Iron restriction with DEF inhibits ALAS2 expression and reduces porphyrin accumulation in UROS-deficient TF1 and erythroid cells from a patient with CEP. Panels A-C represent experiments performed on TF1-UROS^WT, TF1-UROS^E4i, and TF1-UROS^E10i induced to erythroid differentiation. (A) Porphyrin accumulation after treatment at various doses of DEF (10, 20, 50, 100, 200, or 500 μM) for 48 hours. (B) Real-time quantitative analysis of ALAS1 and ALAS2 mRNA after treatment with DEF 200 μM for 48 hours. The relative gene expression was normalized with the ribosomal protein P0 gene and expressed as ratio vs untreated control for each condition. This dose allows the efficient reduction of porphyrin accumulation without cellular toxicity on TF1 cells. (C) Western blot analysis of ALAS2, IRP1, IRP2, and GAPDH proteins after treatment with DEF 200 μM for 48 hours. Iron exerts a posttranscriptional regulation of ALAS2 and IRP2 expression: in the presence of iron, ALAS2 mRNA is translated while IRP2 protein is degraded by ubiquitination. Iron restriction represses ALAS2 and induces IRP2 expression in WT and UROS-deficient TF1 cells. Iron modulates the activity of IRP1 without changing the expression level of the protein. Panels D-G represent experiments performed on the erythroid progeny of CD34⁺ cells. Control (cord blood) and UROS-deficient (steady-state peripheral blood from a patient with CEP) CD34⁺ cells were amplified and subjected to erythroid differentiation in a 3-step protocol over 17 days. (D) The erythroid maturation was analyzed on day 17 by flow cytometry analysis of glycophorin A (FITC), CD36 (APC), and CD71 (APC) expression. Most control and CEP cells had matured fully from stage I to stage III/IV (defined by the sequential gain of CD36 and then GlyA expression and the subsequent loss of CD36 expression) by day 17. (E) Porphyrin accumulation in CD34⁺-derived erythroid cells (normal and CEP) after treatment with DEF (200 μM) for 48 hours (days 15-17). (F) Real-time semi-quantitative analysis of ALAS2 mRNA in the erythroid progeny of normal and CEP CD34⁺ cells after treatment with DEF (200 μM) for 48 hours (days 15-17). The relative gene expression was normalized with the ribosomal protein P0 gene and expressed as ratio vs untreated control for each condition. (G) Western blot analysis of ALAS2 and GAPDH proteins in the erythroid progeny of normal cells and CEP CD34⁺ cells after treatment with DEF (200 μM) for 48 hours (days 15-17). The combination of both UROS deficiency and iron restriction (low ferrochelatase activity) probably affects global heme production and stimulates ALAS2 transcription in UROS-deficient cells. Signal density on western blots was determined by using ImageJ software to perform semi-quantitative analysis of protein expression. Semi-quantitative results are expressed as a ratio vs GAPDH expression. *P < .05 with vs without DEF.