Fig. 1: BH3-only activators induce BAK-dependent autophagy.

a The indicated strains of MEFs were transduced with retroviruses expressing the shown BH3-only molecules and treated with 25 μM zVAD.fmk 7 h later. Cells were lysed 22 h post-transduction for Western blot against the indicated molecules. Expression levels of HA tagged tBID, BIM and PUMA were measured in DKO cells to minimise the possible impact of cell death (right panel). b MEFs expressing GFP-LC3 were retrovirally transduced as in (a) and fixed 22 h later for microscopy. Shown are representative confocal pictures (left panel; green channel: GFP-LC3, blue channel: DAPI; scale bars represent 10 μm), and quantification of the number of GFP-LC3 puncta per cell (right panel, top) and the percentage of cells showing induced GFP-LC3 (right panel, bottom). Graph bars indicate control vector (pink), tBID (red), BIM (blue) or PUMA (green) retroviral transduction. Graphs represent mean values −/+ s.d. of triplicate scoring points (n = 3 microscopy fields, each including at least 25 cells per field; **P < 0.01, ***P < 0.001, ****P < 0.0001 two-tailed Student’s t-test). Numeric P-values are shown. c Bax-/- MEFs were retrovirally transduced as in (a) and treated with E64d and pepstatin (10 μg/ml each) for the last 16 h of culture. Cells were lysed 22 h post-transduction for Western blot using the indicated antibodies. d BAK expression in reconstituted DKO MEFs. The indicated MEF strains along with DKO MEFs retrovirally transduced with WT or L78E human BAK versions and subsequently selected in puromycin were lysed for Western blotting. e The reconstituted MEFs shown in (d) were retrovirally transduced as in (a) and lysed 22 h later for Western blot against the shown molecules. Source data are provided as a Source Data file.