Thapsigargin

Rats were used for the animal model, and the cell line model was mouse derived. Cell culture was used as previously described
[24] (link). Briefly, the MC3T3-E1 subclone 14 cell line (BDCB, Guangzhou, China) was cultured in base medium [Eagle’s medium supplemented with 5.6 mM glucose (Biofroxx, Einhausen, Germany), 2 mM L-glutamine (Solarbio, Beijing, China), 0.5 mM β-glycerophosphate (Beyotime, Shanghai, China), 50 mg/L ascorbic acid (Beyotime), and 10% fetal bovine serum (PAN Biotech, Aidenbach, Germany)] with 5% CO
₂ at 37°C. The (seeding) cell density was 3×10
⁵ cells/mL, and cells at passages 15 to 18 were collected. Before processing, the differentiation capacity of MC3T3-E1 cells was checked at passages 15 to 18.
For high glucose intervention, MC3T3-E1 cells were cultured in high glucose medium (base medium supplemented with 25 mM glucose) for 14 days. For metformin intervention, MC3T3-E1 cells were cultured in medium containing different concentrations of metformin (25, 50, 100 μM metformin in base medium) for 14 days. For thapsigargin intervention, MC3T3-E1 cells were cultured in base medium containing 100 nM β-thapsigargin (Beyotime) for 1 day.

DMEM, RPMI-1640 medium, and fetal bovine serum (FBS) were purchased from HyClone, L-glutamine from Gibco, Propidium Iodide (PI) Solution (421301) from Biolegend (CA, USA), Annexin V-FITC/PI apoptosis detection kit (FA101-02) from TransGen Biotech (Beijing, China), Lipofectamine 3000 (L3000015) from Invitrogen (CA, USA), Thapsigargin (CHXSC0389), MG-132 (S1748), Fura-2 AM (S1052) and Fluo-4 AM (S1060) from Beyotime (Jiangsu, China), Chloroquine (CQ, 105M4035V) from Sigma, Bortezomib (PS-341, S1013), BafilomycinA1 (BAF, S1413) from Selleckchem (PA, USA), Cycloheximide (CHX, C112766) from Aladdin (Shanghai, China).