Figure 5.

NIK Activity Is Essential for NIK to Induce β Cell Failure

INS-1 832/13 cells were infected with β-Gal, NIK, and NIK(KA) adenovirus for 16 h. Insulin secretion, RNA-seq, and qRT-PCR analysis were performed in β-Gal-overexpressing (control), NIK-overexpressing, and NIK(KA)-overexpressing INS-1 832/13 cells. (A) Flag-NIK, NF-κB2, and Tubulin protein levels were measured by immunoblotting. (B) Glucose (16.7 mM)-stimulated insulin secretion (GSIS) was measured by ELISA (n = 3–4 for each group). (C) Insulin content was measured by ELISA (n = 4). (D and E) Cell viability (D) and the number of TUNEL-positive cells (E) were measured after virus infection for 48 h (n = 5–6 for each group). The scale bar represents 100 μm. (F) Heatmap derived from RNA-seq expression data of the differentially expressed genes (DEGs)(|log2(FoldChange)| > 1 and q < 0.005). (G) Top GO biological process terms enriched in downregulated and upregulated genes in NIK-overexpressing INS-1 832/13 cells compared with β-Gal control. (H) The expression of genes related to inflammation (Il1, Il6, Tnfα, Ccl5, Ccl11, Cxcl1, Cxcl10, and Cxcl11), antigen presentation (RT1-CE4, RT1-CE7, RT1-M2, RT1-M3, B2M, RT1-Ba, and RT1-Da), and insulin secretion (Gck, Glut2, Hnf1α, Hnf1β, Hnf4α, Insulin1/2, Neurod1, and Pdx-1) was measured by qRT-PCR (n = 6 for each group). ∗p < 0.05, ∗∗p < 0.01. Data represent the mean ± SEM.