Figure 6
Figure 6. Combination of salubrinal with AZA or HU significantly enhances HbF production post-transcriptionally in differentiating primary erythroid cells. (A) Erythroid progenitors were treated with 3 μM salubrinal (Sal), 6 μM Sal, 200 nM AZA, or in combination with Sal and AZA at these doses. The mRNA expression of γ- and β-globin was evaluated from days 7 through 20 of the culture and the AUC was calculated for each transcript. AZA treatment increases globin expression, whereas AZA in combination with Sal treatment reduces this enhancement. Globin gene expression still remains elevated in the combination-treated cells relative to the untreated control. Error bars represent ± standard error of the mean of 4 independent experiments (3 unique donors). (B) For all treatments, the AUCs were compared as a γ/(γ + β) mRNA ratio. AZA (200 nM) significantly enhances the γ/(γ + β) ratio, and the combination of Sal and AZA treatments did not change this induction. Error bars express ± standard error of the mean of 4 independent experiments (3 unique donors). P values were calculated using an unpaired two-tailed t test. (C) Erythroid progenitors were treated with 10 μM HU or 3 μM Sal alone or in combination. The mRNA expression of γ- and β-globin was evaluated from day 7 through day 20 of the culture and the AUC was calculated for each transcript. Error bars represent ± standard error of the mean of 3 independent experiments (2 different donors). (D) The AUCs were compared as a γ/(γ + β) mRNA ratio. HU treatment increases the γ/(γ + β) ratio but the combination with Sal did not significantly change this enhancement. Error bars represent ± standard error of the mean of 3 independent experiments (2 different donors). P values were calculated using an unpaired two-tailed t test. (E) The percentage of HbF was determined by HPLC on day 20 for cells treated with AZA and Sal alone or in combination. The combination of AZA and Sal dose dependently increases the percentage of HbF. Although the combination of 200 nM AZA and 3 μM Sal did not result in a significant enhancement of HbF relative to AZA alone, the combination with 6 μM Sal was significant. Error bars represent ± standard error of the mean of 4 independent experiments using 3 unique donors. P values were calculated using an unpaired two-tailed t test relative to the 200 nM AZA treatment. (F) HPLC was performed on day 20 and the percentage of HbF was calculated for cells treated with 10 μM HU and 3 μM Sal alone or in combination. The combination of HU and Sal greatly enhances the percentage of HbF relative to HU treatment alone. Error bars represent ± standard error of the mean of 3 independent experiments using 2 different donors. P values were calculated using an unpaired two-tailed t test relative to the 10 μM HU treatment. (G) HPLC was used to evaluate the proportions of HbF and HbA at the end of the differentiation process. These representative HPLC traces show the combination of 200 nM AZA and 6 μM Sal additively increases HbF while also decreasing HbA.

Combination of salubrinal with AZA or HU significantly enhances HbF production post-transcriptionally in differentiating primary erythroid cells. (A) Erythroid progenitors were treated with 3 μM salubrinal (Sal), 6 μM Sal, 200 nM AZA, or in combination with Sal and AZA at these doses. The mRNA expression of γ- and β-globin was evaluated from days 7 through 20 of the culture and the AUC was calculated for each transcript. AZA treatment increases globin expression, whereas AZA in combination with Sal treatment reduces this enhancement. Globin gene expression still remains elevated in the combination-treated cells relative to the untreated control. Error bars represent ± standard error of the mean of 4 independent experiments (3 unique donors). (B) For all treatments, the AUCs were compared as a γ/(γ + β) mRNA ratio. AZA (200 nM) significantly enhances the γ/(γ + β) ratio, and the combination of Sal and AZA treatments did not change this induction. Error bars express ± standard error of the mean of 4 independent experiments (3 unique donors). P values were calculated using an unpaired two-tailed t test. (C) Erythroid progenitors were treated with 10 μM HU or 3 μM Sal alone or in combination. The mRNA expression of γ- and β-globin was evaluated from day 7 through day 20 of the culture and the AUC was calculated for each transcript. Error bars represent ± standard error of the mean of 3 independent experiments (2 different donors). (D) The AUCs were compared as a γ/(γ + β) mRNA ratio. HU treatment increases the γ/(γ + β) ratio but the combination with Sal did not significantly change this enhancement. Error bars represent ± standard error of the mean of 3 independent experiments (2 different donors). P values were calculated using an unpaired two-tailed t test. (E) The percentage of HbF was determined by HPLC on day 20 for cells treated with AZA and Sal alone or in combination. The combination of AZA and Sal dose dependently increases the percentage of HbF. Although the combination of 200 nM AZA and 3 μM Sal did not result in a significant enhancement of HbF relative to AZA alone, the combination with 6 μM Sal was significant. Error bars represent ± standard error of the mean of 4 independent experiments using 3 unique donors. P values were calculated using an unpaired two-tailed t test relative to the 200 nM AZA treatment. (F) HPLC was performed on day 20 and the percentage of HbF was calculated for cells treated with 10 μM HU and 3 μM Sal alone or in combination. The combination of HU and Sal greatly enhances the percentage of HbF relative to HU treatment alone. Error bars represent ± standard error of the mean of 3 independent experiments using 2 different donors. P values were calculated using an unpaired two-tailed t test relative to the 10 μM HU treatment. (G) HPLC was used to evaluate the proportions of HbF and HbA at the end of the differentiation process. These representative HPLC traces show the combination of 200 nM AZA and 6 μM Sal additively increases HbF while also decreasing HbA.

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