Supplementary Information
Materials and Methods
Cell culture
L929sAhFas cells are L929sA cells transfected with cDNA of the human Fas receptor
as described previously 1. The cells were cultured in Dulbecco’s modified Eagle’s medium
(Gibco, Paisly, Scotland) supplemented with 10% fetal calf serum (Greiner, Poitiers, France),
100 IU/ml penicillin (Gibco BRL, Paisley, Scotland), 0.1 mg/ml streptomycin (Gibco BRL)
and 0.03% L-glutamine (Gibco BRL).
Cytokines and reagents
Recombinant murine TNF (6x107 IU/ml) and recombinant murine IFN-β (3x106
IU/ml) were produced in E. coli and purified in our laboratory. LiCl (ICN Biomedicals Inc,
Ohio, USA) was dissolved at 1 M in medium. Polyinosinic-polycytidylic acid (poly(I)poly(C)), a synthetic dsRNA (Amersham Pharmacia Biotech, Rainham, UK), was dissolved
at 3.5 mg/ml in water. Propidium iodide (PI; Becton Dickinson, Sunnyvale, CA, USA) was
dissolved at 3 mM in PBS. Dihydrorhodamine 123 (DHR123; Molecular Probes, Eugene,
OR, USA) was dissolved at 1 mM in DMSO. NADH (Boehringer Mannheim) was dissolved
at 1 mM in water.
The following products were purchased from Sigma Chemical Co. (St. Louis, MO)
and dissolved as indicated: BHA and BHT (100 mM in ethanol), acetylCoA (3 mM in water),
bovine serum albumin (BSA, 1% in water), malonate (0.5 M in water), KCN (30 mM in
water), rotenone (5 mM in ethanol), antimycine A (0.1 mM in DMSO/water 1:2 v/v), horse
cytochrome c (50 mg/ml in 10 mM potassium phosphate buffer, PPB, pH 7.0), 2,6dichloroindophenol sodium salt hydrate (DCPIP, 1 mM in 10 mM PPB, pH 7.5), sodium
succinate (200 mM in 100 mM PPB, pH 7.5), 5,5’-dithiobis (2-nitrobenzoic acid) (DTNB, 1
mM in 0.1 M Tris-HCl, pH 8.1), oxaloacetic acid (10 mM in 0.1 M Tris-HCl, pH 8.1),
decylubiquinone (65 mM in ethanol), AA861 (50 mM in ethanol) and BEL (80 mM in
DMSO). MAFP (Biomol) was dissolved at 50 mM in ethanol and NDGA (Calbiochem) at
100 mM in ethanol. NaBH4 was purchased from Aldrich and heparin from B. Braun
Melsungen AG (50 U/ml stock).
Induction of necrosis for fluorescence-activated cell sorter (FACS) analysis
L929sAhFas cells were seeded at 2x105 cells per well (uncoated 24-well tissue culture
plates, Sarstedt Inc., Newton, NC) and incubated overnight in suspension. Following 24 h
culture at 37°C, cells either received no treatment or were treated with murine IFN-β (200
IU/ml) 2. After another 24 h, the cells were pre-incubated for 0.5 h with different inhibitors or
medium as indicated in the figure legends, and then stimulated with murine TNF (10 000
IU/ml) in combination with 10 mM LiCl 3, or dsRNA (2.5 µg/ml). MAFP was used at 100
μM, AA861 at 50 μM, NDGA at 100 μM, BEL at 30 μM, rotenone at 25 μM, malonate at 5
mM and antimycin A at 50 μM.
Measurement of cell membrane permeability, hypoploidy and ROS production by FACS
analysis
Loss of cell membrane integrity as a measure of cell death was determined by PI
fluorescence. The percentage of cells containing hypoploid DNA was determined by PI
staining following one freeze-thaw cycle to permeabilize cells. PI was used at 30 µM and
fluorescence was detected at 610 nm on a FACScalibur flow cytometer (Becton Dickinson
Biosciences) equipped with a 488 nm argon ion laser. Production of ROS was measured at
525 nm and calculated as mean rhodamine 123 (R123) fluorescence resulting from oxidation
of DHR123 by PI-negative cells. Cells were incubated with 0.1 µM DHR123 for 30 min
before analysis. Relative rhodamine 123 fluorescence is defined as the ratio between
fluorescence at a given time point and the initial fluorescence for the same condition.
