Fig. 4.

Phenprocoumon promotes ferroptosis in vitro. a Murine NIH3T3 cells were treated as indicated at 37 °C for 24 h with a sublethal dose of 0.1 µM RSL3. In the top row (vehicle), the aggravated cell death in the presence of increasing concentrations of phenprocoumon is clearly evident under these conditions. Addition of 1 µM ferrostatin-1 (Fer-1) or 10 µM vitamin K1 (vit.K1) illustrates that cell death mediated by phenprocoumon is based on preventable ferroptosis. FACS dot plots of one representative experiment are shown, the adjacent graph b presents the mean and SD of three independent experiments, and the identical effect of phenprocoumon using a sublethal dose of 0.2 µM erastin. c Phenprocoumon-induced facilitation of ferroptosis in vitro is species-independent, as confirmed using human HT-1080 cells. However, the human cells were treated as indicated at 37 °C for 24 h with a sublethal dose of 1 µM RSL3 and 2.5 µM erastin, respectively. Again, addition of 1 µM Fer-1 or 10 µM vit.K1 proved that the phenprocoumon-dependent effect was based on ferroptotic cell death. Cell death was quantified by FACS analysis using 7-amino-actinomycin D (7-AAD) and phosphatidylserine accessibility (Annexin V staining) as markers. Graphs show mean ± SD (n = 3 independent repeats)