Legends to supplementary figures
Supplementary Figure 1 Bax potential trypsin cleavage sites. Amino acid sequence (a)
and 3D structure (b) of human Bax. Its 9 -helices are underlined, potential trypsin
cleavage sites are depicted in red, and probable accessible sites in oligomerized Bax are
marked with an arrow (R34 or R37). 2D2 (amino acids 3-16) and polyclonal (amino acids
43-61) anti-Bax antibodies’ epitopes are respectively outlined in yellow and in green. The
3D structure of Bax was produced using the UCSF Chimera package from the Resource
for Biocomputing, Visualization, and Informatics at the University of California, San
Francisco (supported by NIH P41 RR-01081).1
Supplementary Figure 2 Tr-Bax formation requires tBid and is inhibited by Bcl-xL.
PC/CL 60/40 liposomes were incubated with recombinant Bax (100 nM) and various
concentrations of tBid (0, 1, 5, 10 and 100 nM) (a) or with tBidmIII-2 (10 nM) (b).
Binding of Bax and of tBid to the liposomes was assessed by immunoblotting (- trypsin)
as was the degree of Tr-Bax formation (+ trypsin). (c) Similarly, PC/CL liposomes were
incubated with recombinant Bax (100 nM) and tBid (10 nM) and various concentrations
of Bcl-xL (0, 10, 25 and 50 nM), and Bax and tBid binding and Bax oligomerization were
assayed. Blots show duplicates and are representative of three independent experiments.
Supplementary Figure 3 Tr-Bax can be completely degraded by trypsin. tBid-induced
Bax oligomerization assays were performed in vitro with PC/CL 60/40 liposomes.
Samples were digested with (a) various trypsin concentrations (from 0.0017 to 17 mg/ml)
or (b) various incubation times (from 30 to 240 min) and analyzed by immunoblotting.
Results are representative of three independent experiments.
Supplementary Figure 4 Only oligomeric Bax is resistant to trypsin digestion. In vitro
Bax activation assays with 100 nM Bax and 10 nM tBid were performed with PC/CL
60/40 liposomes. After the ultracentrifugation, Bax was extracted from the liposomes
with CHAPS and run on a size exclusion chromatography column (a). Individual
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fractions were then digested with trypsin (b). Alternatively, after the ultracentrifugation,
samples were digested with trypsin and then loaded on the column (c). All samples were
analyzed by immunoblotting.
Supplementary Figure 5 CHAPS-solubilized oligomerized Bax is also partially resistant
to trypsin digestion. Bax (100 nM) was activated by tBid (10 nM) using PC/CL 60/40
liposomes, extracted from the liposomes with CHAPS (2%) and incubated with trypsin
(0.17 mg/ml) for 1 or 2 hours. Tr-Bax formation was analyzed by immunoblotting and
showed digestion of Bax to a slightly smaller trypsin-resistant Bax fragment (*), probably
linked to a partial unfolding of the protein in the presence of detergents. Result is
representative of three independent experiments.
Supplementary Figure 6 Cholesterol inhibits Bax oligomerization. tBid-induced Bax
activation was examined in vitro with PC/CL/CHO liposomes with a low CL content
(20% w/w). The binding of tBid and Bax to the liposomes, as well as the resistance of
Bax to trypsin digestion were examined by immunoblotting. Result is representative of
three independent experiments.
Supplementary Figure 7 Crude mitochondria isolated from untreated and U18666Atreated cells show a similar contamination by other intracellular organelles. (a) 48h after
incubation with U18666A, mitochondria were isolated from HeLa cells and analyzed by
immunoblotting for contaminations with endosomes (Rab5), TGN (GS28), lysosomes
(Lamp1), ER (Calnexin) and peroxysomes (SKL and Catalase). Blots are representative
of more than three independent experiments. (b) For each experiment, band intensities
were quantified and normalized by the intensity of the SLP-2 band, and Control to
U18666A ratios were calculated. Values are means of more than three independent
experiments +/- SEM. Using a paired Student’s t-test, differences were not significant.
Supplementary Figure 8 U18666A added to HeLa mitochondria does not delay tBidinduced MOM permeabilisation. Mitochondria were isolated from HeLa cells and
incubated with various concentrations of U18666A for 10 minutes. tBid (20 nM) was
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then added, a 10 minute incubation was carried out at room temperature, and the samples
were spun. Smac and Cytochrome c release were assayed in the supernatants by
immunoblotting. The blot is representative of three independent experiments.
Supplementary Figure 9 U18666A-treatment does not increase the resistance of cells to
mitochondria-independent apoptotic stimuli. 293T cells were incubated with U186666A
for 36 hours and incubated with FasL in presence of cycloheximide. Cell death was
analyzed after 28 hours by Annexin-V and PI staining. The percentage of unstained cells
is displayed. Values represent means of more than six independent experiments +/- SEM.
**: p < 0.009.
References
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Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC et
al. UCSF Chimera--a visualization system for exploratory research and analysis. J
Comput Chem 2004; 25: 1605-12
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Supplementary Figure 1
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Supplementary Figure 2
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Supplementary Figure 3
Supplementary Figure 4
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Supplementary Figure 5
Supplementary Figure 6
Supplementary Figure 7
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Supplementary Figure 8
100
% live cells
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60
40
20
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Supplementary Figure 9
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