HEXIM1 promotes erythroid proliferation and survival. (A) Diagram of the 7SK complex and pTEFb with WT HEXIM1 (top) and HEXIM1 Y271A (bottom). Phosphorylation of WT HEXIM1 results in dissociation of HEIXM1 from the 7SK complex and release of pTEFb. The Y271A mutation prevents phosphorylation at a key residue, impairing HEXIM1 dissociation and pTEFb release. (B) HEXIM1 messenger RNA (mRNA) levels in HUDEP-2 cells transduced with EV, HEXIM1 OE, or Y271A OE. Data are presented relative to 18S ribosomal RNA. (C) HEXIM1 protein levels in HUDEP-2 cells transduced with EV, HEXIM1 OE, or Y271A OE. A representative western blot (top) and quantitation (bottom) are shown. (D) RNA immunoprecipitation assay for HEXIM1 WT and the Y271A mutant, confirming increased affinity of the Y271A mutant for the 7SK complex. (E) Live fold expansion of HEXIM1 WT, Y271A, and EV transduced cells in expansion media (left) and maturation media (right). (F) Imaging flow cytometric analyses of HEXIM1 WT, Y271A, and EV cell lines measuring cell size (left), nuclear size (middle), and CD235a expression (right). (G) Live fold expansion of HEXIM1 heterozygous cells transduced with EV, HEXIM1 WT, or Y271A in maturation media. (H) HEXIM1 messenger RNA levels in CD34⁺ hematopoietic stem and progenitor cells transduced with EV, HEXIM1 WT, or Y271A. Data are presented relative to 18S ribosomal RNA. (I) Erythroid colony-forming ability (burst-forming unit erythroid, and colony-forming unit erythroid) after transduction with EV, HEXIM1 WT, or HEXIM1 Y271A. For all experiments, n = minimum of 3 replicates; ∗P < .05; ∗∗P < .005; ∗∗∗P < .0005; ∗∗∗∗P < .00005.