Figure 3. Pancreatic α cells are more resistant than β cells against CVB5- but not against cytokine- or PIC-induced cell death.

FACS-purified rat β and α cells (>90% purity for both cell types) were treated with interleukin-1β (IL-1β) + type II interferon (IFNγ) (50 or 500 U/ml, respectively) (A) or PIC (1 μg/ml) for 24 hr or infected with CVB5 (multiplicity of infection—M.O.I. 5) for 36 hr (C–G). (A–C) Apoptosis was evaluated by staining with the nuclear dies Hoechst 33342 and PI. (D) VP1 mRNA expression was assayed by RT-PCR and normalized by the housekeeping gene GAPDH. (E and F) The figures show representative Western blots of VP1 protein expression after CVB5 infection and α-tubulin for loading control. (F) The Western blot (E) was overexposed to allow visualization of VP1 expression in α cells. (G) Titration of the supernatants from β and α cells infected with CVB5 for 36 hr. Results of 4–6 experiments are plotted as box plots, indicating lower quartile, median, and higher quartile, with whiskers representing the range of the remaining data points; *p < 0.05, **p < 0.01, and ***p < 0.001 treated vs untreated (A and B) or CVB5 vs mock infection (C, D, and G); ###p < 0.001 as indicated by bars; ANOVA followed by Student's t-test with Bonferroni correction.

DOI: http://dx.doi.org/10.7554/eLife.06990.008

Figure 3—figure supplement 1. Dose-response of cytokine-induced apoptosis in pancreatic α and β cells.

FACS-purified rat β and α cells (>90% purity for both cell types) were treated with different concentrations of IL-1β + IFNγ, as indicated in the figure, for 24 hr. Apoptosis was evaluated by staining with the nuclear dies Hoechst 33342 and PI. Results are mean and SEM of 3 independent experiments; **p < 0.01 and ***p < 0.001 treated vs untreated; ANOVA followed by Student's t-test with Bonferroni correction.

DOI: http://dx.doi.org/10.7554/eLife.06990.009

Figure 3—figure supplement 2. Pancreatic rat α and β cells have similar NO production, cytokine, and chemokine expression following exposure to cytokines.

FACS-purified β and α cells (>90% purity for both cell types) were treated with IL-1β + IFNγ (50 or 500 U/ml, respectively) (A–D). iNOS (A), CXCL10 (C), and CCL2 (D) mRNA expression were assayed by RT-PCR and normalized by the housekeeping gene GAPDH. (B) Cytokine-induced NO production by primary rat β or α cells was evaluated by medium nitrite accumulation. Results of 4–5 experiments are plotted as box plots, indicating lower quartile, median, and higher quartile, with whiskers representing the range of the remaining data points; *p < 0.05, **p < 0.01, and ***p < 0.001 treated vs untreated; #p < 0.05 as indicated by bars; ANOVA followed by Student's t-test with Bonferroni correction.

DOI: http://dx.doi.org/10.7554/eLife.06990.010

Figure 3—figure supplement 3. Pancreatic rat α and β cells have similar cytokine and chemokine expression following exposure to PIC.

FACS-purified β and α cells (>90% purity for both cell types) were treated with intracellular PIC (1 μg/ml) for 24 hr (A–F). IFNβ (A), IFNα (B), STAT1 (C), iNOS (D), CXCL10 (E), and CCL2 (F) mRNA expression were assayed by RT-PCR and normalized by the housekeeping gene GAPDH. Results of 4–5 experiments are plotted as box plots, indicating lower quartile, median, and higher quartile, with whiskers representing the range of the remaining data points; *p < 0.05, **p < 0.01, and ***p < 0.001 treated vs untreated; #p < 0.05 and ##p < 0.01 as indicated by bars; ANOVA followed by Student's t-test with Bonferroni correction.

DOI: http://dx.doi.org/10.7554/eLife.06990.011

Figure 3—figure supplement 4. Prolonged time-course of CVB5-induced apoptosis in pancreatic α and β cells.

FACS-purified rat β and α cells (>90% purity for both cell types) were infected with CVB5 (multiplicity of infection—M.O.I. 5) for 48, 72, or 96 hr. Apoptosis was evaluated by staining with the nuclear dies Hoechst 33342 and PI. Results are mean and SEM of 3 independent experiments; ***p < 0.001 CVB5 vs mock infection; ###p < 0.001 as indicated by bars; ANOVA followed by Student's t-test with Bonferroni correction; h.p.i., hours post-infection.

DOI: http://dx.doi.org/10.7554/eLife.06990.012

Figure 3—figure supplement 5. UV-inactivated CVB5 does not induce cell death in pancreatic α and β cells.

FACS-purified rat β and α cells (>90% purity for both cell types) were infected with CVB5 (multiplicity of infection—M.O.I. 5) or UV-inactivated CVB5 (1000 J/m²) for 36 hr. Apoptosis was evaluated by staining with the nuclear dies Hoechst 33342 and PI. Results are mean and SEM of 3 independent experiments; #p < 0.05 CVB5 vs. UV-inactivated CVB5 as indicated by bars; ANOVA followed by Student's t-test with Bonferroni correction.

DOI: http://dx.doi.org/10.7554/eLife.06990.013

Figure 3—figure supplement 6. Cell counting after CVB5 infection of pancreatic α cells.

FACS-purified rat α cells (>90% purity) were infected with CVB5 (M.O.I. 5) for 36 hr. Supernatants were collected and the cells attached to the well trypsinized and also collected for quantification. The counting of supernatant cells (A) or attached cells (B) was performed in Neubauer chambers, and each point was measured in triplicate by two observers, one of them unaware of sample identity. Results are mean and SEM of 4 independent experiments; Student's t-test with Bonferroni correction.

DOI: http://dx.doi.org/10.7554/eLife.06990.014

Figure 3—figure supplement 7. Pancreatic α cells infected with CVB5 under different medium conditions.

FACS-purified rat α cells (>90% purity) were infected in parallel with CVB5 (multiplicity of infection—M.O.I. 5) for 36 hr in two different media, that is, medium used for β cell culture, with 10 mM glucose and 5% fetal bovine serum, (gray) and the usual medium used in α cells culture (white). Apoptosis was evaluated by staining with the nuclear dies Hoechst 33342 and PI. Results are mean and SEM of 3 independent experiments; ANOVA followed by Student's t-test with Bonferroni correction.

DOI: http://dx.doi.org/10.7554/eLife.06990.015