Variation of HbF and HbS levels in CD34⁺-derived SCD erythroid cells treated with pCL-ZF-Ldb1 LV or HbF inducers in vitro. (A) Schematic representation of the experimental protocol of human SCD CD34⁺ cells treated with the inducer drugs or pCL-ZF-Ldb1. (B) Erythroid cell number (by benzidine staining; N = 2) using the indicated doses of drugs and compared with pCL-ZF-Ldb1 (with VCN ∼1 copy per cell). Black arrows indicate the dose of each drug chosen for the subsequent experiments. (C) Net increase of HbF percentage and (D) net decrease of HbS percentage in SCD erythroblasts (N = 7, 2 of which were replicated once or twice, respectively) treated with the HbF inducers or pCL-ZF-Ldb1 (***P < .005). (E) Erythroid cell count for each treatment normalized to count of untreated samples (*P = .05, **P < .05). (F) Morphological assessment of untreated (ctrl, left) vs pCL-ZF-Ldb1–treated cells (right) in hypoxic conditions. (G) Proportion of sickled cells/total cell count and (H) relative means and SD, measured on 3 replicas on 3 sets of samples treated with the various inducers or pCL-ZF-Ldb1 (**P < .05, ***P < .005) and exposed to hypoxic conditions. P values under and above bars refer to differences between treated and untreated samples and between treated samples, respectively. 5-AZA, 5-aza-2′-deoxy-cytidine; But, butyrate; ND, not determined; PBMC, peripheral blood mononuclear cell; Pom/Pomalid., pomalidomide; SD, standard deviation; TCP, tranyl-cypromine.