Figure 3.
Figure 3. DBA-affected patients also exhibit excess free heme. (A) Expression levels of the major proteins involved in heme metabolism and their regulators in a DBA patient who carried a mutation in the RPS19 gene during terminal erythroid differentiation compared with a healthy control. Immunoblots of 50 000 human primary erythroid cells in each lane obtained from purified peripheral blood CD34+ cells from the affected DBA patient (UPN#35) during terminal erythroid differentiation from day 7 (D7) to day 12 (D12). Protein expression compared with β-actin or GAPDH, depending on the size of the proteins, to optimize the numbers of proteins analyzed on the same western blot. Due the difficulty in obtaining samples from DBA patients, this patient has been studied once; other DBA-affected patients have been studied. We validated the data because the same protein profile on the western blots in all of the mutated RPS19 DBA patients have been seen (as example another mutated RPS19Mut/+ patient, supplemental Figure 5). (B) Same data as in (A) for a DBA-affected patient who carried a mutation in the RPL11 gene (UPN#1099). (C) From the immunoblot in panel A, representation of the level of protein expression of FLVCR1 in the erythroid precursors of DBA patient UPN#35 relative to β-actin and in a healthy control at each day of the studied terminal erythroid differentiation. (D) From the immunoblot in panel B, representation of the level of FLVCR1 expression in the erythroid precursors of DBA patient UPN#1099 relative to β-actin and to the healthy control during terminal erythroid differentiation (value = 1 at day 7 in the control erythroid cells). (E) Increased FLVCR1 and decreased globin expression levels in a DBA-affected patient with a mutated RPL5 gene (RPL5Mut/+) (UPN#845). (F) Relative mRNA expression of GATA1, ALAS2, EIF2α, α and β globins, and TfR1 in a DBA-affected patient (UPN#845) compared with a healthy control (value = 1). (G) Relative ROS production by various DBA-affected patients. These DBA patients carried mutations in the RPS19 or RPL5 gene or even an unknown gene compared with their healthy controls (mean fluorescence intensity = 1). *P < .05 in triplicate experiments.

DBA-affected patients also exhibit excess free heme. (A) Expression levels of the major proteins involved in heme metabolism and their regulators in a DBA patient who carried a mutation in the RPS19 gene during terminal erythroid differentiation compared with a healthy control. Immunoblots of 50 000 human primary erythroid cells in each lane obtained from purified peripheral blood CD34+ cells from the affected DBA patient (UPN#35) during terminal erythroid differentiation from day 7 (D7) to day 12 (D12). Protein expression compared with β-actin or GAPDH, depending on the size of the proteins, to optimize the numbers of proteins analyzed on the same western blot. Due the difficulty in obtaining samples from DBA patients, this patient has been studied once; other DBA-affected patients have been studied. We validated the data because the same protein profile on the western blots in all of the mutated RPS19 DBA patients have been seen (as example another mutated RPS19Mut/+ patient, supplemental Figure 5). (B) Same data as in (A) for a DBA-affected patient who carried a mutation in the RPL11 gene (UPN#1099). (C) From the immunoblot in panel A, representation of the level of protein expression of FLVCR1 in the erythroid precursors of DBA patient UPN#35 relative to β-actin and in a healthy control at each day of the studied terminal erythroid differentiation. (D) From the immunoblot in panel B, representation of the level of FLVCR1 expression in the erythroid precursors of DBA patient UPN#1099 relative to β-actin and to the healthy control during terminal erythroid differentiation (value = 1 at day 7 in the control erythroid cells). (E) Increased FLVCR1 and decreased globin expression levels in a DBA-affected patient with a mutated RPL5 gene (RPL5Mut/+) (UPN#845). (F) Relative mRNA expression of GATA1, ALAS2, EIF2α, α and β globins, and TfR1 in a DBA-affected patient (UPN#845) compared with a healthy control (value = 1). (G) Relative ROS production by various DBA-affected patients. These DBA patients carried mutations in the RPS19 or RPL5 gene or even an unknown gene compared with their healthy controls (mean fluorescence intensity = 1). *P < .05 in triplicate experiments.

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