Metronomic capecitabine inhibits liver transplant rejection in rats by triggering recipients’ T cell ferroptosis

Figure 6 5-fluorouracil increases the pool of labile iron and contributes to ferroptosis in CD3⁺ T cells. Vitro experiments were conducted on rat splenic and human peripheral blood CD3⁺ T cells, which were sorted by immunomagnetic beads. According to predetermined IC50, the rat, and human primary CD3⁺ T cells were treated with 5-fluorouracil (5-FU) (15 μmol/L) and ferrostatin-1 (Fer-1) (2 μmol/L) for 48 h. A: Cell viability was assessed with a Cell Counting Kit-8 kit; the results are expressed as optical density read at 450 nm; B: Transmission electron microscopy images of mitochondria in T cells show increased membrane density (yellow arrows) and a shrunk morphology (Scale bar, 500 nm, and 250 nm); C: Representative FACS data showing FerroOrange staining as the measurement of the level of cytoplasmic labile iron in primary CD3⁺ T cells. The graph shows the statistical analysis of mean fluorescence intensity in the different groups; D: Levels of ferritinophagy-related proteins nuclear receptor coactivator 4 and ferritin heavy chain 1 were assessed by western blot. CON groups: T cells were treated with DMSO for 48 h; 5-FU groups: T cells were treated with 5-FU (15 μmol/L) for 48 h; 5-FU+ Fer-1 groups: T cells were treated with 5-FU (15 μmol/L) and Fer-1 (2 μmol/L) for 48 h. Statistical analysis was done by one-way ANOVA, n = 3. Data are shown as mean ± SD. Data are shown as mean ± SD. ^cP < 0.001 vs the control group, ^dP < 0.05 vs the 5-FU group, ^eP < 0.01 vs the 5-FU group, ^fP < 0.001 vs the 5-FU group. CON: Untreated control groups; 5-FU: 5-fluorouracil; NCOA4: Nuclear receptor coactivator 4; FTH1: Ferritin heavy chain 1; Fer-1: Ferrostatin-1.