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. 2019 Jun 28;33(9):10515–10527. doi: 10.1096/fj.201900154RR

Figure 4.

Figure 4

KDM3C deletion enhances and accelerates activation and translocation of NF-κB in mouse BMDMs. A) NF-κB subunit p65 localization was determined in BMDMs from Kdm3C WT (Kdm3C+/+) and Kdm3C KO (Kdm3C−/−) mice after Pg LPS (1 µg/ml) treatment for 0, 30, and 60 min. Representative images from the immunofluorescence assay. Scale bars, 5 µm. Green, p65; cyan, DAPI staining (nuclei). B) Nuclear-located p65 cells were scored after Pg LPS treatments for 0, 10, 30, and 60 min. Cells were counted in 7 random areas of each image in duplicate experiments and described as a ratio of cells containing nuclear-located p65 for total cells. In each region, at least 150 cells were blindly counted. Statistics were performed using 1-way ANOVA and the Bonferroni method. ***P < 0.001 compared with Kdm3C+/+ BMDMs. C) Western blotting analysis of the levels of phosphorylated p65 (p-p65), total p65, IκB-α, and KDM3C in Kdm3C+/+ and Kdm3C−/− BMDMs upon time-dependent Pg LPS exposure. β-actin was used as a loading control. The normalized intensities of the proteins were calculated and shown as fold change. The p-p65:p65 ratio was calculated and shown as fold change. These data are representative of at least 3 independent experiments. D) Expression levels of NFKBIA/Nfkbia, encoding IκB-α, were determined by real-time qPCR in the Kdm3C+/+ and Kdm3C−/− BMDMs (left) or in the KDM3C–knocked-down (si-KDM3C) and control (si-control) THP-1 cells (right). **P < 0.01, ***P < 0.001 compared with Kdm3C+/+ BMDMs (left) or control THP-1 cells (right). E) KDM3C binding and H3K9me1, H3K9me2 levels to the NFKBIA promoter in control and KDM3C–knocked-down THP-1 cells. Cells were chromatin immunoprecipitated with an antibody against KDM3C, H3K9me1, H3K9me2, or IgG controls. The precipitated DNA was then amplified by qPCR using primers illustrated at the top. *P < 0.05, ***P < 0.001 compared with control cells. TSS, Transcription start site. F) The positions of primer pairs are indicated as R1 to R6. ChIP assays using primers (R1 ∼ R6) to the NFKBIA promoter to determine the location of H3K9me2 binding, which is regulated by KDM3C, on the NFKBIA promoter in THP-1 cells.