Figure 2.
Figure 2. Excess free heme in the shRNA model reproducing DBA (human primary erythroid cells depleted in RPS19, RPL5, or RPL11). (A) Quantification of the total heme in depleted RPS19, RPL5, or RPL11 erythroid cells after CD34+ cord blood infection with specific shRNAs at day 9 of primary human erythroid cell culture. The data are mean ± standard deviation of 3 independent experiments, relative to total heme content of shcontrol (value = 1). (B) Excess free heme in depleted RPS19, RPL5, or RPL11 erythroid cells after CD34+ cord blood infection with specific shRNAs. Pellets of 100 000 RP-depleted erythroid cells were analyzed, and OD scans were measured from 200 to 800 nm with a spectrophotometer. The day-9 measurement is shown. Free heme was calculated as the ratio between 380 nm ± 2 nm (heme band) and 560 nm ± 2 nm (hemoglobin band) (supplemental Figure 2A). The data are mean ± standard deviation of 3 independent experiments, relative to free heme content of shcontrol (value = 1). (C) Major excess free heme after depletion of RPL5 or RPL11 compared with RPS19 lentivirus–depleted erythroid cells. Immunoblots of 100 000 RPL5 or RPL11 depleted erythroid cells revealed a significant decrease in ALAS2 expression levels, whereas ALAS1 was normal, reinforcing the decreased total heme content and the specificity of the defect in erythroid cells. The iron uptake based on TfR1 expression level was decreased significantly under all conditions. Immunoblots also revealed indirect signs of the large amount of excess free heme in depleted RPL5 and RPL11 erythroid cells on the decreased BACH1 and a large increase in FLVCR1 expression levels. Proteins BACH1, FLVCR1, TfR1, ALAS2, and ALAS1 were compared with the β-actin expression level. A western blot representative of 3 experiments at day 9 of the primary erythroid cell culture is shown (statistics are shown in supplemental Figure 3B). (D) Quantification of ROS production in RPS19-, RPL5-, or RPL11-depleted erythroid cells at day 9 of erythroid culture. We show the second method used for ROS production based on flow cytometry with a CellROX Deep Red Reagent kit (Invitrogen). RPL5- and RPL11-depleted erythroid cells at day 9 produced a higher ROS compared with the control, whereas RPS19 ones exhibited a slight increase in ROS production. Data are representative of 3 experiments. (E) Brief reminder of the HRI/EIF2α pathway (adapted from Chen17). (F) HRI/EIF2α protein pathway analysis in DBA erythroid cells after CD34+ cord blood infection with specific shRNA-RPS19, -RPL5, -RPL11. Immunoblots of 100 000 RPS19-, RPL5-, or RPL11-depleted primary erythroid cells compared with β-actin expression and shcontrol. A western blot representative of 3 experiments at day 9 of the primary erythroid cell culture is shown (statistics are shown in supplemental Figure 3B). (G) HRI (left panel) and EIF2α (right panel) mRNA expression in DBA erythroid cells after CD34+ cord blood infection with specific shRNA-RPS19, -RPL5, -RPL11. We observed a significant decrease in HRI and EIF2α mRNA compared with the reporter gene mRNAs at day 9 of terminal erythroid differentiation. The data are mean ± standard deviation of 3 independent experiments relative to mRNA expression level of shcontrol compared with β-actin (value = 1). *P < .05, **P < .01. NS, nonsignificant.

Excess free heme in the shRNA model reproducing DBA (human primary erythroid cells depleted in RPS19, RPL5, or RPL11). (A) Quantification of the total heme in depleted RPS19, RPL5, or RPL11 erythroid cells after CD34+ cord blood infection with specific shRNAs at day 9 of primary human erythroid cell culture. The data are mean ± standard deviation of 3 independent experiments, relative to total heme content of shcontrol (value = 1). (B) Excess free heme in depleted RPS19, RPL5, or RPL11 erythroid cells after CD34+ cord blood infection with specific shRNAs. Pellets of 100 000 RP-depleted erythroid cells were analyzed, and OD scans were measured from 200 to 800 nm with a spectrophotometer. The day-9 measurement is shown. Free heme was calculated as the ratio between 380 nm ± 2 nm (heme band) and 560 nm ± 2 nm (hemoglobin band) (supplemental Figure 2A). The data are mean ± standard deviation of 3 independent experiments, relative to free heme content of shcontrol (value = 1). (C) Major excess free heme after depletion of RPL5 or RPL11 compared with RPS19 lentivirus–depleted erythroid cells. Immunoblots of 100 000 RPL5 or RPL11 depleted erythroid cells revealed a significant decrease in ALAS2 expression levels, whereas ALAS1 was normal, reinforcing the decreased total heme content and the specificity of the defect in erythroid cells. The iron uptake based on TfR1 expression level was decreased significantly under all conditions. Immunoblots also revealed indirect signs of the large amount of excess free heme in depleted RPL5 and RPL11 erythroid cells on the decreased BACH1 and a large increase in FLVCR1 expression levels. Proteins BACH1, FLVCR1, TfR1, ALAS2, and ALAS1 were compared with the β-actin expression level. A western blot representative of 3 experiments at day 9 of the primary erythroid cell culture is shown (statistics are shown in supplemental Figure 3B). (D) Quantification of ROS production in RPS19-, RPL5-, or RPL11-depleted erythroid cells at day 9 of erythroid culture. We show the second method used for ROS production based on flow cytometry with a CellROX Deep Red Reagent kit (Invitrogen). RPL5- and RPL11-depleted erythroid cells at day 9 produced a higher ROS compared with the control, whereas RPS19 ones exhibited a slight increase in ROS production. Data are representative of 3 experiments. (E) Brief reminder of the HRI/EIF2α pathway (adapted from Chen17 ). (F) HRI/EIF2α protein pathway analysis in DBA erythroid cells after CD34+ cord blood infection with specific shRNA-RPS19, -RPL5, -RPL11. Immunoblots of 100 000 RPS19-, RPL5-, or RPL11-depleted primary erythroid cells compared with β-actin expression and shcontrol. A western blot representative of 3 experiments at day 9 of the primary erythroid cell culture is shown (statistics are shown in supplemental Figure 3B). (G) HRI (left panel) and EIF2α (right panel) mRNA expression in DBA erythroid cells after CD34+ cord blood infection with specific shRNA-RPS19, -RPL5, -RPL11. We observed a significant decrease in HRI and EIF2α mRNA compared with the reporter gene mRNAs at day 9 of terminal erythroid differentiation. The data are mean ± standard deviation of 3 independent experiments relative to mRNA expression level of shcontrol compared with β-actin (value = 1). *P < .05, **P < .01. NS, nonsignificant.

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