Figure 4 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

<p>TTK inhibition reduces NF-κB RELA nuclear localization and activity in HNSCC cells. <b>A,</b> Representative immunofluorescence images of RELA localization in UMSCC1 and UMSCC47 cells. Cells were transfected with TTK siRNA or control siRNA for 72 hours, with TNFα (20 ng/mL) added for 30 minutes. DAPI was used as a nuclear counterstain. <b>B,</b> Quantification of percentage nuclear RELA from <b>A</b>. Data represent the percentage nuclear localization of RELA from 15 cells and were analyzed using ImageJ as previously (<a href="#bib12" target="_blank">12</a>). Bars represent means ± SD. <b>C,</b> UMSCC1 and UMSCC47 cells were treated with 20 nmol/L BAY1217389 (B389) for 24 hours. Cells were then analyzed for the percentage of phosphorylated histone H3 (Ser10), a marker of mitosis. <b>D,</b> NF-κB reporter activity after treatment with increasing doses of B389 in UMSCC1^κB cells. Cells were treated with increasing doses of B389 or vehicle control for 24 hours, with TNFα (20 ng/mL) added for the final 16 hours. <b>E,</b> Representative immunofluorescence images of RELA localization in UMSCC1 and UMSCC47 cells. Cells were treated with B389 (20  nmol/L) or vehicle control for 6 hours, with TNFα (20 ng/mL) added for 30 minutes. DAPI was used as a nuclear counterstain. <b>F,</b> Quantification of percentage nuclear RELA from <b>E</b>. Data represent the percentage nuclear localization of RELA from 15 cells and were analyzed using ImageJ as previously (<a href="#bib12" target="_blank">12</a>). All experiments are representative of at least three biological replicates. NS, not significant; ***, <i>P</i> < 0.001.</p>