Interfering with the SUMOylation E1 ligase components SAE1 or SAE2 impairs proliferation and provokes G2/M arrest and polyploidy of Myc-induced lymphoma. (A) Human Raji BL cells carrying the ecotropic receptor were infected with retrovirus that expressed shRNAs directed against SAE1 or SAE2 and that also expressed a GFP reporter gene. Following GFP sorting for infected cells by flow cytometry, qPCR analyses (left) of the indicated transcripts and immunoblotting (right) of the indicated proteins were performed. (B) GFP-positive Raji cells from panel A were assessed for SUMO1 expression by immunoblotting. (C) Raji cells from panel A were followed for GFP expression in a competitive repopulation assay at the indicated time points (n = 3). (D) Eµ-Myc lymphoma cells were transduced with lentivirus expressing shRNAs targeting murine Sae1 and Sae2 or a scramble control shRNA. Cells were then followed for GFP positivity in a competitive repopulation assay by flow cytometry at the indicated time points (n = 3). (E) Colony-forming unit assay of GFP-sorted Eµ-Myc cells after knockdown of Sae2. A total of 2 × 10⁴ cells were plated and colony number determined at day 10. (F-I) Human Raji BL cells engineered to express the ecotropic receptor and rtTA were transduced with retroviruses expressing Dox-inducible shRNAs targeting human SAE1 and SAE2 and the dsRed reporter gene, as well as a constitutively expressed GFP reporter. GFP-sorted cells were incubated for the indicated intervals with 1 µg/mL Dox. (F) Flow cytometric cell-cycle analysis measuring DAPI uptake (n = 3). (G) Quantification of polyploid cells (n = 3). (H) Quantification of cells in G2/M. (I) Forward scatter low/side scatter low as a measure of pycnotic cell death is shown (n = 3).