FIGURE 5.

Transgenic Bcl-xL is neutralized by Noxa and inhibits bortezomib-induced apoptosis, whereas MCL1 has no impact on death sensitivity. A, SH-EP and STA-NB15 cells were retrovirally infected with vectors coding for Bcl-xL and MCL1. The ectopic expression was verified by immunoblot analysis. Cells were treated with 50 nm bortezomib for the times indicated and subjected to PI-FACS analyses. Statistical significance between mock-infected controls and Bcl-xL transgenic cells was assessed by the unpaired Student's t test (*, p < 0.05; ***, p < 0.001). B, NB15-BclxL cells were treated for 18 h with 50 nm bortezomib, lysed, and subjected to co-immunopurification using rabbit anti-MCL1 and rabbit anti-Bcl-xL as precipitating antibodies, rabbit IgG was used as negative control. Immunoprecipitates were analyzed for presence of Noxa, Bcl-xL, and MCL1 by immunoblot. C, single cell clones were isolated by limiting dilution from SH-EP-Mcl1 cells. MCL1 expression in mock-infected SH-EP-ctr, SH-EP-Mcl1 clone1, and clone10 cells was determined by immunoblot analysis. SH-EP-ctr, SH-EP-Mcl1 clone1, and clone10 cells were treated with 25 and 50 nm bortezomib and subjected to PI-FACS analysis for 48 and 72 h.