Fig. 6. Induced expression of hBIMs was not sufficient to death of untreated cells, but did restore sensitivity of Bax^−/−Bak1^−/−Bim^−/− WEHI7 cells to treatment with dexamethasone.

a Bax^−/−Bak1^−/−Bim^−/− WEHI7 cells were transduced with a lentiviral construct expressing human BIMs from a doxycycline-inducible promoter. Five independent ihBIM Bax^−/−Bak1^−/−Bim^−/− WEHI7 clonal cell lines were cultured for 3 days in the presence or absence of 1 µg/ml doxycycline (Dox). Bim expression was monitored by immunoblotting, using ACTIN as loading control. b Bax^−/−Bak1^−/−Bim^−/− parental cells (gray bars) and five independent Bax^−/−Bak1^−/−Bim^−/− inducible hBIMs (ihBIM) WEHI7 cell clones (white bars) were cultured for 6 days in the presence or absence of 1 µg/ml Dox and/or 1 µM Dex. Cell death was assessed by PI uptake using flow cytometry. For cell death to occur, both treatment with Dex, as well as induction of BIMs, were necessary. c Dot plots show one of the five independent clones as shown in b, numbers represent the percent of PI-negative cells of a total of 10,000 cells analyzed per condition. d Bax^−/−Bak1^−/−Bim^−/− WEHI7 cells and those from ihBIM Bax^−/−Bak1^−/−Bim^−/− WEHI7 lines were cultured for 6 days in the presence or absence of 1 µg/ml Dox and/or 1 µM Dex. Cytoplasmic extracts were subjected to western blot analysis, with antibodies specific for CYTC and ACTIN. For CYTC to be released into the cytosol, both treatment with Dex, as well as induction of BIMs, were necessary.