Fig 1.
Immunoblot analysis of Nox2 and Nox4 in human islets (A-C) and EndoC-βH1 cells (D). Nox2 (A) and Nox4 (B) expression was assessed after 4h and 24h of IL-1β (20 ng/ml) + IFN-γ (20 ng/ml) (cyt) and palmitate (1.5 mM in 2% BSA) + high glucose (20 mM) (PH) exposure. The expression of Nox2 and Nox4 was normalized to total ERK, which was used as a loading control. Results are means ± S.E.M for 5 independent experiments. * denotes P<0.05 using Student’s paired t-test. (C) Representative immunoblot image for Nox2, Nox4 and total ERK in human islet cells. (D) Representative immunoblot image for Nox2 (green), Nox4 (red), tubulin (green) and total ERK (red) in EndoC-βH1 cells.
Fig 2.
Immunofluorescence analysis of Nox2 and Nox4 expression in human islet cells and EndoC-βH1 cells.
Islets and EndoC-βH1 cells were analyzed for Nox2/Nox4 immunofluorescence and counterstained with DAPI. Some cells were incubated for 24h with IL-1β (20 ng/ml) + IFN-γ (20 ng/ml) (cyt). Arrows point to EndoC-βH1 cells in late telophase with increased cytoplasmic Nox4 accumulation. Results are representative for 3 independent experiments.
Fig 3.
ROS production in human islet cells.
(A) Quantification of ratio of DCF fluorescence to DAPI fluorescence intensity. Human islets were incubated at control condition (Ctrl) or with palmitate (1.5 mM + 2% BSA) + high glucose (20 mM) (PH) for 24h. During the last 3h of the 24h incubation Nox inhibitors: DPI (0.2 μM), Dapsone (10 μM), GLX351322 (10 μM) and GLX481372 (6 μM) were added to the culture medium. each treatment group, 5 images (100–250 cells in each image) were taken. The intensities of green (DCF) and blue (DAPI) signals were quantified by Image J software. Results are means ± S.E.M for 4 donors. * denotes P<0.05 compared with PH using repeated measurements one-way ANOVA and Fisher LPD. For (B) Representative photographs showing DCF and DAPI fluorescence. M22 depicts GLX351322 and M72 depicts GLX481372.
Fig 4.
Effects of DPI, GLX351322, GLX481372 and Dapsone on human islet cell viability and caspase 3 activation.
(A) Quantification of relative cell death (ratio PI/Bisbenzimide). Human islets were incubated at control conditions or with palmitate (1.5 mM + 2% BSA) + high glucose (20 mM) (PH) for 24h with Nox inhibitors: DPI, Dapsone, GLX351322 and GLX481372 (same concentrations as described in Fig 3 and present during the entire 24h exposure period). For each treatment group, 5 images (100–250 cells in each image) were taken. Islets were photographed in an inverted fluorescence microscope and the intensities of red (PI) and blue (Bisbenzimide) signals were quantified using Image J software. Results are means ± S.E.M for 7 human islet donors. * denotes P<0.01 compared with PH using repeated measurements one-way ANOVA and Fisher PLD. (B) Representative images of cleaved caspase 3 staining (red) after a 24h palmitate + high glucose incubation with and without Nox inhibitors.
Table 1.
Summary of Nox inhibitor characteristics.
Fig 5.
Effects of ML-171, Phos-I2 and GLX7013114 on human islet cell viability.
Human islets were incubated at control condition, with IL-1β (20 ng/ml) + IFN-γ (20 ng/ml) (A), or with palmitate (1.5 mM + 2% BSA) + high glucose (20 mM) (PH) (B) for 2 days with or without Nox1 inhibitor ML-171 (2 μM), Nox2 inhibitor Phos-I2 (2 μM) and Nox4 inhibitor GLX7013114 (1 μM). Islets were photographed in an inverted fluorescence microscope and the intensities of red (PI) and blue (Bisbenzimide) signals were quantified using Image J software. Results are means ± S.E.M for 7 human islet donors. * denotes P<0.05 compared with cyt or PH, respectively, using repeated measurements one-way ANOVA and Fishers PLD post-hoc test.
Fig 6.
Effects of ML-171, Phos-I2 and GLX7013114 on EndoC-βH1 cell ROS production and viability.
(A) Flow cytometry quantification of DCF (total ROS) and MitoSOX (mitoROS) intensities at control conditions. Results are means ± S.E.M for 3 independent experiments. * denotes P<0.05 compared with control using Student’s paired t-test. (B) Flow cytometry quantification of cell death after a 2-day incubation with cytokines (cyt) supplemented with 0.2 μM, 0.5 μM or 1.0 μM of Nox1 inhibitor ML-171, Nox2 inhibitor Phos-I2 or Nox4 inhibitor GLX7013114, respectively. Results are means ± S.E.M for 7 independent experiments. (C) Flow cytometry quantification of cell death after 24h incubation with palmitate (1.5 mM + 2% BSA) + high glucose (20 mM) (PH) supplemented with 0.2 μM, 0.5 μM or 1.0 μM of Nox1 inhibitor ML-171, Nox2 inhibitor Phos-I2 or Nox4 inhibitor GLX7013114, respectively. Results are means ± S.E.M for 8 independent experiments.