Fig. 2.

Vitamin K1 inhibits RSL3-induced ferroptosis in vitro. a Murine NIH3T3 cells, MCTs, MEFs, and PTCs were left untreated or stimulated at 37 °C for different durations with 5 µM RSL3 in the presence or absence of 1 µM ferrostatin-1 (Fer-1) or 10 µM vitamin K1 (vit.K1), as indicated. Cell death was quantified by FACS analysis using 7-amino-actinomycin D (7-AAD) and phosphatidylserine accessibility (Annexin V staining) as markers. b Examination of lipid ROS production preceding ferroptosis using BODIPY™ (581/591) C11. Indicated cells were treated at 37 °C for 4 h with 5 µM RSL3 (± 1 µM Fer-1 or 10 µM vit.K1). Overlay images show detection of accumulated ROS over time. A minimum of 10,000 cells were analyzed per condition. Notably, a shorter stimulation with RSL3 than in a was required for this assay, since ROS production in ferroptosis typically precedes triggering of cell death by several hours. Representative results of one of three independent experiments are shown