Fig 1: NLRC4 mosaic variants on the domain organization model and a partial 3D structure of the protein. (a) Domain organization model of the NLRC4 protein on which are shown the NLRC4 mosaic variant p.Thr133Ile (in red) identified in this study and the previously reported variants p.Ser171Phe, p.Thr177Ala and p.His443Gln (in blue); CARD (caspase activation and recruitment domain), NBD (nucleotide-binding domain), HD1 (helical domain 1), WHD (winged-helix domain), HD2 (helical domain 2) and LRR (leucine-rich repeats domain). (b) Clustal Omega multiple sequence alignment of NLRC4 protein across various species, indicating the position of the Thr133Ile variant within a highly conserved region of NLRC4. (c) Ribbon plot of the mouse Nlrc4 protein highlighting Thr133 (in red) and Ser171, Ser177 and His443 (in blue). The adenosine diphosphate (ADP) molecule is shown in green
Fig 2: ASC-GFP speck formation and NF-κB activity induced by NLRC4 variants. a. Percentage of ASC-GFP speck-positive cells upon transfection with empty vector (pEV), pNLRC4_WT, pNLRC4_Thr133Ile, pNLRC4_Ser171Phe or pNLRC4_Thr177Ala (n = 4). b. NF-κB activity in cells co-transfected with a NF-κB reporter construct, Renilla, pNLRC4_WT and each of the pNLRC4 mutants, with pNAIP when indicated (n = 4). Statistically significant P values (< 0.05) are denoted as *, ** and *** for < 0.05, < 0.01 and < 0.001, respectively. The data are presented with SEM error bars
Fig 3: Patient’s biological and molecular data. (a) Patient’s CRP, Hb (hemoglobin) and ferritin blood levels are plotted. In each chart, the blue bars represent the periods of treatment with anakinra. The horizontal dashed lines represent the normal ranges for CRP, Hb and ferritin. (b) Plasma cytokine levels during and immediately following the flare and compared to five healthy donors. The results from healthy individuals were presented as median. ND: not detected. (c) Sanger chromatograms showing the NLRC4 genotype at codon 133 of the patient (right) who carries the c.398C>T, p.Thr133Ile variation in a mosaic state and of a healthy individual (left)
Supplier Page from Sino Biological, Inc. for Human NLRC4 Gene ORF cDNA clone expression plasmid, N-Flag tag