Fig. 5.

FTH protects RPTEC from heme cytotoxicity. (A) Human RPTEC were transduced in vitro with Rec.Ad. (10 pfu/cell) encoding β-galactosidase (LacZ), human FTH, or mutated human FTH (FTH^m) lacking ferroxidase activity and exposed to heme (5 µM) and/or H₂O₂ (50 µM). Cytotoxicity was measured using a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide-based cell viability assay, as detailed in SI Appendix. Data (mean ± SD) are from two independent experiments with a similar trend. (B) Relative expression of FTH and β-actin in human RPTEC transduced with LacZ, FTH, or FTH^m Rec.Ad, detected by Western blot in one experiment representative of two with a similar trend. (C) ROS accumulation (mean ± SD) in human RPTEC exposed to heme (5 µM) and/or H₂O₂ (50 µM), detected using the CM-H₂DCFDA probe. Data are from two independent experiments with a similar trend. (D) Time course of ROS accumulation (mean ± SD) in human RPTEC transduced and treated as in A. Data are from two independent experiments with a similar trend. (E) Quantification of data from D. AUC: area under the curve. P values in A determined using two-way ANOVA with Tukey’s multiple comparison test; in C and E using one-way ANOVA with Tukey’s multiple comparison test. NS, not significant; ***P < 0.001.