Overexpression of miR-92a leads to altered expression of genes involved in erythroleukemogenesis. (A) p53 protein levels in miR-92a–induced erythroleukemic spleens 1 to 5 (leukemic LSB1-5), cell lines derived from these tumors (SB1-SB5), and the miR-17-92–overexpressing cell lines CSC1 and CSC2. Levels of p53 were compared with normal spleens (Normal) and the TK6 cell line, known to express p53. (B) p53 mRNA levels were detected in SB1-SB5 and control TK6 cells by RT-PCR. (C-D) Expression of Spi-1/PU.1 (C) c-Myc, and Fli-1 (D) in erythroleukemias LSB1-LSB5 and cell lines SB1-SB5. Normal splenocytes and erythroleukemic cell lines HB60-5 and CB3 were used as controls. (E) Quantitative RT-PCR analysis of precursor miR-92a (left panel) and miR-17 (right panel) expression in pooled-populations of CSC1 cells infected with miR-92a or vector control retroviruses. Results were normalized relative to β-actin expression in all experimental samples. Each experiment was performed in quadruplicate, where error bars represent SD; *P < .05, ***P < .0005. (F) Flow cytometric analysis revealed that miR-92a overexpressing CSC1 cells contain a significantly higher number of CD71⁺ erythroid cells relative to vector alone-infected cells. (G) CSC1 cells infected with miR-92a or vector alone retroviruses were subjected to Western blot analysis using the indicated antibodies.