Legends to supplementary figures
Supplementary Figure 1 Incubation of isolated HeLa mitochondria with PLA2 does not
lead to tBid or Bax degradation. Isolated mitochondria were incubated without or with
PLA2 (0.3 or 1.5 µg/ml) and further incubated in the absence or presence of tBid
(20 nM). The amounts of Bax and tBid associated with the mitochondrial membranes
after centrifugation were assessed by immunoblotting. An antibody directed against
SLP-2 was used as a loading control. Blot is representative of two independent
experiments.
Supplementary Figure 2 Effects of tBid and of PE on Bax activation. PC/CL 60/40 or
PC/CL/PE 40/40/20 liposomes were prepared and in vitro Bax activation assays were
performed in the absence (-) or presence (+) of tBid. The binding of Bax (- trypsin) and
its oligomerization (+ trypsin) were assessed by immunoblotting.
Supplementary Figure 3 Size distribution of extruded liposomes. (a) The size
distribution of liposomes extruded through 800, 400 or 200 nm pore filters was analyzed
by dynamic light scattering. Data represented is the relative distribution in surface weight
of vesicles. (b) For each size, the amount of liposomes pelleting during in vitro Bax
activation assays was checked by TLC. Experiment was done in duplicates. (a, b) Results
are representative of three independent experiments.
Supplementary Figure 4 Down-regulation of CLS using shRNAs does not lead to a
significant reduction in CL level. (a) Two different shRNAs (pR CLS 1 and pR CLS 2)
designed against hCLS were transfected in HeLa cells, and after 120 hours, the level of
CLS mRNA was assessed by quantitative real-time PCR. Both of them led to a specific
reduction in the mRNA level of CLS of about 90% in comparison to cells transfected with
an shRNA designed against the Firefly Luciferase (pR Luc). Results for pR Luc and
pR CLS 1 correspond to two independent experiments; result for pR CLS 2 corresponds
to a single experiment. (b) 120 hours post-transfection, the level of CL in mitochondria of
HeLa cells transfected with pR Luc, pR CLS 1 or pR CLS 2 was assayed by extracting
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lipids and analyzing them by TLC (CHCl3/MeOH/acetic acid 32.5/14/4) and iodine
staining. (c) The intensities of the CL spots were quantified and normalized relative to the
pR Luc control. We did not detect any significant change in the total CL level. Values are
means of four independent experiments +/- SEM. (d) 96 hours after transfection, HeLa
cells were incubated with [3H]-palmitic acid, and after 24 hours, lipids were extracted,
separated by TLC, and visualized by film exposure. (e) The radioactivity of the spots was
counted. The intensities of the CL and phosphatidylglycerol (PG) spots were normalized
with the intensities of the spots corresponding to PE and PC. We observed a 30%
decrease in the amount of radioactive CL and a respective increase in the level of
radioactive PG. Values are means of two independent experiments +/- STDEV. Whereas
total PG levels were negligible in comparison to CL levels1 an important fraction of PG
was labeled with [3H], probably reflecting the low rate of conversion of PG to CL.
Supplementary Figure 5 HeLa cells were transfected with pR CLS 1 (C) or with
pR Luc (L) as a control. (a, b) 120 hours after transfection, mitochondria were isolated
and incubated in the absence (-) or presence (+) of recombinant tBid (20 nM) for 5, 10 or
15 minutes. The insertion of Bax and tBid, assayed by immunoblotting the pellets
obtained after incubation in sodium carbonate, was similar using both types of
mitochondria (a). Similarly, the release of Smac/Diablo and of cytochrome c was
unchanged by the expression of the CLS shRNA (b). (c-e) 120 hours post-transfection,
HeLa cells were incubated with actinomycin D for various time points (0, 2.5, 3.5 or 5
hours). Cytosolic fractions were purified and assayed by immunoblotting for caspase-3
activation (c) and for Smac/Diablo and cytochrome c release (d). Mitochondria were
purified and incubated in sodium carbonate to assay Bax insertion in the membranes (e).
