Streptozotocin (stz)

MIN6 cells were obtained from Shanghai Bogoo Biological Technology Co., Ltd., and were maintained in Dulbecco's modified Eagle medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% heat inactivated FBS (Beijing Solarbio Science & Technology Co., Ltd.), 2 mM L-glutamine, 100 U/ml penicillin and streptomycin (Beijing Solarbio Science & Technology Co., Ltd.) at 37°C in a 5% CO₂ humidified incubator. MIN6 cells were seeded in a 6-well plate at a density of 3×10⁵ cells/well and cultured to 80–90% confluence. Cells were stimulated with 5 µM STZ (Biosharp Life Sciences) for another 24 h at 37°C.

D-glucose (DG), glucose oxidase (GO), wheat germ agglutinin (WGA), and DHE were purchased from Sigma-Aldrich (St. Louis, MO). The LDH assay kit was from Solarbio (Beijing, China). PI was from Beyotime Biotechnology (Shanghai, China). Hoechst33342 reagent was obtained from Invitrogen (Carlsbad, CA). 4', 6-diamino-2-phenylindole reagent was from Cell Signaling Technology (Boston, MA). The Bicinchoninic Acid Assay protein assay kit was obtained from Pierce (Rockford, IL). The primary antibody for HSPA12A was from Abcam (Cambridge, MA). Primary antibodies for Akt, phosphor-Akt, FOXO1, phosphor-FOXO1, and Cleaved-caspase3 were from Cell Signaling Technology (Beverly, MA). The primary antibody for HO-1 was from Proteintech (Chicago, CA). Anti-α-Tubulin antibody was from Sigma-Aldrich. MK-2206 was from MedChemExpress (Monmouth Junction, NJ). Bovine serum albumin was from Roche (Basel, Switzerland). Dulbecco’s Modified Eagle’s medium and fetal bovine serum were from Gibco (Shelton, CT). High-sig enhanced chemiluminescence western blotting substrate was from Tanon (Shanghai, China). STZ was from Biosharp (Hefei, China).

Sixteen male C57BL/6J mice (18–20 g, 4 weeks of age) were obtained from the Experimental Animal Center of Anhui Medical University and used in compliance with the US National Institutes of Health guideline (publication no. 8523). The mice were randomly placed in one of two groups: a normal control group (n=8) and a mouse model of type 2 diabetes group (n=8). Mice in the experimental group received a single intraperitoneal injection of streptozotocin (STZ, 40 mg/kg, dissolved in pH 4.5 citrate buffer; Biosharp). Control mice received an equivalent volume of citrate buffer. After the STZ injection, both groups were fed a standard mouse chow diet and were housed in a standard temperature-controlled (25±2℃) environment. Mice were considered a successful model of diabetes when their fasting blood glucose levels reached 16.7 mM, approximately 1 week after the STZ injection. Immunohistochemical assays were performed 8 weeks after the STZ injection.