Plag1 and PLAGL2 are overexpressed in RIM-AML samples. (A) Representation of Plag1-RIM insertions (V62, Vc6, V84, V85, VC4, V61, VC2, V89). Thin arrows indicate location of retroviral insertions and direction of LTR-transcription. Triangles indicate regions of chromosomal breakpoints in pleomorphic adenomas.²²  Boxes indicate exons, including untranslated (▦) and translated (□) regions, and alternatively spliced exon 2b (▤). (B) RT-PCR analysis of Plag1 (top panel), Cbfb-MYH11 (middle panel), andActb (β-actin) (bottom panel), including whole embryo day 14.5 (E14.5), testis (te), bone marrow (bm), hematopoietic progenitor-enriched bone marrow (bmp; bone marrow 6 days after 5-fluorouracil treatment), spleen (sp), peripheral blood white blood cells (pb), Plag1-associated RIM-AML samples (V62, Vc6, V85, Vc4, Vc2, V89, and V84), Vc6 transplant (Vc6T), Plagl2-associated AML sample (V65), and 0.1 ng Plag1 plasmid control (+c). (C) Representation of Plagl2-RIM insertions (Vc1 and V65). Arrows indicate location of retroviral insertions and direction of LTR-transcription. Boxes indicate exons, including untranslated (▦) and translated (□) regions. (D) RT-PCR analysis of Plagl2 (top panel), Plagl2 transcribed from viral LTR (middle panel), and Actb (bottom panel); including hematopoietic tissues, Plag1-associated RIM-AML samples (V62, Vc6, V85, Vc4, Vc2, V89, and V84), Plagl2 RIM-AML sample V65, and a representative RIM-AML sample with no viral insertion near Plag1 or Plagl2 (V68). (E) Quantitative PCR analysis of Plagl2, using Plagl2 specific primers, in normal and leukemic samples. Expression levels were normalized to Actb and shown relative to sample E14.5 (E14.5 = 1). Error bars indicate standard errors from duplicate experiments.