Figure 4.

CHOP controls Puma expression in islet cells undergoing ER stress. (a and b) Quantitative RT-PCR of wild-type C57BL/6 islets cultured for 3 days in a control medium or in a medium containing 50 mM ribose, 0.5 mM TUDCA or 1.0 mM NAC. Relative mRNA expression levels for Puma (a) and Bim (b) were calculated by normalizing to the signal for β-Actin mRNA in each sample and comparison with islets cultured in a control medium. Treatment with thapsigargin (5 μM for 1 day) was used as a positive control. Results represent mean±s.e.m. of n=3–5 independent experiments. NS=not significant. *P<0.05, ***P<0.001 (one-way ANOVA). (c) Quantitative RT-PCR of islets from wild-type C57BL/6 and Chop^−/− mice that had been cultured for 3 days either in a control medium or in a medium containing 50 mM ribose. Relative mRNA expression levels for Bim, Puma, Bax and Bip were calculated by normalizing to the signal for β-Actin mRNA in each sample and comparison with wild-type islets cultured in a control medium. Results represent mean±s.e.m. of n=5 independent experiments. *P<0.05, **P<0.01 when comparing islets cultured in ribose with those cultured in a control medium (two-way ANOVA). (d) Western blot analysis with antibodies to phosphorylated Akt (p-Akt, 60 kDa) and total Akt (loading control), and phophorylated FoxO3a (p-FoxO3a, 95 kDa) and total FoxO3a (loading control). A total of 400–700 islets were cultured for 2 days in a control medium or in a medium containing 50 mM ribose. Results are representative of four independent experiments