Evidence that the exosome complex creates a barrier to erythroid maturation. (A) Crystal structure of the human exosome complex26 demonstrating the interaction between Exosc8 and Exosc9. (B) Real-time RT-PCR analysis of Exosc8 and Exosc9 mRNA levels in control vs Exosc8 or Exosc9 knockdown in expanding primary murine erythroid precursor cells sorted into distinct, R1, R2, R3, and R4/5 cell populations (mean ± SE; 5 independent experiments). Values were normalized to 18S rRNA level and the expression relative to the control R1 shRNA condition. (C) Semiquantitative western blot analysis of Exosc9 protein in expanding primary murine erythroid precursor cells 24 hours postinfection with Exosc8- or Exosc9-specific shRNA retroviruses. Representative image from 3 independent experiments. (D) Real-time RT-PCR analysis of selected GATA-1 target genes and erythroid transcription factors mRNA levels in control vs Exosc8 or Exosc9 knockdown in expanding primary murine erythroid precursor cells sorted into distinct, R1, R2, R3, and R4/5 cell populations (mean ± SE; 5 independent experiments). Values were normalized to 18S rRNA level and the expression relative to the control R1 shRNA condition. (E) Flow cytometric quantitation of erythroid maturation stage by CD71 and Ter119 staining upon Exosc8 or Exosc9 knockdown in primary erythroid precursor cells. Representative flow cytometry data, with the R1-R5 gates denoted from 4 independent experiments. The percentage of the cell populations in R1-R5 stages (mean ± SE; 4 independent experiments). (F) Representative images of Wright-Giemsa–stained cells infected with control vs Exosc8 or Exosc9 shRNA-expressing virus. Cells were cultured under expansion conditions (scale bar = 10 µm). (G) Cell number fold change during the 72-hour expansion of primary murine erythroid precursor cells postinfection with control, Exosc8, or Exosc9 shRNA-expressing retroviruses (mean ± SE; 8 independent experiments). (H) Flow cytometric quantitation of erythroid maturation stage by CD71 and Ter119 staining upon Exosc8 or Dis3 knockdown in primary erythroid precursor cells. Representative flow cytometry data, with the R1-R5 gates denoted. The percentage of the cell populations in R1-R5 stages (mean ± SE; 6 independent experiments. *P < .05, **P < .01, ***P < .001.
