PMC

RT-PCR analysis of mRNA transcripts specific for xCT and 4F2hc in wild type and Hfe^−/− RPE cells. HPRT1 was used as an internal control.

(A) MTT assay to determine hyperproliferation of Hfe^−/− (KO) primary RPE cells compared to wild type (WT) cells. n = 12; *, p < 0.001. (B) [³H]-Thymidine incorporation assay with wild type and Hfe^−/− RPE cells. n = 14; *, p < 0.001.

Ferritin levels were determined by ELISA. (A) Ferritin levels in sera (n = 4) obtained from 18-month-old wild type mice and age-matched Hfe^−/− mice. (B) Ferritin levels in retinal tissues (n = 6) and RPE cell preparations (n = 4) obtained from 18-month-old wild type mice and age-matched Hfe^−/− mice. *, p < 0.05; **, p < 0.001.

(A) Cellular content of glutathione was measured in wild type and Hfe^−/− RPE cells. n = 10; *, p < 0.001. (B) RT-PCR analysis of c-Fos and c-Jun mRNA levels in wild type and Hfe^−/− RPE cells. HPRT1 was used as an internal control.

(A) Saturation kinetics of glutamate uptake in wild type and Hfe^−/− RPE cells was evaluated by monitoring the uptake in a Na⁺-free medium with varying concentrations of glutamate. (B) Eadie-Hofstee transformation of the data. The experiment was repeated three times.

(A) Cells were treated with or without ferric ammonium citrate (100 μg/ml) for 72 h. RNA was isolated from these cells and used for RT-PCR. (B) x_c⁻ -specific transport activity in ARPE-19 cells with and without exposure to ferric ammonium citrate. n = 3; *, p < 0.001.

(A) Uptake of glutamate in wild type and Hfe^−/− RPE cells. Uptake of [³H]-glutamate (2.5 μM) was measured for 15 min in control and Hfe^−/− cells at 37°C in the absence of Na⁺. The values represent transport activity specific for system x_c⁻. n = 9; *, p < 0.001. (B) Substrate selectivity of glutamate uptake in wild type and Hfe^−/− RPE cells. Uptake of [³H]-glutamate (2.5 μM) was measured in wild type and Hfe^−/− RPE cells in the absence of Na⁺ for 15 min at 37°C in the absence or presence of unlabeled amino acids glutamate, cystine, valine and aspartate, each at a concentration of 1 mM. n = 6; *, p < 0.001.

(A) Hematoxylin and eosin stained wild type and Hfe^−/− retinas from 8-week-old mice. (B) Hematoxylin and eosin stained wild type and Hfe^−/− retinas from 18-month-old mice. (C) Electron microscopic analysis of RPE from 18-month-old wild type and Hfe^−/− retinas. (D) Nuclear stained (DAPI) retinal sections of 18-month-old wild type and Hfe^−/− retinas as analyzed by laser-scanning confocal microscopy. Inset is a higher magnification of the RPE cell layer showing characteristic hyperplasia. A total of 5 mice each for wild type and Hfe^−/− genotype were used for the 18-month age group with similar results. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium.

(A) Giemsa staining or colony formation assay in a 12-well plate for wild type and Hfe^−/− RPE cells. 8,000 cells per well (left two columns) or 4000 cells per well (right two columns) were seeded in triplicates. The cells were cultured for 12 days, with medium change every 2 days. Cells were then fixed with methanol for 30 min followed by staining with KayroMax Giemsa for 1 h. (B) Wild type and Hfe^−/− RPE cells were seeded at varying cell density in a 12-well plate and cultured for 12 days. Medium was changed every 2 days. On the 12^th day, cells were fixed with methanol and than stained with KayroMax Giemsa. The contents of the wells were solubilized and the dye released was quantified. n = 6; *, p < 0.001. (C) Wild type and Hfe^−/− RPE cells were seeded at a density of 10,000 cells per well and cultured for varying time periods. At the end of the indicated time period, cells were stained with Giemsa, and the dye quantified as described above. n = 6; *, p < 0.001.