Table 1.
Primers used for RT-PCR.
Fig 1.
Single cytokine treatment is not sufficient to induce apoptosis in isolated mouse islets.
Islets were examined microscopically following labeling with YO-PRO-1 (green) and propidium iodide (red). (A) Islets were treated with a triple cytokine cocktail of IL-1β/TNF-α/IFN-γ (PIC), or dual cytokine combinations of IL-1β/TNF-α, IL-1β/IFN-γ, or TNF-α/IFN-γ. (B) Islets were treated with a single cytokine of either IL-1β, TNF-α, or IFN-γ at a 1-fold (1x) or 4-fold (4x) dose. Graph shows quantified apoptosis (C) from dual cytokine treatment or (D) from single cytokine treatment for 1x and 4x doses for all islets per experiment * p < 0.05, ** p < 0.01, *** p < 0.001 relative to Ctl, ## p < 0.01, ### p < 0.001 relative to PIC and n = 3.
Fig 2.
Cytokine induced MCP-1 in mouse islets.
Expression of MCP-1 gene following stimulation of mouse islets with single cytokines (A) IL-1β, TNF-α, or IFN-γ at 1x, 2x, or 4x or dual cytokines with (B) IL-1β/TNF-α, IL-1β/IFN-γ, or TNF-α/IFN-γ relative to stimulation with PICs. ## p < 0.01, ### p < 0.001 relative to PIC and n = 3.
Fig 3.
Cytokine treatment affects IL-12 gene expression in mouse islets.
(A) IL-12 p40 and (B) IL-12 p35 gene expression was measured in islets treated with PICs or the dual cytokine combinations of IL-1β/TNF-α, IL-1β/IFN-γ, or TNF-α/IFN-γ. # p < 0.05 relative to PIC and n = 3.
Fig 4.
A neutralizing antibody to IL-12 p40 protects β-cells from PIC-induced apoptosis.
(A) MCP-1 gene expression in PIC-treated βTC-3 cells without or with IL-12 p40 neutralizing antibody. (B) Caspase-3 activity in PIC-treated βTC-3 cells without or with IL-12 p40 neutralizing antibody. Graph (B) shows pro-caspase-3 cleavage (RFU). # p < 0.05, ## p < 0.01 relative to PIC and n = 3.
Fig 5.
STA-5326 inhibits PIC-induced IL-12 gene expression in β-cells and improves β-cell survival.
(A) IL-12 p40, (B) IL-12 p35, and (C) IL-23 p19 gene expression in βTC-3 cells treated with PICs in the absence or presence of 100nM STA-5326. (D) Pro-caspase-3 cleavage in PIC-treated βTC-3 cells without or with 1nM or 100nM STA-5326. *** p < 0.001 relative to Ctl, # p < 0.05, ### p < 0.001, #### p < 0.0001 relative to PIC and n = 3.
Fig 6.
Lisofylline protects β-cells from PIC-induced apoptosis.
(A) IL-12 p40 and (B) IL-12 p35 gene expression in PIC-treated βTC-3 cells without or with 20μM Lisofylline (LSF). (C) PIC-induced caspase-3 activation in the absence or presence of 20μM LSF or 50μM c47 in βTC-3 cells. Graph shows pro-caspase-3 cleavage relative fluorescent units (RFU). (D) Apoptosis was measured in PIC-treated βTC-3 cells without or with 20μM LSF or 50μM c47. Cells were examined microscopically following labeling with the viability dye, YO-PRO-1 (green). Representative images are shown for untreated (Ctl), PIC-treated, PIC with 50μM c47, or PIC with 20μM LSF respectively. (E) Graph shows quantified apoptotic index. ** p < 0.01, *** p < 0.001 relative to ctl, ## p < 0.01 relative to PIC and n ≥ 3.
Fig 7.
c47 confers protection to mouse islets from PIC-induced apoptosis.
(A) PIC-induced caspase-3 activation in the absence or presence of 50μM c47 in mouse islets. Graph shows pro-caspase-3 cleavage. (B) Apoptosis was measured in PIC-treated islets without or with 50μM c47. Cells were examined microscopically following labeling with YO-PRO-1 (green) and PI (red). Representative images are shown for untreated (Ctl), PIC-treated, or PIC with 50μM c47 respectively. (C) Graph shows quantified apoptotic index. * p < 0.05, *** p < 0.001 relative to ctl, # p < 0.05, ### p < 0.001 relative to PIC and n > 3.
Fig 8.
Inhibition of the IL-12 pathway reduces PIC-induced IFN-γ gene expression in β-cells.
The β-cell line βTC-3 was treated with PICs in the absence or presence of (A) 1μg/mL IL-12 p40 neutralizing antibody, (B) 100nM STA-5326, (C) 20μM LSF, or (D) 50μM c47 prior to determination of IFN-γ gene expression. # p < 0.05 relative to PIC and n = 3.
Fig 9.
Islets from STAT4 deficient mice are resistant to PIC-induced apoptosis.
(A) Fluorescence microscopy was used to image apoptotic islets labeled with YO-PRO-1 (green) and PI (red). Islets from WT mice (top row) or islets from STAT4ko mice (bottom row) were treated with PICs, IL-1β/TNF-α, IL-1β/IFN-γ, or TNF-α/IFN-γ. (B) Graph shows quantified apoptosis from cytokine treated STAT4ko islets. (C) Graph shows quantified apoptotic index from cytokine treated WT islets vs STAT4ko islets. * p < 0.05, ** p < 0.01, *** p < 0.001 relative to ctl, +++ p < 0.001, ### p < 0.001 relative to WT PIC treatment. Three groups of each condition were treated and analyzed.