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. 2022 Mar 15;132(6):e128808. doi: 10.1172/JCI128808

Figure 2. NEMO-Δex5 blocks NF-κB activation in response to poly(I:C) but TNF-induced NF-κB remains intact.

Figure 2

(A) NEMO/TANK/TBK1 interaction and function. (B) HEK293T cells transfected with TANK, TBK1, and NEMO followed by TANK IP and Western blot as indicated. (C and D) Patient and healthy control (HC) dermal fibroblasts (DFs) were stimulated with poly(I:C) in a time course followed by Western blot as indicated. Quantitation of phospho-p65/IκBα by optical densitometry, below. Dashed line at 20%. (E) P1 and HC DFs were stimulated with TNF or poly(I:C) for 60 minutes and stained for NF-κB p65 visualized at 200× magnification by immunofluorescence (Keyence BZ9000), quantitation of p65 nuclear intensity, right. Statistical significance by 2-way ANOVA with Tukey’s multiple-comparison test. (F) DFs from HC and P1 were stably transduced with NEMO-Δex5 or FL-NEMO, respectively, and stimulated with poly(I:C) in a time course; lysates probed by Western blot as indicated. Different exposure intensity of NEMO panels (HC/EV, HC/Δex5, P1/EV, and P1/WT) was required to reveal all relevant bands, relative quantitation by optical densitometry, below. (G) P1 and HC DFs stimulated with poly(I:C), TNF, or cultured in media for 3 hours. Average gene expression by NanoString, ratio in stimulated/untreated from n = 3 independent experiments. (H) DFs treated as in G with relevant differentially expressed genes by RNA-Seq and totals shown, n = 3 technical replicates per media and TNF condition, n = 2 for poly(I:C) treatment. (I) P1 and HC DFs stimulated with TNF or poly(I:C) for 12 hours, cytokine production by capture bead assay, representative experiment of n = 3. (J) DFs from patients with NDAS, NEMO controls, and HCs incubated with hPIV3-GFP. Quantitation of total GFP intensity per cell, error bar indicates SEM. Two-way ANOVA applying Dunnett’s multiple-comparison test for adjusted P values, pertaining to 48-hour point. ***P < 0.001; ****P < 0.0001. Poly(I:C) dose 10 μg/mL and TNF 20 ng/mL throughout. All results representative of at least 2 independent experiments.