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. 2022 Jul 31;298(9):102312. doi: 10.1016/j.jbc.2022.102312

Figure 2.

Figure 2

Nos2 promoter activity, iNOS protein level, and NO production in INS-1E cells and mouse islets.A, INS-1E cells were transiently co-transfected with a Nos2 promoter-luciferase plasmid and a Renilla plasmid to control for transfection efficiency for 4 h. The following day, cells were exposed to IL-1β (12.5 pg/ml) for 6 h and/or butyrate (But, 0.4 mM) or left unexposed (Ctr). Luciferase activity is shown after normalizing for Renilla luciferase activity. Data are shown as fold increase relative to Ctr. B, iNOS protein expression measured in whole cell extracts from INS-1E cells using Western blotting. Cells were exposed to IL-1β for 1, 6, or 72 h with or without butyrate. Cells exposed to IL-1β for 1 h were pre-exposed to butyrate for 1 h. Cells exposed to butyrate alone were exposed for 2 h. A representative blot is shown, and β-actin was used as loading control. Band intensities were quantified using Image Studio, and data are shown as fold increase relative to Ctr. C, NO production was measured as nitrite accumulation in culture medium from INS-1E cells or mouse islets (50 islets/ml) using Griess reagent. Cells were cultured for 6 or 72 h. Mouse islets were exposed to IL-1β (50 pg/ml) with or without butyrate (0.2 mM) for 5 days or left unexposed. Bars show means ± SEM of n = 4 to 6. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. iNOS, inducible nitric oxide synthase; IL-1β, interleukin-1β; NO, nitric oxide; ND, not detectable.