Figure 5
Figure 5. Complementary pharmacologic and genetic methods augment p-eIF2α and increase HbF. (A) Human primary erythroid cells were treated with 5 μM BT and 25 μM GBZ on days 15 and 18 of differentiation. The mRNA expression for γ- and β-globin was assessed from day 7 through day 20 and the AUC was calculated for each. BT and GBZ treatments did not change the γ/(γ + β) AUC mRNA ratio relative to untreated control. The error bars represent ± standard error of the mean of 3 independent experiments assessing BT treatment and 2 independent experiments analyzing the effects of GBZ. (B) HPLC was used to determine the amount of HbA and HbF present on day 20 from lysates prepared from erythroid progenitors treated with 5 μM BT and 25 μM GBZ. Both BT and GBZ significantly increase the percentage of HbF relative to the untreated control. Error bars represent ± standard error of the mean of 3 independent experiments assessing BT treatment and 2 independent experiments analyzing GBZ-treated cells. P values were determined using an unpaired two-tailed t test. *P < .05; **P < .01. (C) Differentiating human primary erythroid progenitors were infected with a control shRNA (shCTRL) and shRNAs targeting either GADD34 (shGADD34) or CReP (shCReP) on days 12 and 13. On day 16, transcript levels were evaluated for GADD34 and CReP using quantitative PCR. Both shGADD34 and shCReP resulted in approximately 50% knockdown when normalized to the shCTRL. Error bars denote ± standard error of the mean of 3 independent experiments (3 different donors). (D) Western blot analysis shows shGADD34 and shCReP result in significant knockdown of GADD34 and CReP protein, respectively, in comparison with shCTRL. Protein lysates were taken on day 16 of differentiation after 2 infections on days 12 and 13. As a loading control, β-actin was used. (E) Protein lysates were taken on days 16 and 18 of differentiation from erythroid progenitors infected with shCTRL, shGADD34, or shCReP. Western blot analysis reveals both shGADD34 and shCReP significantly increase p-eIF2α levels compared with shCTRL. Total eIF2α and β-actin were used as loading controls. (F) The mRNA expression of γ- and β-globin was assessed throughout differentiation. The AUC was compared as a γ/(γ + β) mRNA ratio. shGADD34 and shCReP did not change the γ/(γ + β) ratio relative to the shCTRL. Error bars express ± standard error of the mean of 3 independent experiments (3 unique donors). (G) HPLC was performed on day 20 of differentiation to assess the percentage of HbF in shCTRL-, shGADD34-, and shCReP-infected cells. Due to the differences in HbF levels at baseline among donors, the percentage of HbF is shown as fold increase over the shCTRL. Both shGADD34 and shCReP significantly enhances the percentage of HbF relative to the control. Error bars express ± standard error of the mean of 3 independent experiments (3 different donors). P values were determined using 1 sample, two-tailed t test. *P < .05; **P < .01.

Complementary pharmacologic and genetic methods augment p-eIF2α and increase HbF. (A) Human primary erythroid cells were treated with 5 μM BT and 25 μM GBZ on days 15 and 18 of differentiation. The mRNA expression for γ- and β-globin was assessed from day 7 through day 20 and the AUC was calculated for each. BT and GBZ treatments did not change the γ/(γ + β) AUC mRNA ratio relative to untreated control. The error bars represent ± standard error of the mean of 3 independent experiments assessing BT treatment and 2 independent experiments analyzing the effects of GBZ. (B) HPLC was used to determine the amount of HbA and HbF present on day 20 from lysates prepared from erythroid progenitors treated with 5 μM BT and 25 μM GBZ. Both BT and GBZ significantly increase the percentage of HbF relative to the untreated control. Error bars represent ± standard error of the mean of 3 independent experiments assessing BT treatment and 2 independent experiments analyzing GBZ-treated cells. P values were determined using an unpaired two-tailed t test. *P < .05; **P < .01. (C) Differentiating human primary erythroid progenitors were infected with a control shRNA (shCTRL) and shRNAs targeting either GADD34 (shGADD34) or CReP (shCReP) on days 12 and 13. On day 16, transcript levels were evaluated for GADD34 and CReP using quantitative PCR. Both shGADD34 and shCReP resulted in approximately 50% knockdown when normalized to the shCTRL. Error bars denote ± standard error of the mean of 3 independent experiments (3 different donors). (D) Western blot analysis shows shGADD34 and shCReP result in significant knockdown of GADD34 and CReP protein, respectively, in comparison with shCTRL. Protein lysates were taken on day 16 of differentiation after 2 infections on days 12 and 13. As a loading control, β-actin was used. (E) Protein lysates were taken on days 16 and 18 of differentiation from erythroid progenitors infected with shCTRL, shGADD34, or shCReP. Western blot analysis reveals both shGADD34 and shCReP significantly increase p-eIF2α levels compared with shCTRL. Total eIF2α and β-actin were used as loading controls. (F) The mRNA expression of γ- and β-globin was assessed throughout differentiation. The AUC was compared as a γ/(γ + β) mRNA ratio. shGADD34 and shCReP did not change the γ/(γ + β) ratio relative to the shCTRL. Error bars express ± standard error of the mean of 3 independent experiments (3 unique donors). (G) HPLC was performed on day 20 of differentiation to assess the percentage of HbF in shCTRL-, shGADD34-, and shCReP-infected cells. Due to the differences in HbF levels at baseline among donors, the percentage of HbF is shown as fold increase over the shCTRL. Both shGADD34 and shCReP significantly enhances the percentage of HbF relative to the control. Error bars express ± standard error of the mean of 3 independent experiments (3 different donors). P values were determined using 1 sample, two-tailed t test. *P < .05; **P < .01.

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