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. 2023 Oct 2;19(16):5160–5173. doi: 10.7150/ijbs.86207

Figure 3.

Figure 3

AQP3 deficiency inhibits NF-κB activation in keratinocytes. (A) Top-ranked enriched KEGG terms in genes that were differentially regulated respectively in the two comparisons (WT (LL37) vs WT (Control), and WT (LL37) vs Aqp3-/- (LL37)) revealed by GSEA. NF-κB signaling pathway was highlighted in red box. (B) GSEA on RNA-sequencing data from the two comparisons both shows enrichment for NF-κB signaling pathway in LL37 group. Significance was calculated by permutation test. (C) The mRNA expression levels of NF-κB family of transcription factors (Nfkb1, Nfkb2, Rela and Relb) in mouse skin lesions (n=5/group). (D) Immunostaining of phospho-p65 (p-p65) in skin sections. Scale bar: 50 μm. (E) Quantitative result of fluorescent intensity for (D) is shown. Data represent the mean ± SEM. (F) Immunoblotting of p-p65 and p65 in cell lysates from scrambled and AQP3 shRNA-transfected HaCaT keratinocytes. Cells were stimulated with TNF-α for 0.5 hr. p-p65 protein levels were analyzed relative to p65. Data are representative of at least three independent experiments. (G) Immunofluorescence of p65 in scrambled or AQP3 shRNA-transfected HaCaT keratinocytes. Cells were stimulated with TNF-α for 1 hr. DAPI staining (blue) indicates nuclear localization. Scale bar: 50 μm. (H) Percentage of p65 positive cells in the nucleus. All results are representative of at least 3 independent experiments.