Resistance of Bax, Bak doubly deficient murine embryonic fibroblasts (MEFs) to tBID-induced apoptosis. (A) Bright-field microscopy (×20 magnification) of wild-type, Bax−/−Bak−/−, Bax−/−Bak+/+, and Bax+/+Bak−/− primary embryonic day 13.5 MEFs 24 hours after infection with a tBID-expressing vector. Murine p15 BID and BAX were cloned into the retroviral expression vector MSCV-IRES-GFP (pMIG) (11). Retroviruses were generated by transfecting 293GPG packaging cell line as described (33). Retrovirus-containing supernatant was collected 3 and 5 days after transfection and used to infect MEFs in the presence of polybrene (8 µg/ml). (B) Bright-field and GFP fluorescence of the same field (×40 magnification) of wild-type (wt) and DKO MEFs 24 hours after infection with the tBID vector. (C) Quantitation of apoptosis in MEFs shown at the 24-hour time point after infection with pMIG (empty vector control), tBID, and/or BAX vectors. Cell death was quantitated by flow cytometric detection of Annexin-V staining (Bio-vision). Values shown are mean ± 1 SD from three independent experiments. (D) Quantitation of GFP-positive cells 24 hours after infection with pMIG, tBID, and/or BAX vectors. GFP-positive cells were detected by flow cytometry. Values shown are mean ± 1 SD from three independent experiments. (E) Immunoblot with antibody to Bid of whole-cell lysates from SV40-transformed (34) wild-type and DKO MEFs at 17 hours after infection with pMIG or tBID vectors.