Nonspecific, genetic strain-dependent inhibition of erythroblast enucleation by Trim58 shRNAs. (A) Immature lineage (lin⁻) erythroid precursors from E14.5 fetal livers (CD-1 strain) were purified by immunomagnetic bead selection; expanded for 2 days in medium containing dexamethasone, stem cell factor, and erythropoietin; transduced with retroviral vectors encoding Trim58 shRNAs; then induced to undergo terminal maturation by culture in erythropoietin alone. Reticulocytes generated after 36 hours maturation were quantified as described in Figure 1C. Data show mean ± SD from three biological replicate experiments. *P < .05, 1-way analysis of variance (ANOVA). (B) Trim58 mRNA levels determined by qRT-PCR of shRNA vector-transduced CD-1 erythroblasts described in panel A after 36 hours maturation. Data are normalized to Actb mRNA and are shown as mean ± SD for three biological replicate experiments. *P < .05 by 1-way ANOVA. (C) Reticulocytes generated after 36 hours maturation of mixed strain Trim58^+/+ and Trim58^−/− erythroblasts after transduction with 2 different Trim58 shRNAs, as described in panel A. Data show mean ± SD from three biological replicate experiments, with nonsignificance (n.s.) determined by 1-way ANOVA. (D) Trim58 mRNA levels determined by qRT-PCR of mixed strain erythroblasts described in panel C after 36 hours maturation. Data are normalized as in panel B and show mean ± SD for three biological replicate experiments. *P < .05 by 1-way ANOVA. (E) Percent reticulocytes generated after 36 hours maturation from C57Bl/6 Trim58^+/+ and Trim58^−/− erythroblasts after transduction with 2 different Trim58 shRNAs. Data are presented as mean ± SD for three biological replicate experiments. *P < .05 by 1-way ANOVA. (F) Trim58 mRNA levels determined by qRT-PCR of C57Bl/6 erythroblasts described in panel E after 36 hours maturation. Data are normalized as in panel B and are presented as mean ± SD for three biological replicate experiments. *P < .05 by 1-way ANOVA.