Fig 2: KPC2 and p65, two novel interactants of CD95(A) The activation status of the NF-?B signaling pathway was analyzed in wild-type (WT) and CD95 knock-out (KO5 and KO9) MDA-MB-231 cells by immunoblotting with the indicated antibodies. Tubulin immunoblot serves as a loading control. Images are representative of three independent experiments.(B) The presence of p65, p105, and p50 in the whole lysate (cytosol) or the nucleus fraction of wild-type and CD95 k.o. cells was evaluated by immunoblotting. Lamin A and tubulin serve as loading controls for nucleus and cytosolic fractions, respectively. Images are representative of three independent experiments.(C) Nuclear extracts from WT and CD95 k.o. cells were subjected to the indicated ELISA to quantify activation of NF-kB. Mouse c-Rel DNA binding cannot be assessed with this kit. Mean ± SEM (n = 3), p values were calculated using nonparametric Mann-Whitney test.(D) RelA activity was measured in indicated tumor cells using luciferase reporter assay (n = 6–8). ** and **** stand for p < 0.001 and p < 0.00001, respectively, using unpaired and nonparametric Mann-Whitney t test.(E) Schematic representation of the BioID experiment.(F) Schematic representation of BirA-fused CD95 constructs.(G and H) BioID assay was conducted in CD95 k.o. HEK/293T cells (G) and MDA-MB-231 cells (H) reconstituted with indicated constructs and after streptavidin pull-down indicated immunoblotting was performed. Images are representative of three independent experiments.

Fig 3: Shared peaks in ATAC-seq and chromatin immunoprecipitation with sequencing (ChIP-seq) experiments between paired subjects.Percentage of shared peaks between paired samples in ATAC-seq compared to NFKB1 ChIP-seq experiments.

Fig 4: Aberrant expression of Tweak and Fn14 in skeletal muscle of Smn2B/-SMA mice. a–g qPCR analysis of parvalbumin (a), Tweak (b), Fn14 (c), Pgc-1a (d), Mef2d (e), Glut-4 (f), and HKII (g) in TA muscles from P0 (birth), P2 (pre-symptomatic), P4 (pre-symptomatic), P11 (early symptomatic), and P19 (end stage) Smn2B/- and WT mice. Normalized relative expressions are compared to WT P0. Data are mean ± SEM, n = 3–4 animals per experimental group, two-way ANOVA, Sidak’s multiple comparison test between genotypes, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. h Quantification of Tweak protein levels normalized to total protein in the triceps of late-symptomatic (P18) Smn2B/- mice and age-matched WT animals. Images are representative immunoblots. Data are mean ± SEM, n = 6–7 animals per experimental group, unpaired t-test, p = 0.014. i Quantification of NF-?B1 p50 and p105 protein levels normalized to total protein in the triceps of late-symptomatic (P18) Smn2B/- mice and age-matched WT animals. Images are representative immunoblots. Data are mean ± SEM, n = 6–7 animals per experimental group, unpaired t-test, ns, not significant (p50), p = 0.0354 (p105). j Quantification of NF-?B2 p52 and p100 protein levels normalized to total protein in the triceps of late-symptomatic (P18) Smn2B/- mice and age-matched WT animals. Images are representative immunoblots. Data are mean ± SEM, n = 3–4 animals per experimental group, unpaired t test, ns, not significant (p52), p = 0.0532 (p100)

Fig 5: NFKB1 deletion alters the transcriptional profile of THP1 macrophages. (A) Principal component analysis plot showing sample similarity of RNAseq analysis of control (WT) and NFKB1 -/- (KO) cells untreated (Unt) or treated with LPS. (B) Heatmap showing differentially expressed genes, Z-score normalized. Relative expression is shown as the average of replicates ranging from maximum to minimum values. (C) Venn diagram showing the overlap of LPS-inducible genes and genes differentially expressed in NFKB1 -/- cells relative to WT cells in unstimulated and LPS stimulated conditions (p-value and q-value < 0.05). (D) Selected gene expression data plotted as normalized RNAseq readcounts. Data presented as mean ± SD of biological replicates, p values adjusted for Benjamini-Hochberg are indicated. (E) Patterns of expression for selected genes confirmed in an independent experiment using QPCR. Data presented as mean ± SEM of technical replicates. (F) PMA-induced macrophages from WT and NFKB1-/- THP1 cells were stimulated with LPS, and the supernatant was analysed by IL-6 ELISA. (n = 4, data presented as mean ± SD of four independent experiments). **p < 0.003 using T-test analysis.