Triad1 controls Gfi1 stability. (A) GFP or Gfi1-GFP was transfected with different amounts of Triad1 in HEK293 cells. At 24 hours after transfection, the Gfi1-GFP protein levels per cell were measured by flow cytometer. The mean fluorescence intensity (MFI) of transfected cells without Triad1 was set at 100%. The relative Gfi1-GFP protein levels increased when Triad1 was coexpressed, while Triad1 coexpression had no effect on the MFI of GFP transfected cells. Triad1 expression was checked in Western blot analysis (right panel). (B) HEK293 cells were transfected with 1 μg Flag-Gfi1 with or without Triad1 at indicated ratios. At 48 hours after transfection, cells were transferred to 6-well plates and allowed to adhere for 16 hours. Next, cells were treated with cycloheximide for the indicated time points. Total cell lysates were analyzed by Western blotting followed by an α-Flag staining. The Gfi1 half-life increased in a Triad1 concentration–dependent manner. The same samples were were reloaded for actin and Triad1 staining on 1 and the same blot to demonstrate equal loading and Triad1 expression. (C) U937 cells were transduced with Triad1- or Triad1dR2-encoding viruses. At 48 hours after transduction, cells were sorted based on NGFR expression using MACS. Equal amounts of NGFR⁺ cells were lysed and used in Western blot analysis to detect endogenous Gfi1 (N20 staining). To show equal loading, in the left panel, the samples were reloaded and stained for actin. The blot was reprobed with a Triad1 antibody to demonstrate Triad1 expression. In the right panel, the same blot was restained for actin to show equal loading. As a control to demonstrate retroviral Triad1 and Triad1dR2 expression, we transduced NIH3T3 cells followed by a Triad1 staining (Figure S3). (D) U937 cells were transduced with Gfi1, Triad1, Triad1dR2, or control viruses (encoding GFP and tNGFR). After transduction, cells were maintained as a mixed culture (containing transduced and nontransduced cells). At 4 days after transduction, the transduction efficiencies ranged from 6% to 67% based on either GFP positivity (Gfi1 and control vector) or NGFR positivity (Triad1 and Triad1dR2). At that time, the amount of GFP⁺ or NGFR⁺ cells was set at 100%. The relative amount of positive cells relative to this value was plotted in time. Both Gfi1⁺ and Triad1⁺ (based on GFP and NGFR positivity, respectively) cells were rapidly overgrown by nontransduced cells, while the relative amount of control and Triad1dR2⁺ cells remained stable during the culturing period. Gfi1 expression in Gfi1-transduced cells was confirmed at day 6 by Western blot analysis (Figure S4).