Figure 6 from Functional RNAi Screening Identifies G2/M and Kinetochore Components as Modulators of TNFα/NF-κB Prosurvival Signaling in Head and Neck Squamous Cell Carcinoma
Authors: Morgan Ethan L. , Saleh Anthony D. , Cornelius Shaleeka , Carlson Sophie G. , Toni Tiffany , Cheng Hui , Jeon Jun , Viswanathan Ramya , Yang Xinping , Silvin Christopher , Clavijo Paul E. , Sowers Anastasia L. , Mitchell James B. , Ormanoglu Pinar , Lal Nag Madhu , Martin Scott E. , Chen Zhong , Van Waes Carter 
Published: November 2024

<p>TTK inhibition induces mitotic abnormalities and genome instability in combination with radiation treatment. <b>A,</b> Cell-cycle analysis of UMSCC1 and UMSCC47 cells were treated with 20 nmol/L B389 for 24 and 72 hours. Red asterisks indicate a significant difference in the >4N population compared with control; black asterisks indicate a significant difference in the sub-G1 population compared with control. Bars indicate a significant difference in the sub-G1 population between B389 and the combination treatment. Statistics are shown in Supplementary Table S5. <b>B,</b> UMSCC1 and UMSCC47 cells were treated with 20 nmol/L B389 for 24 and 72 hours. Cells were then analyzed for the percentage of phosphorylated histone H3 (Ser10), a marker of mitosis, and their DNA content using PI. Red asterisks indicate a significant difference in the >4N population compared with control; black asterisks indicate a significant difference in the sub-G1 population compared with control. Bars indicate a significant difference in the >4N population between B389 and the combination treatment. Statistics are shown in Supplementary Table S5. <b>C,</b> left, Clonogenic survival assays of UMSCC1 and UMSCC47 treated with B389 at the indicated doses for 2 hours before irradiation. Cells were then immediately harvested and replated after irradiation. Right, Survival fraction at 4  Gy from each cell line. DMF values were calculated as the vehicle radiation dose for 10% survival divided by radiation dose for 10% survival with the indicated treatment. <b>D,</b> UMSCC1 and UMSCC47 cells were treated with B389 (20 nmol/L) for 2 hours before irradiation. Cells were then analyzed for γH2AX expression by flow cytometry after 24 hours. For nonirradiated samples, cells were treated with vehicle or B389 (20  nmol/L) for 24 hours before analysis. Data are presented as the relative MFI compared with the nonirradiated control samples. <b>E,</b> left, Representative immunofluorescence images of γH2AX foci in UMSCC1 and UMSCC47 cells after treatment with B389 (20 nmol/L) and/or radiation. Cells were treated with B389 (20 nmol/L) for 2 hours before irradiation. Cells were then analyzed for γH2AX foci by immunofluorescence analysis at the indicated time points. For nonirradiated samples, cells were treated with vehicle or B389 (20 nmol/L) for 24 hours before analysis. DAPI was used as a nuclear counterstain. Scale bar, 10 μmol/L (right). Quantification of γH2AX foci. For each condition, foci from at least 200 cells were counted. <b>F,</b> Cells were treated as in <b>D</b>. After 24 hours, mitotic cells were analyzed by confocal microscopy for the appearance of mitotic aberrations. Cells were probed for phosphorylated histone H3 (Ser10) and α-tubulin, and DAPI was used as a nuclear counterstain. The percentage of each type of mitotic aberrations is presented from at least 30 individual cells from duplicate experiments. <b>G,</b> Cells were treated as in <b>D</b>. After 24 hours, cells were analyzed by confocal microscopy for the appearance of micronuclei using DAPI as a nuclear counterstain. The percentage of cells with 0, 1, 2, or ≥3 associated micronuclei is presented at least 200 individual cells from duplicate experiments. Bars represent means ± SD. All experiments are representative of at least three biological replicates, except (<b>F</b> and <b>G</b>), which are from duplicate experiments. *, <i>P</i> < 0.05; **, <i>P</i> < 0.01; ***, <i>P</i> < 0.001. NS, not significant; MFI, median fluorescence intensity; propidium iodide; DMF, dose modification factor, XRT, X-ray therapy.</p>