Preparation of muscle homogenates
Skeletal muscle was isolated from C57BL/6 mice. Twenty volumes of
homogenization buffer (10 mM Tris-HCl, 0.25 M sucrose, 2 mM EDTA, 50 U/ml heparin,
pH 7.4) were added to one volume of tissue sample (50 mg) for homogenization in a
glass/glass pestle. The homogenate was centrifuged (5 600 g) for 1 min. The supernatant
(whole tissue homogenate) was kept on ice and used for spectrophotometric analysis. Protein
concentrations were in the range of 3-5 mg/ml. Immediately before complex activity
measurements, the homogenate was sonicated to disrupt mitochondrial membrane integrity
and allow uptake of essential substrates.
Spectrophotometric analysis of electron transport chain complex activities
Spectrophotometric assays were performed to measure the activities of NADH-CoQ
reductase (complex I), succinate CoQ oxidoreductase (complex II), cytochrome c-ubiquinole
reductase (complex III), cytochrome c oxidase (complex IV) and citrate synthase, as
previously described 4. For each experiment, the results are presented as the mean activity of
the complex in 10 independent skeletal muscle samples, with error bars to indicate standard
error of the mean (SEM). Briefly, each assay was set up as follows:
NADH-CoQ reductase activity (complex I):
A 50 µl sample of skeletal muscle homogenate was incubated in 25 mM potassium
phosphate buffer (PPB), pH 7.4, containing 2.5 mg/ml BSA, 5 mM MgCl2, 100 µM NADH
(substrate complex I), and 0.6 mM KCN (to block oxidation by complex IV). Inhibitors were
added where appropriate. Treatment with 10 µM rotenone was used as a positive control of
inhibition. After stirring and stabilization for 1 min at 30 °C, decylubiquinone (commercial
quinone substitute) was added to a final concentration of 130 µM, and the rate of NADH
oxidation at 340 nm (ε = 6 290 M-1cm-1) was integrated over consecutive intervals of 16
seconds for a period of 4 min. Results are corrected for mitochondrial input as determined by
citrate synthase activity.
Succinate CoQ oxidoreductase activity (complex II):
A 20 µl sample of skeletal muscle homogenate was incubated in 10 mM PPB, pH 7.4,
containing 20 mM sodium succinate (substrate for complex II) and 3 mM KCN (to block
oxidation by complex IV), with or without inhibitors. Treatment with 5 mM malonate was
used as a positive control of inhibition. After stirring and stabilization for 5 min at 37 °C,
DCPIP (50 µM final) and decylubiquinone (130 µM final) were added. DCPIP reduction was
determined at 600 nm (ε = 19 100 M-1cm-1) over consecutive intervals of 16 seconds for a
period of 4 min. Results are corrected for mitochondrial input as determined by citrate
synthase activity.
Cytochrome c-ubiquinole reductase activity (complex III):
A mixture of 900 µl of complex III buffer (31 mM KH2PO4, 3 mM EDTA, 0.6 mM
KCN in DMSO/water (1:9 v/v), pH 7.5), 10 µl decylubiquinole (synthesized by reducing 13
mM decylubiquinone with NaBH4 5) and 100 µl cytochrome c (1% in 10 mM PPB, pH 7.0),
with or without inhibitors, was stabilized for 3 min at 37 °C. Afterwards, a 10 µl sample of
skeletal muscle homogenate was added. Treatment with 1 µM antimycin A was used as a
positive control of inhibition. Reduction of cytochrome c was monitored at 550 nm (ε = 18
500 M-1cm-1) over consecutive intervals of 16 seconds for a period of 4 min. Results are
corrected for mitochondrial input as determined by citrate synthase activity.
Cytochrome c oxidase activity (complex IV):
A 990 µl mixture of 10 mM PPB, pH 7.0, containing 0.1% reduced cytochrome c
(stock solution of 1% in 10 mM PPB pH 7.0 reduced with sodium dithionit) was incubated
for 10 min at 37 °C, with or without inhibitors. Afterwards, a 10 µl sample of skeletal muscle
homogenate was added. Treatment with 0.6 mM KCN was used as a positive control for
inhibition of complex IV. Oxidation of cytochrome c was monitored at 550 nm (ε = 18 500
M-1cm-1) over consecutive intervals of 30 seconds for a period of 5 min. Results are corrected
for mitochondrial input as determined by citrate synthase activity.
Citrate synthase activity:
A 10 µl sample of skeletal muscle homogenate was incubated in 0.1 mM Tris-HCl
buffer, pH 8.1, containing 60 µM acetylCoA, 100 µM DTNB and 0.05% Triton X-100. After
stirring and stabilization for 2 min at 37 °C, oxaloacetic acid was added (0.5 mM final).
Reaction with DTNB was measured at 412 nm (ε = 13 600 M-1cm-1) over consecutive
intervals of 33 seconds for a period of 4 min 6.
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Supplementary figure 1: Structure of BHA and BHT