Constitutive activation of Notch1 in ZMYM2-FGFR1 lymphomas is ligand independent. (A) Western blot analysis showing that the Notch ligands Jag1, Jag2, DLL1, and DLL4 were either not changed or decreased in lymphoma (Lym) samples compared with normal thymuses (Nor. Thy) and lymph nodes (Nor. LN). (B) When ZNF112 cells were labeled with PKH26 (see “Cell cultures and proliferation assay”) and cultured on DLL1 or GFP retrovirally transduced murine OP9 stromal cells (OP9-DLL1 or OP9-GFP, respectively), similar growth patterns (left) were seen, as were levels of the activated form of Notch1, as demonstrated by flow cytometry and Western blotting. (C) Mutations were not detected in the Notch1 heterodimerization (HD) domain (n = 10). A single base insertion (arrow) and a single base substitution (asterisk) were identified near the PEST domain of Notch1 in ZMYM2-FGFR1 lymphomas, which would produce a frameshift with the generation of a predicted downstream stop codon at codon 2490. (D) Notch1 gene schematic showing the relative locations of the primers used to analyze the deletion mutants (top). A 5′ deletion (brackets) creates a 500-bp fragment using the P1/P2 primers. In normal cells, the wild-type ∼11.5-kb fragment cannot be amplified (bottom). (E) RT-PCR showed that the alternative Notch1 transcript (exon 1a-exon 4) was only found in tumor samples (top), whereas real-time RT-PCR showed that Notch1 transcription levels for exons 30-31 was higher than that for exons 1-3 (bottom) in the majority of lymphomas compared with normal cells.