Fig 1: Simvastatin promotes ferroptosis in Ishikawa cells. (a–d): Western blot for detection of the effect of simvastatin on the expression of ferroptosis-related proteins (SLC7A11, FPN, and TRF 1) (a) and quantitative analysis based on Image J (b–d); (e): ELISA for detection the effect of simvastatin on Fe2+ level in Ishikawa cells, **P < 0.01 vs. control group; ##P < 0.01 vs. 5 µM group. SLC7A11, solute carrier family 7 member 11; TRF1, transferrin receptor 1; FPN, ferroportin.
Fig 2: RAS agonist reverses the promoting effects of simvastatin on ROS level and ferroptosis in Ishikawa cells. (a–b): flow cytometry detected the effects of simultaneous treatment of ML-098 and simvastatin on DCFH-DA-labeled ROS level in Ishikawa cells; (c–d): ELISA detected the effects of simultaneous treatment of ML-098 and simvastatin on the level of MDA (c) and GSH (d) in Ishikawa cells; (e): ELISA to detect the effect of simultaneous treatment of ML-098 and simvastatin on the Fe2+ level in Ishikawa cells; (f-g): Western blot to determine the effect of simultaneous treatment of ML-098 and simvastatin on the expression of ferroptosis-related proteins (SLC7A11, TRF1, and FPN) in Ishikawa cells, **P < 0.01 vs. control group; ##P < 0.01 vs. simvastatin group. ROS, reactive oxygen species; MDA, malondialdehyde; GSH, glutathione; solute carrier family 7 member 11; TRF1, transferrin receptor 1; FPN, ferroportin.
Fig 3: NCOA4-mediated FTH1 degradation led to pathologically high intraocular pressure (ph-IOP)-induced iron accumulation in retinas.a–d Western blotting detection of the retinal levels of DMT1, Fpn1, and TR (normalized to that of actin) in control and ph-IOP injured mice (n = 3 in each group). e–g Western blotting detection of the retinal levels of FTH1 and NCOA4 (normalized to that of actin) in control and ph-IOP injured mice (n = 3 in each group). h Representative photomicrographs of immunofluorescence staining for FTH1 (red) in retinal ganglion cells (counterstained with RBPMS; green). I, j Co-immunoprecipitation showing the endogenous interaction between NCOA4 and FTH1 in control and ph-IOP injured mice. k, l Western blotting detection of the retinal levels of NCOA4 (normalized to that of actin) in mice receiving pAAV-spgRNA-EGFP (control) and pAAV-shNcoa4-EGFP injection for 3 weeks (n = 3 in each group). m–o Western blotting detection of the retinal levels of NCOA4 and FTH1 (normalized to that of actin) in ph-IOP injured mice, with or without AAV-mediated Ncoa4 knockdown at 1 h after modeling (n = 3 in each group). p Retinal iron contents in ph-IOP injured mice, with or without AAV-mediated Ncoa4 knockdown, at 1 h after modeling (n = 5 in each group). DMT1, divalent metal transporter 1; Fpn1, ferroportin 1; FTH1, ferritin heavy polypeptide 1; NCOA4, nuclear receptor coactivator 4; TR, transferrin receptor. Data are shown as the mean ± SD; *p < 0.05, **p < 0.01 (compared with the control group using one-way analysis of variance); ##p < 0.01 (compared with the shNcoa4 group using one-way analysis of variance). Bar = 50 µm.
Supplier Page from Abcam for Anti-SLC40A1 antibody