Bulk RNA sequencing of SV40-fibroblasts and RT-qPCR and western blot of the canonical NF-κB pathway. (A) Heatmap showing the normalized and scaled expression (vst) of IRF3 target genes significantly upregulated in controls after stimulation with poly I:C 10 µg/ml for 6 h. Each column represents a patient or a control. RNA sequencing has been performed in biological duplicates for each control or patient (as indicated by _1 or _2 at the end of each column name). (B) Z-score of the different “molecules” predicted to be inhibited (top 10 based on the z-score) based on IPA (Qiagen Inc.) after poly TNFα stimulation for the NFKB2 p.R853* cells. A negative score indicates that the molecule is predicted to be inhibited based on the DEGs that have been processed. (C) Heatmap showing the relative quantity normalized to GAPDH of each genes (scaled between 0 and 1, in column), for each control or the patient’s cells (in row) after stimulation with TNFα 10 ng/ml for 6 h or poly I:C 1 µg/ml for 6 h (as indicated by TNF or PIC respectively), as assessed by RT-qPCR. (D) Western blot assessing the quantity of phospho-p65 (S536), p65, phospho-IκBα (S32), and IκBα on cell lysates from SV40-fibroblasts stimulated with TNFα at indicated time points. Tubulin was used as a loading control. (E) Western blot of cytoplasmic and nuclear extracts from SV40-fibroblasts after 2 h of TNFα (T) (10 ng/ml) or poly I:C (P) (1 µg/ml) stimulation. RELA/p65 and p50 were revealed using a specific antibody to assess for translocation in the nucleus. α-tubulin was used as a loading control of cytoplasmic extract while Lamin A/C was used for nuclear extracts. Source data are available for this figure: SourceData FS5.