Fig. 6.

DAT increases lipid ROS and sensitizes cancer cells to ferroptosis in vitro and in vivo. a Increasing concentrations of RSL3 were used to treat a panel of lung, breast and colorectal epithelial cancer cell lines. Cell death curves were plotted from percentage PI-positive cells quantified by flow cytometry. c Lipid ROS was uantified using the BODIPY-C11 lipid probe by flow cytometry after cells were treated with 2 μM of RSL3 for 24 h, or RSL3 with 1 μM of ferroptosis inhibitor Ferrostatin-1 for 24 h. d GPX4-iKO was induced using CRISPR/doxycycline-inducible Cas9. Knockout was confirmed by western blot. e–g DAT sensitizes H292 cells to GPX4 KO induced ferroptosis in vivo. In e, Six- to eight-week old nude mice were inoculated with 3 × 10⁶ doxycycline-inducible GPX4 iKO cells and randomly divided into 5 groups: vehicle (injected with PBS only), DAT only, doxycycline only, DAT and doxycycline and DAT, doxycycline and Liproxstatin-1. Tumor volume was measured daily using a Vernier caliper. Error bars represent standard error. ** Between – DAT + Dox and + Dox + DAT groups is P = 0.0049. ** Between – Dox + DAT and + Dox + DAT groups is P = 0.0044. P-values were calculated using a two-tailed t-test with 95% confidence interval. In f, the xenografted tumors were dissected and their size was measured as shown. In g, xenografted tumors were sectioned, fixed and stained with H&E, or immunohistochemically stained for GPX4 and proliferation marker Ki67