Fig 1: Effects of FAC, erastin and other treatments in the endometriosis mice model. (A) Protocol to generate the endometriosis model in Balb/c nude mice. Balb/c nude mice were injected subcutaneously with the endometrial tissues of people with endometriosis and treated with erastin (15 mg/kg/i.p. injection), FAC (15 mg/kg/i.p. injection), vehicle, FAC+Fer-1 (1 mg/kg/i.p. injection), FAC+DFO (100 mg/kg/i.p. injection), FAC+vehicle at day 14 once a day for 2 weeks. Tumor volume was calculated on Day 28 (n = 5 mice/group). (B) Representative photographs of the endometriosis mice model and visible nodules growth on day 14 after treatment were shown. (C) HE staining of endometriotic-like lesions to confirm the success of the endometriosis model, the typical endometrioid gland including highly cylindrical epithelium could be seen. (D) Macroscopic aspect of visible lesions on Day 28 was different among groups. The volume of ectopic lesions was enhanced after treatment with 15 mg/kg FAC and tend to reduce after treatment with 15 mg/kg erastin for 2 weeks. In the circumstance of iron overload, Fer-1 and DFO reduce the volume of ectopic lesions. (E) Summary data of volume sizes are presented as the mean ± SEM. (F) Representative 4-HNE immunohistochemical results of endometriosis ectopic tissues in each group. FAC-treated group and erastin-treated group revealed higher levels of 4-HNE in comparison to other groups. (G) Quantitative analysis of 4-HNE in each group. (H) MDA content was assayed using a Lipid Peroxidation MDA Assay Kit (Abcam, ab118970). MDA was significantly increased in the FAC and erastin-treated groups. Fer-1 and DFO could reduce the MDA content. (I) Representative Collagen1 and α-SMA immunohistochemical results. Fibrosis-related protein immunohistochemical results revealed markedly higher levels of Collagen 1 and α-SMA in the FAC-treated group. In the circumstance of iron overload, the improved effect of iron on endometriosis fibrosis was damaged by treatment with Fer-1 and DFO. (J) Quantitative analysis of Collagen 1 and α-SMA. (K) Quantitative analysis of Collagen 1 and α-SMA. Scale bars: 50 μm, Significance in (E,G,H) was calculated using a one-way analysis of variance; Significance in (J) was calculated using a TWO-way analysis of variance;*p < 0.05, **p < 0.01, ***p < 0.001. qd, once a day, i.p., intraperitoneal injection.