copper and 20 mM N-ethylmaleimide to block unreacted cysteine residues, and the samples analyzed by non-reducing SDS-PAGE. Isolation of mouse liver mitochondria Mouse liver mitochondria (MLM) were isolated from approx- imately 20-week old C57Bl-6 wild type or bak2/2 mice as previously described [41]. MLM were resuspended at 0.5 mg/ml in MEF cytosolic extracts and incubated with or without caspase- 8-cleaved Bid (tBid) for 60 min at 37uC. Mitochondrial outer membrane permeabilization by tBid Permeabilized cells, or combinations of membrane and cytosol fractions generated as above, were incubated with tBid for 30 or 60 min at 30uC. Where indicated, cytosol derived from MEFs was incubated with MLM and incubated with tBid as above. Supernatant and membrane fractions were separated by centri- fugation (13,000 g for 5 min) and fractions transferred to nitrocellulose for immunoblotting for cytochrome c, or to PVDF for immunoblotting for Bak. IASD labelling Cytosol and membrane fractions were separated and treated with 1 mM IASD (4-acetamido-49-((iodoacetyl) amino) stilbene-2,29- disulfonic acid, Invitrogen) in the presence of 0.1 mM TCEP (Tris(2-Carboxyethyl)phosphine; Pierce) for 30 mins at room temperature. Labelling was quenched with 1 mM dithiothreitol and labelling assessed by SDS-PAGE and immunoblotting for Bak. Preferential Bak activation by Noxa overexpression bak2/2bax2/2 MEFs expressing Bak, Bax, Bak/BaxCS or Bax S184L were retrovirally infected with BimS or BimS containing the BH3 domain from Bad or Noxa (provided by D.C.S. Huang, and described in [46]). After 36 hours, cell death was assessed by propidium iodide uptake. Percentage cell death was calculated by normalizing against the rate of infection (GFP-positive cells) of the three BimS constructs for bak2/2bax2/2 cells. Supporting Information Figure S1 Mitochondrial cytochrome c release mediat- ed by Bak variants. bak2/2bax2/2 MEFs expressing the indicated Bak variants were left untreated (black line) or treated with UV (red lines) as in Figures 1B, 2B and 3B and examined for mitochondrial cytochrome c (Cyt c) content by flow cytometry (Waterhouse and Trapani, Cell Death Differ 2003 Jul; 10 (7): 853– 855) using anti-cytochrome c antibody (6H2.B4; Pharmingen) and R-phycoerythrin-conjugated secondary antibody (1031-09; South- ern Biotech, AL, USA). Untreated cells were incubated with secondary antibody only (hatched). Cells with low mitochondrial cytochrome c content lie to the left of the marker line. Results are representative of two independent experiments. (TIF) Figure S2 Cell death mediated by Bak/BaxCS is apo- ptotic. (A) Cell death mediated by Bak/BaxCS is blocked by caspase inhibition. bak2/2bax2/2 MEFs expressing either wild-type Bak or Bak/BaxCS were treated or not with etoposide (10 mM) in the presence or absence of caspase inhibitor Q-VD.oph (50 mM) for 24 h prior to assessment of cell death by propidium iodide uptake. Results are expressed as mean +/2 SEM of three independent experiments. (B) Cell death mediated by Bak/BaxCS involves cytochrome c release. bak2/2bax2/2 expressing Bak/BaxCS treated as in (A) were harvested, and cytosolic (Cyt) and membrane (Memb) fractions immunoblotted for cytochrome c, or for VDAC1 (31HL; Mutagenesis of the Bak Membrane Anchor Calbiochem) as a mitochondrial membrane marker. Results are representative of two independent experiments. (TIF) Figure S3 Bak/BaxCS oligomerizes predominantly in the membrane fraction during apoptosis. bak2/2bax2/2 MEFs expressing Bak/BaxCS were treated with actinomycin D (1 mM) in the presence of caspase-inhibitor Q-VD.oph (50 mM) for 24 h. Cells were fractionated into cytosol (Cyt) and membrane (Memb) fractions prior to addition of oxidant (CuPhe) to induce disulphide bonds. Samples were electrophoresed under non- reducing (upper) or reducing (bottom) conditions prior to immuno- blotting for Bak and for HSP70 as a cytosolic marker. MX, non- activated intramolecular cross-linked monomer; M, non-cross- linked monomer; D, intermolecular crosslinked dimers. Results are representative of two independent experiments. (TIF) Figure S4 A variant of Bak/BaxCS containing an extra tryptophan residue also retains full pro-apoptotic func- tion. (A) C-terminal sequence of Bak and Bax variants, in particular demonstrating the two Bak/BaxCS variants containing either four (KKMG) or five (WKKMG) residues from Bax. (B) Both Bak/ BaxCS variants retain proapoptotic function. bak2/2bax2/2 MEFs expressing Bak, Bak/BaxCS or Bak/BaxCSb were left untreated or treated with increasing doses of etoposide. Percentage cell death is expressed as the mean 6 SEM from three independent experi- ments. Statistical significance for the 10 mM dose when compared to Bak is shown; *p,0.05. Panels on the right are cell lysates immunoblotted for Bak, and for HSP70 as a loading control. (TIF) Figure S5 Bak/BaxCS redistributes to mitochondria during apoptosis. (A) FLAG-tagged Bak/BaxCS is predomi- nantly cytosolic. Cytosol (Cyt) and membrane (Memb) fractions were generated from bak2/2bax2/2 MEFs stably expressing FLAG-Bak or FLAG-Bak/BaxCS and immunoblotted for FLAG. * is a non-specific band. (B) FLAG-Bak/BaxCS translocates to mitochondria following etoposide treatment. FLAG-Bak or FLAG-Bak/BaxCS expressing cells were left untreated or treated with etoposide (10 mM) in the presence of Q-VD.oph (50 mM) for 24 h, and fixed in 4% paraformaldehyde for 20 mins at room temperature. Following staining with an anti-FLAG primary antibody (M2; Sigma) and FITC-conjugated secondary antibody (Southern Biotech, AL, USA), immunofluorescence was detected by confocal microscopy (Leica SP2). All images were obtained using the same parameter settings. Bar represents 20 mm. Note that staining with secondary antibody alone gave essentially no signal (not shown). Images were from a single experiment, representative of two experiments. (C) FLAG-Bak/BaxCS colo- calizes with mitochondria. Cells were treated as in (B), except that cells were incubated with with MitoTracker Deep Red FM (0.5 mM; Invitrogen, CA) for 30 mins at 37uC prior to fixation and immunostaining. Bar represents 20 mm. Images were from a single experiment, representative of two experiments. (TIF) Figure S6 Cysteine variants of Bax and of Bak/BaxCS retain proapoptotic function. bak2/2bax2/2 MEFs expressing the indicated cysteine variants were left untreated or treated with etoposide. Percentage cell death is expressed as the mean 6 SEM from three independent experiments. Statistical significance for the 10 mM dose when compared to wild-type (wt) protein is shown; *p,0.05. Panels on the right are cell lysates immunoblotted for Bax or Bak, and re-blotted for HSP70 as a loading control. (TIF) PLoS ONE | www.plosone.org 11 March 2012 | Volume 7 | Issue 3 | e31510