Fig. 4.

Intracellular ferroptosis evaluations. a) Relative GPX activities of 4T1 cells after incubation with various concentrations of CMS (32, 64 and 128 μg/mL, n = 3), and b) the corresponding Western blotting analysis of GPX4 protein in 4T1 cells after CMS treatment. c) MDA levels of 4T1 cells after CMS treatment (n = 3). d) CLSM images of C11-BODIPY^581/591-stained 4T1 cells after CMS treatment (scale bar = 20 μm). e) The viabilities of 4T1 cells after co-incubation with CMS and varied concentrations of Fer-1 (n = 3). f) Schematic diagram of CMS inducing ferroptosis in tumor cells by GSH depletion, ROS generation and Ca²⁺ overload. Data represented as mean values ± SD. *, ** and *** represent statistically significant differences at P < 0.05, P < 0.01 and P < 0.001, respectively.