Fig. 2.

CAFs induce ferroptosis by promoting intracellular iron overload in NK cells.

A, The viability of NK92 cells co-cultured with CAFs was measured by CCK8 assay in the presence of specific inhibitors Z-VAD (caspase-3 inhibitor), Nec1 (necroptosis inhibitor), or Fer1 (ferroptosis inhibitor) for 48 h. B–C, The effects of CAFs on NK92 and pb-NK cytotoxicity against MKN45 and SNU16 GC cells were measured by DELFIA EuTDA cell cytotoxicity assay (Fer1 5 μM, Lip1 50 nM). D-E, Lipid ROS in NK92 cells co-cultured with CAFs was detected using C11 BODYPI 581/591 probe. NK92 cells treated with 0.5 μM RSL3 for 48 h served as a positive control (scale bar = 20um). F, MDA in NK92 cells co-cultured with CAFs was detected using an MDA assay kit. NK92 cells treated with 0.5 μM RSL3 for 48 h served as a positive control. G, The mitochondria of pb-NK cells were analyzed by transmission electron microscopy (scale bar = 1μm/500 nm). H–I, Ferrous iron in pb-NK cells was detected by ferroorange assay in untreated cells or co-culture with CAFs (+CAFs) (scale bar = 20um). J, Ferrous iron in NK92 cells was detected by ferroorange assay in untreated cells after co-culture with CAFs in the absence or presence of DFO (10 μM, 48 h). K, Lipid ROS in NK92 cells co-cultured with CAFs only or CAFs plus DFO (10 μM) was detected by C11 BODYPI 581/591 probe. L, MDA in NK92 cells co-cultured with CAFs only or CAFs plus DFO (10 μM) was detected by MDA assay. M, 4-HNE in NK92 cells co-cultured with CAFs only or CAFs plus DFO (10 μM) was detected by ELISA. Data are representative of at least three independent experiments. Student t-test was used to analyze the data (mean ± SD; *P < 0.05, **P < 0.01).