HEXIM1 OE phenotype is dependent on pTEFb activity. (A-B) Growth curves of HEXIM1 OE and EV cell lines and live fold amplification on day 7 in (A) expansion and (B) maturation media with and without NVP2 treatment. (C) Cytospins on maturation day 10 in EV, HEXIM1 OE, and HEXIM1 OE plus NVP2. (D) γ-globin expression in HEXIM1 OE via quantitative polymerase chain reaction with and without NVP2 treatment. Data are presented relative to 18S ribosomal RNA. (E) Levels of serine-2 (Ser2) RNAPII and (hemoglobin subunit gamma (HBG) in HEXIM1 OE cells with and without NVP2 treatment. (F) HbF-expressing HUDEP-2 cells at maturation day 5 in the indicated cell lines with and without NVP2 treatment via FACS analysis. (G) Quantification of F-cell percentage in the indicated cell lines with and without NVP2 treatment. (H) Distribution of HbF expression level in the indicated cell lines with and without NVP2 treatment. (I) Quantification of the median HbF expression level in the HbF⁺ population in the indicated cell lines with and without NVP2 treatment. (J) Proportion of green fluorescent protein–positive (GFP⁺) HEXIM1 OE cells after 2 days of treatment with either NVP2 or dimethyl sulfoxide (DMSO). The number of GFP⁺ HEXIM1 OE cells was identical before treatment. (K) Proportion of F-cells in GFP+ HEXIM1 OE primary erythroblasts treated with NVP2 or DMSO. (L) Distribution of HbF expression level in the indicated cell lines with and without NVP2 treatment. (M) Median HbF expression levels in GFP⁺ EV or HEXIM1 OE primary erythroblasts with and without NVP2 treatment. n = minimum of 3 replicates. ∗P < .05; ∗∗P < .005; ∗∗∗P < .0005.