Ht22 cells

Mouse hippocampus-derived neuronal HT22 cells (Merck KGaA, Darmstadt, Germany) were cultured in MEM medium containing 10% fetal bovine serum and 1% penicillin/streptomycin and incubated under standard conditions of 5% CO₂ at 37 °C. The cells were sub-cultured or treated with drugs when they reached approximately 80% confluence. In the experiment, the HT22 cells were treated with D-gal at a concentration of 250 mM and 1,25(OH)₂D₃ at a concentration of 200 nM 24 h. Prior to treatment, the HT22 cells were pretreated with 20 μM ML385 (MedChemExpress, Piscataway, NJ, USA) for 1 h. At the end of each experiment, the total cellular protein was extracted and used to determine protein expression.

The HT22 cells were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) at 37 °C. For oxygen-glucose deprivation (OGD) insult, the neurons were maintained in a hypoxic chamber (Thermo Fisher Scientific Inc., Waltham, MA, USA) at 94% N₂, 1% O₂, and 5% CO₂ and then cultured in glucose-free DMEM solution at 37 °C for 0, 2, 4, 6, or 8 h, as previously described (Lai et al. 2020 ). After OGD, the neurons were cultured in normal DMEM solution with 10% FBS for 24 h. In the CE (MedChemExpress, NJ, USA) treatment groups, HT22 cells were pretreated with CE at doses of 1, 2, 4, or 8 µg/mL for 4 h prior to OGD/R (Tian et al. 2019 (link); Wang et al. 2020a (link)).