Effect of proteasome inhibition on HSPC transduction by LVs. (A,C) Percentage of GFP-positive cells and (B,D) mean fluorescence intensity (MFI) of CD34⁺ cells transduced with 10⁸ TU/mL LV-GFP with (▴) or without (▪) cytokine stimulation in the presence of increasing doses of the proteasome inhibitors MG132 (A-B) and PS-341 (C-D) and analyzed 2 weeks after transduction. Each panel represents the average of 2 experiments. (E) Percentages of GFP-positive cells in suspension culture (left panel) and GFP-positive colonies from CFC assays (right panel) from CD34⁺ cells transduced with or without cytokine (Cyt) stimulation with increasing doses of LV-GFP (TU/mL) and in the absence or presence of 1μM MG132 (MG), as indicated, and analyzed 2 weeks after. Toxicity was calculated from the ratio between the number of colonies arising from MG132-exposed and not exposed cells and shown as percentage of CFCs killed. Average (—) and individual values (▴, ▪) from 2 experiments. Statistical analysis is reported in “Results.” (F) Quantitative real-time PCR analysis of DNA extracted after 3 weeks of suspension culture from HSPCs stimulated or not with cytokines (Cyt), transduced with different doses of LV-GFP (TU/mL), and treated or not with different doses of PS-341 (PS) or MG132 (MG), as indicated. Vector sequences were amplified with a system annealing to the ψ of the vector (PSI system) and a system annealing to the GFP sequence (GFP system). For each condition, an average of 3 detections is shown.