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. 2024 Nov 7;4(11):2903–2918. doi: 10.1158/2767-9764.CRC-24-0274

Figure 4.

Figure 4

TTK inhibition reduces NF-κB RELA nuclear localization and activity in HNSCC cells. A, 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, Quantification of percentage nuclear RELA from A. Data represent the percentage nuclear localization of RELA from 15 cells and were analyzed using ImageJ as previously (12). Bars represent means ± SD. C, 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. D, NF-κB reporter activity after treatment with increasing doses of B389 in UMSCC1κB 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. E, 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. F, Quantification of percentage nuclear RELA from E. Data represent the percentage nuclear localization of RELA from 15 cells and were analyzed using ImageJ as previously (12). All experiments are representative of at least three biological replicates. NS, not significant; ***, P < 0.001.