Fig. 4: KRT32 inhibits the activation of NF-κB signaling pathway.

A Selected KEGG signal transduction pathway enrichment analysis with significant DEGs (FDR < 0.05 and log₂FC < −0.585) through RNA sequencing of Ker-CT cells with KRT32 overexpression. The statistical test was hypergeometric test, and the level of significance was set at a two-sided P < 0.05 without multiple comparisons. B Volcano plot illustrating significant DEGs between KRT32 overexpression and negative control in Ker-CT cells. Upregulated genes were labelled by red dots with FDR < 1e⁻²⁰, log₂FC > 0.585; and downregulated genes was labelled by blue dots with FDR < 1e^−20, log₂FC < −0.585. Selected NF-κB pathway-associated genes are highlighted. FC denotes fold change. The statistical test was Wald test and the level of significance was set at a two-sided FDR < 0.05 with multiple comparisons by Benjamini-Hochberg method. C NF-κB-dependent luciferase activation assay in HEK293T cells to analyze concentration-dependent role of KRT32 in NF-κB activation under the stimulation with TNF (20 ng/mL). D The phosphorylated p65 expression in KRT32 knockdown Ker-CT cells with and without TNF (20 ng/mL) treatment for the indicated time. E–G Detection of IL­1β, IL-6, IL­8 secretion in supernatant of KRT32 knockdown Ker-CT cells with or without TNF treatment by MSD assay. H–I The phosphorylated p65 expression in primary keratinocytes and Ker-CT cells with Flag-tagged KRT32 overexpression. J–L MSD assay detection of IL­1β, IL-6, IL­8 secretion of Ker-CT cells overexpressing KRT32. M The phosphorylated p65 expression in Ker-CT cells overexpressing Flag-tagged KRT32 wildtype and mutations. N, O MSD assay detection of IL­1β, IL-8 secretion of Ker-CT cells overexpressing KRT32 wildtype and mutations with and without TNF treatment. P Analysis of luciferase activity after transfection with expression vectors encoding KRT32 wildtype and the variants (6 rare (MAF < 0.0005) and predicted to be damaging variants, 3 rare (MAF < 0.0005) and predicted to be benign variants, and 12 common (MAF > 0.0005) variants) in stimulated with TNF (20 ng/mL) for 12 h. Data are shown as means ± SEM of three independent biological repeats in (C, E–G, J–L and N–P). The P value was calculated using ordinary one-way ANOVA with Dunnett’s multiple comparisons test in (C, P); a two-sided unpaired Student’s t test in (E–G, J–L); and two-ANOVA with Dunnett’s multiple comparisons test in (N) and (O). Floating bars show the minimum, average, and maximum values within each group in (P). One representative experiment from two independent experiments with similar results is shown in (D, H, I and M). Source data are provided as Source Data file.