Fig. 3.

IκBζ regulates a subset of psoriasis-related IL-36 target genes. Primary KCs or HaCaT cells were transduced with a control or NFKBIZ-specific shRNA. Triplicates of each time point and shRNA were analyzed by RNA-seq or qPCR and were normalized to the reference gene RPL37A. (A) Control of NFKBIZ-knockdown efficiency. (Upper) IκBζ protein was detected in primary KCs treated for 1 h with 100 ng/mL IL-36α. (Lower) NFKBIZ mRNA levels were measured after 1.5 h and 24 h of IL-36α stimulation. (B) After library preparation from total RNA, primary KC samples were sequenced, and reads were aligned to the human genome hg19. Depicted are two separate heatmaps with normalized z-scores of IκBζ target genes after 1.5 h and 24 h of IL-36α treatment. As a cutoff, genes with a minimum fold change of 1 and a P value < 0.05 were considered. (C) Venn diagrams showing the fraction of IκBζ target genes among IL-36α–regulated genes 1.5 and 24 h after stimulation of primary KCs. (D) GO term analysis of significantly enriched IκBζ-dependent gene sets after 1.5 and 24 h of IL-36α treatment. (E) Validation of selected IκBζ target genes by qPCR in primary KCs after 1.5 and 24 h of incubation with 100 ng/mL IL-36α. (F) Gene-expression analysis of IκBζ target genes in primary KCs stimulated with 100 ng/mL IL-17A and 10 ng/mL TNFα for 1.5 and 24 h. *P < 0.05; **P < 0.01; ***P < 0.001.