Fig. 3.

Low-dose pancreatic endoplasmic reticulum kinase inhibitor (PERKi) improved 3-methyladenine (3-MA)-suppressed autophagy and insulin synthesis through autophagy related 7 (ATG7). (A-E) Mouse islets or dispersed cells were treated with 3-MA (0.5 mM) for 24 hours with/without GSK2606414. Autophagy was induced by fetal bovine serum deprivation by further incubation for 5 hours (A, B). Each treatment group was composed of 25 to 30 islets, resulting in total 120 islets from 3 to 4 mice per experiment, by hand-picking of healthy islets estimated by their morphology. The islets were randomly assigned to the groups, without blinding to the researchers. Western blotting of autophagy and unfolded protein response markers was conducted, and representative blots are demonstrated (A, C, D, E). For (B), dispersed islet cells were transduced with BacMam tandem red fluorescent protein (RFP)-green fluorescent protein (GFP)-light chain 3B (LC3B) reagent, incubated overnight, then treated as described above. Before fixation with 4% formaldehyde, live cell nuclei were stained with Hoechst 33342, and GFP or RFP puncta were manually counted in cytoplasm of live cells. About 100 cells were counted in each treatment group by 3 to 6 experiments. Representative pictures are presented: yellow puncta indicating quenching of acid-sensitive GFP by lysosome (in the control), a green punctate (3-MA group) and red puncta indicating autophagolysosome (3-MA with PERKi group) marked by arrows. (F, G, H) Mouse islets were dispersed to single cells, and transfected with 100 nM of siRNA for Atg7 and negative control. Then autophagy was inhibited by 3-MA (0.5 mM) with/without GSK2606414 (20 nM). Each treatment group was composed of 10⁵ cells, resulting in total 3 × 10⁵ cells from 5 to 6 mice per experiment. The cells were randomly assigned to the groups, without blinding to the researchers. After 30 to 48 hours, quantitative reverse transcription-polymerase chain reaction was conducted (Atg7 and insulin 1 [Ins1]), and protein levels were measured by Western blot (ATG7 and tubulin) and enzyme-linked immunosorbent assay (ELISA; insulin contents and secretion). Dot numbers in each group represent experimental numbers. The lines and error bars are median (interquartile ranges) (E) and mean ± standard error of the mean (all the others). One-way analysis of variance (ANOVA) with Bonferroni posttest was applied, except for (B, E) where Kruskal-Wallis with Dunn's multiple comparison test was applied excluding the serum-free control in (E), and for (H) where two-way repeated measures ANOVA with Dunnett's multiple comparisons test was applied. C, control; V, vehicle; NS, no significant differences; EIF2A, eukaryotic translation initiation factor 2 alpha; BIP, binding immunoglobulin protein; LG, low glucose; HG, high glucose; siAtg7, Atg7 siRNA. ^aP < 0.05, ^bP < 0.01 in the post hoc analyses.