Figure 5.

HEXIM1 disruption impairs erythroid cell proliferation and viability. (A) Schematic demonstrates the genetic mutations in the alleles of HEXIM1 for the 3 clonal KITCAT cell lines. (B) Western blot showing HEXIM1 protein levels in KITKAT HEXIM1 ± and KITCAT control lines (KC; i). Western blot showing HEXIM1 protein levels in HUDEP2 cells expressing shRNA targeting HEXIM1 (ii). For both blots, H4 is used as a loading control. (C) HEXIM1 and 7SK levels in KITKAT HEXIM1 ± lines and HUDEP2 cell lines expressing pooled shRNA targeting HEXIM1 or luciferase control. (D) Live fold expansion of clonal HEXIM1 ± mutant lines (i) and shRNA HEXIM1 lines (ii) in expansion conditions. (E) Live fold expansion of HEXIM1 ± mutant lines in maturation conditions. (F) Viability of clonal HEXIM1 ± mutant lines in maturation conditions. (G) Live fold expansion of HUDEP2 lines expressing shRNA targeting HEXIM1 in maturation conditions. (H) Viability of HUDEP2 lines expressing shRNA targeting HEXIM1 in maturation conditions. (I) Imaging flow cytometric quantification of cell size (i) and representative cytospins (ii). (J) Live fold expansion of cells expressing indicated mCherry-HEXIM1 shRNA or luciferase control. Cells were transduced on day 3 (D3) following CD36 selection in the CD34⁺ erythroid culture system shown in Figure 1A. Data are presented as % of mCherry positive cells relative to day 3 after infection (D3PI). Error bars for all figures represent standard error of the mean of 3 independent cultures. *P < .05 compared with control.