Ht22 cells

NIH3T3 and HUVECs were obtained from American Type Culture Collection (Manassas, VA), and the HT22 cells were purchased from the Procell Life Science Technology (Wuhan, China). The cell lines were mycoplasma negative and authenticated by STR profiling. All cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 4.5 g/l glucose and 10% fetal bovine serum (FBS). To synchronize cells in G0 by SS, cells were plated at a density of 1 × 10⁴ cells per cm² overnight and allowed to attach to the tissue culture plate. Cells were washed three times with phosphate-buffered saline and starved in DMEM with 0.1% FBS for 30 hr (Coller et al., 2006 (link)). Then, the cells were induced into the cell cycle with 15% FBS and collected at the indicated times.
For cells arrested by CI, cells were plated at high density (1 × 10⁵ cells/cm²), grown to confluence, and maintained at confluence for up to 3 d (Wallbaum et al., 2009 (link)). During this time, the cells undergo CI entering G0 arrest. The G0-phase cells were then plated at a density of 2 × 10⁴ cells per cm² and cultured with 10% FBS. After attachment, cell samples were collected at 24, 48, and 72 hr. Subsequent analyses using qPCR, WB, IF, and cell cycle analysis were performed.

Mouse myoblasts C2C12 cells and mouse neuron HT22 cells (Procell Life Science & Technology, Shanghai, China) were cultured in 10% FBS (Gibco, Grand Island, NY, USA) supplemented with 1% penicillin/streptomycin (Gibco). The C2C12 cell culture medium was removed completely at 90% cell confluence, and the DMEM medium containing 2% horse serum was added to induce differentiation of myoblasts. Subsequent experiments were carried out after 5 days of cell differentiation. Cells were incubated at 37 °C in a humidified incubator with 5% CO₂. The 5‐aza‐2′‐deoxycytidine (DAC) and the procaine hydrochloride were purchased from Sigma (St. Louis, MO, USA).

For the in vitro assays, we utilized the OGD/R assay. HT-22 cells (obtained from Procell Co., Ltd.) were exposed to 1% oxygen in glucose-free DMEM at 37°C for a duration of 4 hours to induce OGD. The medium was allowed to equilibrate to 1% hypoxia within two hours under hypoxic conditions. To ensure that the effects of hypoxia were observed, an adequate amount of sugar-free DMEM was added to the dishes of the hypoxia workstation four hours prior to OGD. Oxygen-glucose deprivation manipulations were performed in a hypoxic workstation during which HT-22 cells were incubated in complete medium and saturated with a humidified atmosphere consisting of 5% CO₂ and 95% air for eight hours before harvesting. Control cells underwent similar washing and medium changes but remained at a constant temperature of 37°C in complete medium with an atmosphere consisting of 5% CO₂ and 95% air throughout the experiment. Melatonin was dissolved in absolute ethanol and further diluted into basal medium for final concentrations. In this study, we used MT-L (20 μM) and MT-H (40 Μm), both administered thirty minutes prior to OGD/R treatment followed by treatment as planned.

HT-22 cells, a mice hippocampal neuronal cell line (CAT# CP-H042), were purchased from Procell (Wuhan, Hubei, China) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Biological Industries, Kibbutz Beit Haemek, Israel) containing 10% fetal bovine serum (FBS, AusGeneX, Brisbane, Australia) as well as 50 μg/mL streptomycin/penicillin (Hyclone, Logan, UT, United States) at 37°C in an atmosphere of 5% CO₂. Cells at ∼90% confluence were treated in serum-free medium.