RNAi-induced Mcl-1 silencing is lethal in Jurkat T cells. (A) 293 cells were transiently transfected with pLL3.7 and derivatives of it for the delivery of anti-Mcl-1 shRNAs (Mcl-1-)sh1, (Mcl-1-)sh2, (Mcl-1-)sh3. Transfection efficiency was more than 90%. At 48 hours after transfection, cells were added 0.5 μM MG132 for 4 hours or left unstimulated. Subsequently, cell lysates were prepared and Mcl-1 and γ-tubulin levels were assayed by immunoblotting. (B) Bcl-2-overexpressing Jurkat and control cells underwent a double spin infection with lentivirus generated with pLL3.7 plasmid or derivatives of it for Mcl-1 silencing (mean IFU/infection was 2.5 × 10⁶ for pLL3.7 [♦], 2.8 × 10⁶ for Mcl-1-sh2 [•], 3.1 × 10⁶ for Mcl-1-sh1 [▪], and 1.3 × 10⁶ for Mcl-1-sh3 lentivirus [▴]). Cell viability was determined with propidium iodide staining and flow cytometry at the indicated time points after the second infection. Results are presented as mean of triplicates with standard deviation (SD). (C) Jurkat cells were infected with lentivirus generated with pLL3.7 plasmid or a derivative of it for Bcl-2 silencing. Cells were sorted for EGFP expression and used for cell lysate preparation. Bcl-2 and γ-tubulin levels were determined by immunoblotting. (D) Bcl-2-overexpressing Jurkat and control cells underwent a double spin infection with lentivirus generated with pLL3.7 plasmid (, ), the Bcl-2 (, ) or Mcl-1(-sh1) silencing vector (, ; mean IFU/infection was 4.8 × 10⁶ for pLL3.7, 3.4 × 10⁶ for Bcl-2, and 2.6 × 10⁶ for Mcl-1-sh1 lentivirus). Cells were harvested at the indicated time points after the second infection and stained with propidium iodide. Cell viability (, , ) as well as the rate of EGFP⁺ cells within the propidium iodide-negative population (, , ) were determined by flow cytometry. Means of triplicates with SD are shown.