Fig 2: Cell apoptosis and expression of pro-inflammatory cytokine genes are upregulated in Wfs1-deficient pancreatic β-cells. (A, B) INS-1E cells were transfected with scrambled siRNA (siCont) or siRNA targeting rat Wfs1 (siWfs1). WFS1 and cleaved caspase-3 protein expression levels were analyzed by immunoblot. Representative images are shown in (A), and quantitative analysis results are shown in (B) (n=3, normalized to GAPDH). (C) mRNA expression levels of Chop, Il-1β, Il-6, and Cxcl1 (Il-8) in INS-1E cells treated with siCont or siWfs1 normalized to 18srRNA (n=5-6). Data are shown as mean ± SEM, *P < 0.05, **P < 0.005, ****P < 0.0001.

Fig 3: Hyperglycemia upregulates pro-inflammatory cytokine genes expression in Wfs1-deficient pancreatic β-cells. (A) Immunoblot images of WFS1 in Wfs1 wild-type (WT) and knockout (KO) INS-1 832/13 cells. (B) Left panel: Immunoblot images of proinsulin in WT and KO INS-1 832/13 cells treated with 25 mM glucose for 1 h. Right panel: Proinsulin band intensity was quantified and normalized to tubulin (n=3). (C) Left panel: Caspase-3/7 activity normalized to cell viability in WT and KO INS-1 832/13 cells treated with 11 mM or 25 mM glucose for 48 h (n=8). Right panel: Cell viability of WT and KO INS-1 832/13 cells treated with 25 mM glucose for 48 h (n=8) (D) mRNA expression levels of Chop, sXbp1, Bip, and Txnip in WT or KO INS-1 832/13 cells treated with 11 mM or 25 mM glucose for 48 h (n=3-5), normalized to 18srRNA. (E) The mRNA expression level of Il-1β, Il-6, Ccl2, and VegfA in WT and KO INS-1 832/13 cells treated with 11 mM or 25 mM glucose for 48 h (n=3-5), normalized to 18srRNA. Data are shown as mean ± SEM, *P < 0.05, **P < 0.005, ***P < 0.001, ****P < 0.0001.

Fig 4: Cytokine treatment causes ER-stress induced cell death and pro-inflammatory cytokines gene upregulation in Wfs1-deficient pancreatic β-cells. (A) Caspase-3/7 activity normalized to cell viability in Wfs1 wild-type (WT) and knock-out (KO) INS-1 832/13 cells treated with IFN-γ (50 ng/mL) and IL-1β (50 ng/mL) for 24 h (n=5). (B) mRNA expression level of Chop, Il-1β, Il-6, and Ccl2 in WT and KO INS-1 832/13 cells treated with IFN-γ (50 ng/mL) and IL-1β (50 ng/mL) for 24 h (n=6-8), normalized to 18srRNA. Data are shown as mean ± SEM, *P < 0.05, **P < 0.005, ***P < 0.001.