-lapse microscopy of H2B/α-Tubulin fluorescently tagged cell lines from Fig. . ( D ) Top KIF18A co-dependency relationships from the DepMap RNAi dataset. N > 600 cell lines. ( E ) Live-cell wide-field time-lapse microscopy of endogenously tagged Cyclin B1-eYFP at the metaphase-to- anaphase transition for RPE1 and HeLa cells. Scale bar = 10 µm. ( F ) Quantification of Cyclin B1 degradation rates as in ( E ) for untreated endogenously tagged fluorescent cells. The median (colored line) of individual traces (gray lines) is plotted, with the shaded region encompassing the first and third quartile of each population. Cyclin B1 signal is normalized to the metaphase inflection point, and median t 1/2 values are listed. Full sample size information is listed in Dataset . ( G ) Maximum slope of Cyclin B1 degradation at metaphase for untreated endogenously tagged fluorescent cells in ( F ). Rates are calculated relative to the total Cyclin B1 signal at mitotic entry. Data are represented as mean ± SD. N ≥ 30 cells per condition. ( H ) Media normalized 5-day MTT endpoint viability assay of sensitive cell lines overexpressing either WT or M43 CDC20 in KIF18Ai. Data are represented as mean ± SD. N ≥ 3 independent experiments per cell line, n = 3 technical replicates per experiment. Media normalization was used rather than DMN normalization since CDC20 overexpression generated growth rescue in DMN. Statistical significance was determined using a one-way ANOVA with post hoc Dunnett’s multiple comparisons test between each overexpression pair and WT. Full statistical results are listed in Dataset . ( I ) Five-day MTT endpoint viability assay of UBE2S-overexpressing cell lines in KIF18Ai. Data are represented as mean ± SD. N ≥ 1 independent experiment, n = 3 technical replicates per experiment. Statistical significance was determined using an unpaired two-tailed Student’s t test between WT and UBE2S-overexpressing cells. Full statistical results are listed in Dataset . ( J ) Titration of the MPS1 inhibitor Reversine in a 5-day MTT endpoint viability assay in DMSO or KIF18Ai-treated HCC1806 WT or UBE2S-overexpressing cells. Open squares are omitted from the curve fit. N = 3 technical replicates from a single experiment. Data are represented as mean ± SD. Data information: * P < 0.05, ** P < 0.01, and **** P < 0.0001 ( H , I ). .

Article Snippet: To generate HeLa endogenously tagged CCNB1-EYFP (Cyclin B1) cells, an sgRNA targeting the Cyclin B1 translational stop codon (5′-gtgtaacttgtaaacttgagt-3′) was cloned into a pX459 vector (#62988; Addgene).

Techniques: Time-lapse Microscopy, Viability Assay, Over Expression, Generated, Two Tailed Test, Titration

Journal: The EMBO Journal

Article Title: Weakened APC/C activity at mitotic exit drives cancer vulnerability to KIF18A inhibition

doi: 10.1038/s44318-024-00031-6

Figure Lengend Snippet: ( A ) Top 10 RNAi co-dependency relationships from DepMap dataset for KIF18A. Red and blue points represent sensitive and insensitive cell lines respectively from the panel in Fig. . Bolded table entries are APC/C or SAC genes. ( B ) Quantification of Cyclin B1 degradation rates for endogenously tagged fluorescent cells treated with Reversine, KIF18Ai, or Nocodazole. The median (colored line) of individual traces (gray lines) is plotted, with the shaded region encompassing the first and third quartile of each population. Cyclin B1 signal is normalized to the metaphase inflection point and median t 1/2 values are listed. Full sample size information is listed in Dataset . ( C ) Maximum slope of Cyclin B1 degradation at metaphase for endogenously tagged fluorescent cells treated with Reversine, KIF18Ai, or Nocodazole in ( B ). Rates are calculated relative to the total Cyclin B1 signal at mitotic entry. Data are represented as mean ± SD. N ≥ 20 cells per condition (KIF18Ai, Reversine), N ≥ 5 cells per condition (nocodazole).

Article Snippet: To generate HeLa endogenously tagged CCNB1-EYFP (Cyclin B1) cells, an sgRNA targeting the Cyclin B1 translational stop codon (5′-gtgtaacttgtaaacttgagt-3′) was cloned into a pX459 vector (#62988; Addgene).

Techniques: