Ub ligase activity of Triad1 inhibits clonogenic growth of primary murine bone marrow cells. (A) Empty vector (EV) or Triad1 retrovirally transduced murine bone marrow cells with increasing expression (Triad1 I, II, and III, respectively) were used in CFU-GM, revealing a Triad1 concentration-dependent inhibition of clonogenic growth (n = 3). Triad1 staining using lysates from 100 000 transduced bone marrow cells revealed no detectable levels in EV-transduced cells and increasing expression in cells with increasing GFP positivity (fractions II and III, respectively). NC indicates negative control. (B) Morphologic analysis of cells grown in CFU-GM shows a modest increase in the percentage of granulocytes and decrease in erythroblasts on Triad1 expression. (C) Triad1 RING mutants H158A and C161A do not inhibit clonogenic growth, showing that the N-terminal RING finger is essential for the clonogenic inhibition exerted by wild-type Triad1 (n = 2). Comparable expression of wild-type Triad1 and mutants was shown by Western blotting using lysates from 100 000 transduced bone marrow cell (equal loading was checked by Ponceau staining, not shown). (D) Treatment of murine bone marrow cells grown in liquid medium with MG132 indicates maximal tolerated dose with limited toxicity of 1 × 10^-8 M. Cells (5000) were seeded in 96 wells and cultured for 3 days. Relative cell numbers were determined by flow cytometry using fluorescent beads (Beckman Coulter). (E) Treatment of primary murine bone marrow cells with 1 × 10^-8 M MG132 for 2 days resulted in an increase in ubiquitinated proteins (x-(ub)_n). (F) Proteasome activity measurements (indicated as fluorescence units [FUs]) on lysates taken from primary murine bone marrow cells that were treated with 1 × 10^-8 M MG132 for 1 or 2 days showed clear proteasomal inhibition. (G) Proteasome inhibition (MG132) relieves the suppressive effect of Triad1 in CFU-GM (n = 3). MG132 was added to cells prior to cell seeding. For all tested conditions observed numbers of colonies were indicated relative to number of empty vector (EV) obtained colonies (set at 100). (H) Treatment of Triad1-transduced cells with independent proteasome inhibitors counteracted the suppressive effects of Triad1 on colony formation. The number of obtained colonies of mock-treated Triad1 transduced cells was set at 100, and relative colony numbers obtained for cells treated with indicated proteasome inhibitors are indicated (1 × 10^-8 M MG132 [n = 3], 1 × 10^-9 M PS341 [n = 2], 5 × 10^-10 M epoxomycin [epox; n = 1], and 1 × 10^-8 M lactacystin [lacta; n = 1]). Error bars indicate standard deviations.