Cell Culture and Transfection

Murine HT22 cells from the American Type Culture Collection (Manassas, VA, USA) and then grown in Dulbecco’s modified Eagle’s medium (DMEM) plus 10% fetal bovine serum (FBS; Solarbio, Beijing, China) and 1% penicillin-streptomycin. HT22 cells were kept in a 5% CO₂ humidified incubator at 37°C.

In order to construct an in vitro model of cerebral I/R injury, HT22 cells were cultured in glucose-free DMEM for 4 h under hypoxic condition (37°C, 5% CO₂/95% N₂), followed by incubation for 6, 12, or 24 h in reoxygenation condition (37°C, 5% CO₂/95% air).

Lentiviral TNIP2-overexpressing plasmid (LV-TNIP2) and the empty lentiviral vector (LV-Scramble) were purchased from GenePharma (Shanghai, China). After transfection with LV-TNIP2 or LV-Scramble for 48 h, HT22 cells were transferred to fresh culture medium. The transfection efficiency was verified using Western blot.