WT, IKKα KO, IKKβ KO and IKKα/β DKO HCT116 cells were seeded in normal growth medium for 48 h, prior to treatment with 10 ng/ml TNFα for 15, 30 and 120 min. Immunofluorescence staining of cells was performed with anti-c-Rel (red) and nuclei were stained with DAPI (blue). (A) Representative images are shown for time zero, 15, 30 and 120 min of TNFα stimulation. (B) Cells were seeded in 96 well plates in normal growth medium for 48 h, prior to treatment with 10 ng/ml TNFα for the indicated timepoints. Cells were stained with anti-c-Rel and DAPI and confocal images captured using the In Cell Analyzer 6000 high-content imaging system (20× objective). c-Rel staining intensity analysis was performed using CellProfiler software. Data is presented as the log₂ transformed fold change in nuclear:cytoplasmic c-Rel staining intensity. Data are mean ± SD of three independent CRISPR–Cas9 KO clones in each case. Significance testing relative to WT control cells performed using two-way ANOVA (repeated measures) with Dunnett post-hoc test. P < 0.0001 (***). Scale bar, 10 µm.