*4.3. Human Trophoblast Cell Culture*

Human trophoblast cell line JEG-3 was purchased from the cell bank of the Chinese Academy of Sciences. They were cultured in Dulbecco's modified Eagle's medium (Hyclone, China) containing 10% FBS and 1% penicillin/streptomycin, and incubated at standard culture conditions of 5% CO<sup>2</sup> in air at 37 ◦C.

### *4.4. mTORC1 Inhibition in Placental Trophoblast*

Downregulation of mTORC1 activity was conducted as described in a previous study [4]. Briefly, human JEG-3 cells were cultured with 100 nM rapamycin (Gene Operation, USA), a specific mTORC1 inhibitor, or transfected of small interference RNAs (GenePharma, Shanghai, China) targeting raptor

(100 nM; sense: 50 -CGAGAUUGGACGACCAAAUTT-30 ). At 48 h in culture, cells were collected for RNA and protein extraction.
