Skip to main content
. 2022 Apr 19;13(4):374. doi: 10.1038/s41419-022-04826-5

Fig. 4. The NF-κB pathway is a direct target of the NOB–RORs axis.

Fig. 4

A MDA-MB-231 cells were treated with NOB. IkBα mRNA expression was measured by qRT-PCR. Data represent mean ± SEM. Two-tailed Student’s t-test, *p < 0.05. B MDA-MB-231 cells were treated with NOB for 24 h. IkBα protein expression was measured by western blotting. In all, 4.4-fold change, p < 0.01 compared to DMSO. C Schematic representation of putative RRE sites found in the IκBα promoter (TRANSFAC). The distance in bp from the transcription start site of IkBα gene is shown. MDA-MB-231 cells were treated with 20 µM NOB for 12 h. Samples were normalized to input chromatin and expressed as % input. Error bars represent mean ± SD; n = 2; two-tailed Student’s t-test. *p < 0.05; **p < 0.01. D MDA-MB-231 cells were treated with 10 µM NOB for 12 h prior to TNF-α treatment (10 ng/ml). Whole-cell lysates were subjected to western blot analysis using anti-IκBα, phospho-p65 (Ser536), total p65, and GAPDH antibodies. E MDA-MB-231 cells were transfected with pGL4.32 [luc2P/NF-κB-RE/Hygro] vector and treated with NOB (+, 10 µM; ++, 20 µM; +++, 30 µM) for 12 h prior to TNF-α treatment. Data represent mean ± SEM. Two-tailed Student’s t-test, ***p < 0.001. Compared with the TNF-α control, ##p < 0.01, ###p < 0.001. F p65 nuclear localization by TNFα was inhibited by NOB in MDA-MB-231. Representative immunofluorescence images of endogenous p65 in MDA-MB-231 with NOB pretreatment 12 h prior to TNF-α treatment (×400 magnification, scale bar = 10 µm). Two-tailed Student’s t-test, ***p < 0.001. G MDA-MB-231 cells were transfected with control siRNA, siRORA or siRORC and treated 10 µM NOB for 12 h prior to TNF-α treatment. Representative confocal images are shown (×400 magnification, scale bar = 10 µm). Cell extracts were subjected to dual luciferase assays. N nucleus; C cytoplasm.