Exosc8-dependent barrier to erythroid maturation. (A) Real-time RT-PCR analysis of Exosc8 and selected GATA-1 target gene mRNA levels in control vs Exosc8 knockdown primary murine erythroid precursor cells cultured under expansion (–) or differentiation conditions (+) (mean ± SE; 3 independent experiments). Values were normalized to 18S rRNA expression and the expression is shown relative to control shRNA under expansion conditions. (B) Flow cytometric quantitation of erythroid developmental stage by CD71 and Ter119 staining upon Exosc8 knockdown in primary erythroid precursor cells. Representative flow cytometry data, with the R1-R5 gates denoted from 3 independent experiments. The percentage of live cells from each condition and the cell populations in R1-R5 stages (mean ± SE; 3 independent experiments). E, expansion; D, differentiation. (C) Representative images of Wright-Giemsa staining in control vs Exosc8 shRNA–infected primary erythroid precursor cells cultured in expansion or differentiation media (scale bar = 10 µm) and quantitation of cell size by measuring forward scatter using flow cytometry. (D) Flow cytometric cell-cycle analysis of primary erythroid precursor cells infected with retrovirus expressing control or Exosc8 2 shRNA. Representative cell-cycle profile is shown from 3 independent experiments. The percentage of the cell population in each cell-cycle stage is from 3 independent experiments (mean ± SE). Blue, S phase; red, G₀/G₁ or G₂/M phase. (E) Quantitative ChIP analysis of serine 5-phosphorylated RNA Polymerase II occupancy at Exosc8-regulated GATA-1 target and control genes in control and Exosc8-knockdown primary murine erythroid precursor cells (mean ± SE; 3 independent experiments). *P < .05, **P < .01, ***P < .001.