The collection of placentas used for isolation and culture of PHT and PPF cells was reviewed and approved by the Institutional Review Board at the University of Pittsburgh. PHT cells were isolated from placentas of uncomplicated pregnancies, according to our previously published, exempt protocol (Mouillet et al., 2010). They were maintained in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich, St Louis, MI) containing 10% bovine growth serum (HyClone, Logan, UT) and 1% antibiotics at 37°C in a 5% CO2 air atmosphere. Cell quality was monitored by microscopy and by examining production of human chorionic gonadotropin, quantified by enzyme immunoassay (EIA, DRG, Marburg, Germany). BeWo cells (ATCC, Rockville, MD) were maintained in F12K Kaighn's modified medium supplemented with 10% SuperCalf serum (GemCell, Sacramento, CA). PPF cells were isolated during the preparation of PHT cells as we previously detailed (Li et al., 2020) and were cultured in DMEM (Sigma) supplemented with 10% SuperCalf serum. Human MDA-MB-231, U2OS, Hela and Caco2 cells (all from ATCC) were cultured in DMEM supplemented with 10% SuperCalf serum. HUVECs (ATCC) were maintained in endothelial cell basal medium-2 (Lonza, San Diego, CA) supplemented with 5% fetal bovine serum. All cell lines are routine tested in our lab for mycoplasma, and monitored for infections. Some of the cultures were performed in the presence of RSL3 (Selleck Chemicals, Houston, TX), which was used at variable concentrations and times, adjusted to experimental goals. Other ligands included staurosporine (Cell Signaling, Danvers, MA), forskolin (Sigma) and blebbistatin (Sigma). Ferrostatin-1 (Selleck) was used to block ferroptosis or to inhibit spontaneous ferroptosis in GPX4 KD BeWo cells.