Ectopic expression of Gfi1b inhibits proliferation and survival of T-cell lymphoma. (A) Schematic of Gfi1b, ΔSNAG, and N290S. (B) EMSA using in vitro-translated Gfi1b, ΔSNAG, and N290S and labeled oligos containing consensus Gfi1/1b-binding sequence (B30). Anti-Gfi1b antibody was used to confirm the presence of Gfi1b proteins. The complex shifted by Gfi1b protein and supershifted with anti-Gfi1b antibody is shown. (C) Western blot analysis of 0531 cell extracts 24 hours after infection with the indicated virus. The blot was probed with anti-Gfi1b antibody. Two Gfi1b isoforms are detected in MEL cells. (D) Time course of GFP expression in 0531 cells infected with MigR1(♦), MigR1-Gfi1b (▪), MigR1-ΔSNAG (▴), or MigR1-N290S (×) as determined by flow cytometry. Identical results were obtained with 2 other E2A^−/− lymphoma lines. (E) Average relative percent GFP⁺ cells 5 days after infection combining data from 3 experiments. Relative percent GFP⁺ was determined by dividing the percent GFP⁺ on day 5 by the percent GFP⁺ on day 1 (× 100). The decrease in GFP⁺ cells in Gfi1b virus-infected cultures is greater than in all other experimental groups (P < .05, paired Student t test). (F) 0531 cells were infected with MigR1 or MigR1-Gfi1b retrovirus and BrDU was added to cultures for 30 minutes 35 or 54 hours after infection. The GFP⁺ cells were isolated and stained with anti–BrDU-FITC and propidium iodide and examined by flow cytometry.³¹  G₁ = 2N DNA, BrDU⁻; S = BrDU⁺; G₂+M = 4N DNA, BrDU⁻; apoptotic cells = < 2N DNA. (G) 0531 cells infected with MigR1 or MigR1-Gfi1b retrovirus were stained 48 hours after infection with an antibody that detects active caspase-3. Caspase-3 staining on GFP⁺ cells is shown. Similar results were observed with 1.F9 cells.