Sal does not change mRNA stability, mRNA cellular localization, or total HbA to induce HbF. (A) Neither γ-globin nor β-globin relative mRNA half-life is changed in cells treated with Sal in combination with 5 μg/mL actinomycin D (Act D) versus actinomycin D alone. Expression is reported as fold change relative to the untreated control (Untx) on day 15 prior to treatment. Actinomycin D and 6 μM Sal were simultaneously applied on day 15. mRNA expression was quantified by using primers located at either the 5′ or 3′ end of each mRNA. Error bars represent ± standard error of the mean (SEM) of 3 independent experiments. (B) Sal does not change the cytoplasmic (Cyto):nuclear (Nuc) ratio of γ- or β-globin mRNA compared with the untreated control. Cells were treated with 6 μM Sal on days 15 and 18. Cytoplasmic and nuclear RNA were isolated on days 15, 18, and 20, mRNA expression for γ- and β-globin was quantified in each compartment by using primers that spanned at least 1 exon-exon junction, and the cytoplasmic mRNA:nuclear mRNA ratio was compared. Error bars represent ± SEM of 4 independent experiments. (C) Western blotting shows that short hairpin HBB (shHBB) causes 50% knockdown of β-globin protein compared with short hairpin control (shCTRL). Protein lysates were taken on days 13, 15, and 17 of differentiation after infections on days 8 and 9. glyceraldehyde-3-phosphate dehydrogenase was used as a loading control. (D) High-performance liquid chromatography (HPLC) was performed on day 20 of culture to assess the proportions of HbF, HbA, and HbA₂ in shCTRL- and shHBB-infected cells. These HPLC traces from a representative experiment reveal that shHBB enhances the percentage of HbF and percentage of HbA₂ compared with shCTRL. (E) β-globin knockdown does not increase total HbF, but it does enhance total HbA₂. After analysis by HPLC, the area under the curve for HbF, HbA, and HbA₂ was corrected for the total Hb concentration in 2 × 10⁶ cells. Total amounts of HbF, HbA, and HbA₂ are depicted as fold change relative to the shCTRL. Error bars represent ± SEM of 3 independent experiments. P values were determined by using an unpaired 2-tailed Student t test. NS, not significant. *P < .05; **P < .001.