Fig. 4

NFIL3 enhances the activity of NF-κB signaling and inhibits the transcription of NFKBIA in TNBC cells. A. Translocation of NF-κB from the cytoplasm to the nucleus was detected using immunofluorescence to indicate the activation of NF-κB signaling upon changing the expression of NFIL3 in Hs578T cells with TNF-α (25 ng/mL) stimulation (magnification 400×, scale bars = 50 μm). B. A luciferase reporter assay was used to detect the activity of NF-κB signaling in Hs578T cells upon NFIL3 overexpression or knockdown with or without TNF-α (25 ng/mL) stimulation (**p < 0.01, ***p < 0.001). The experiment was repeated in triplicate. C. The binding of NFIL3 in the promoter region of the NFKBIA gene was validated in Hs578T cells using a ChIP assay. D. A dual luciferase reporter assay was performed to test the suppressive effect of NFIL3 on the transcription of luciferase driven by the NFKBIA gene promoter in Hs578T cells (**p < 0.01). This experiment was repeated in triplicate. E-F. The expression change of NFKBIA at the mRNA and protein levels in Hs578T cells was detected using qPCR and western blots, respectively, upon NFIL3 overexpression or knockdown. (*p < 0.05, **p < 0.01, ***p < 0.001). G. Pearson correlation analysis between the expression of NFIL3 and NFKBIA in breast cancer tissues using IHC (magnification, 200×, scale bars = 100 μm, p < 0.01). H. Pearson correlation analysis between NFIL3 and NFKBIA in breast cancer tissues based on the TCGA dataset