Fig. 2: FAC supplementation of culture media is sufficient to alter iron (Fe) metabolism and impair POS uptake by hiPSC-RPE cells. | Cell Death Discovery

Fig. 2: FAC supplementation of culture media is sufficient to alter iron (Fe) metabolism and impair POS uptake by hiPSC-RPE cells.

From: Environmental stress impairs photoreceptor outer segment (POS) phagocytosis and degradation and induces autofluorescent material accumulation in hiPSC-RPE cells

Fig. 2

a Representative images of parallel cultures of Prussian blue-stained untreated and FAC-treated (50 µg/ml, ~1 month) hiPSC-RPE cells showing increased presence of Fe deposits in FAC-treated hiPSC-RPE cells (scale bar = 50 µm). b Quantitative analyses of real-time PCR demonstrating increased expression of iron-regulatory genes (CP, HFE, GSS, and TF) in untreated versus FAC-treated (50 µg/ml, ~1 month) hiPSC-RPE cultures. Data are represented as mean ± SEM. *P ≤ 0.05, ***P ≤ 0.001, n = 3 independent trials. c A schematic depicting the protocol utilized for assessing the impact of acute FAC exposure (200 µg/ml, 24 h) on POS uptake (0 h time point) and degradation (24 h time point) post 2 h POS feeding (20 POS/RPE cell)36. Of note, the amount of POS in hiPSC-RPE cells was evaluated indirectly by measuring the protein levels of RHO, a POS-specific protein. d–f Representative western blot images (d) and corresponding quantitative analyses (e, f) showing reduced uptake of RHO/POS at the 0 h time point (d, e) but similar degradation of ingested RHO at the 24 h time point (d, f) in untreated versus FAC-treated hiPSC-RPE cultures. Data are presented as mean ± SEM. **P ≤ 0.01, n = 3 independent trials. Of note, to represent the rate of POS degradation, the quantification of RHO levels at 24 h time point is calculated relative to the amount of POS uptake (0 h time point). Note: ACTN served as loading control for western blot analyses. g Representative fluorescent microscopy (top panel) and confocal microscopy images (bottom panel) showing no difference in cell viability (calcein-AM) and localization of tight junction marker, ZO-1, in untreated versus FAC-treated (200 µg/ml, 24 h) hiPSC-RPE cultures (scale bar = 20 µm). h Quantitative analyses of TER measurements showing similar TER values with TER ≥ 150 Ω cm2 (dotted line; the reported in vivo TER threshold40) at baseline and the 24 h time point in both untreated and FAC-treated (200 µg/ml, 24 h) hiPSC-RPE cultures. Data are presented as mean ± SEM. n = 3 independent trials

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