Fig. 4.

Hypoxia caused apoptosis, autophagy necrosis, and ferroptosis in trophoblasts.

The in vitro PE model was established by culturing HTR-8/SVneo and TEV-1 cells under hypoxic conditions. (A) Inhibitors of apoptosis (20 μM Z-DEVD-FMK), autophagy (0.5 mM 3-Methyladenine), necrosis (0.5 μM Necrostatin-1) and ferroptosis (60 nM Ferrostatin-1 and 100 μM Deferoxamine mesylate) were added to the cells cultured under hypoxia. Twenty-four hours later, the CCK-8 agent was added to determine the cell viability. (B) Flow cytometer was used to assess the apoptosis rate of HTR-8/SVneo and TEV-1 cells. (C) Extracellular concentration of LDH was used to evaluate the percentage of cells that undergo necrosis. LC3B protein levels in cells were assessed using IF (D) and western blot assays (E). Nor: normoxia; Hyp: hypoxia; API: apoptosis inhibitor; NI: necrosis inhibitor; AUI: autophagy inhibitor; FIF: ferroptosis inhibitor, ferrostatin-1; FID: ferroptosis inhibitor Deferoxamine mesylate. *p < 0.05, **p < 0.01, compared with the HTR-8/SVneo or TEV-1 cells under normoxia; ^#p < 0.05, ^##p < 0.01, compared with the HTR-8/SVneo or TEV-1 cells under hypoxia.