The three processes displayed a similar time course in control cells and in those
expressing pR CLS 1. SLP-2 was used as a loading control for the mitochondrial
fractions and actin and aldolase were used for the cytosolic fractions. In a-e, blots are
representative of three independent experiments. (f) 120 hours post-transfection, HeLa
cells were incubated with actinomycin D (Act D) for 3.5 and 6 hours or with
staurosporine (STS) for 2 and 6 hours. Cell survival was assayed by Annexin-V and PI
staining and FACS analysis. The percentage of unlabelled cells is indicated. No
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significant difference between the susceptibility of the cells transfected with pR CLS 1
(grey) and pR Luc (black) could be detected. Values are means of at least four
independent experiments +/- SEM.
Supplementary material and methods
shRNA design, cloning and transfection
Two shRNAs, specific for the hCLS mRNA (Accession number NM_019095) were
designed (CLS1 sequence: 5’-CCAGTTCCACTTACTTACA-3’, nt 585-603; CLS2
sequence:
5’-AGACTGTTCAGGTGATAAA-3’,
nt
895-913),
and
cloned
into
BglII/HindIII sites of pSUPER-RETRO (gift from Rewen Agami), as previously
described.2,3 As a control, pSUPER-RETRO expressing an shRNA directed against
Firefly Luciferase (sequence: 5’-CGTACGCGGAATACTTCGA-3’) was used. 1 h after
plating, these mammalian expression plasmids were transfected in HeLa cells following a
calcium phosphate protocol. 24 h after transfection, transfected cells were selected
overnight with puromycin (3 µg/ml, Calbiochem). Cells were further cultured for 48 or
72 h until experiments were undertaken.
RNA purification, RT and quantitative real-time PCR analysis
120 h post-transfection, total RNA from HeLa cells expressing pR Luc, pR CLS 1 or
pR CLS 2, was extracted using the Trizol reagent (Invitrogen). Total RNA was then
incubated with RQ1 RNase-free DNase (Promega) in the presence of RNase OUT
inhibitor (Invitrogen) according to the manufacturer’s instructions. RNA was then
purified using a phenol:CHCl3 extraction procedure. In brief, the RNA solution was
mixed for 1 min with an equal volume of citrate-saturated phenol pH 4.3/CHCl3/isoamyl
alcohol (125/24/1), incubated 1 min at room temperature, centrifuged 5 min at 12 000 g,
4 °C. The top phase was recovered and mixed with an equal volume of CHCl3/isoamyl
alcohol (24/1) for 1 min, incubated 1 min at room temperature, and centrifuged 5 min at
12 000 g, 4 °C. The top phase was recovered and mixed with a half volume of
ammonium acetate 7.5 M and 2.5 volumes of EtOH. The solution was incubated 1 h at
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-80 °C, and centrifuged 15 min at 12 000 g, 4 °C. The pellet was washed in EtOH 75 %,
centrifuged 5 min at 7 500 g, 4 °C. Purified RNA was dried at room temperature and
resuspended in water. cDNA was synthesized with M-MLV reverse transcriptase
(Invitrogen) with random hexamer oligonucleotides (Microsynth) according to the
instructions of the manufacturer. Quantitative real-time PCR analysis was performed
using the iQ SYBR Green Supermix (Bio-Rad), iCycler (Bio-Rad) and analyzed with
iCycle iQ Version 3.1 (Optical System Software). The concentration of CLS mRNA was
normalized using primers designed against TFRC (NM_003234; 5’-CATTTG
TGAGGGATCTGAACCA-3’; 5’-CGAGCAGAATACAGCCACTGTAA-3’), EEF1A1
(NM_001402; 5’-AGCAAAAATGACCCACCAATG-3’; 5’-GGCCTGGATGGTTCAG
GATA-3’), and TBP (NM_003194; 5’-GCCCGAAACGCCGAATATA-3’; 5’-CGTGG
CTCTCTTATCCTCATGA-3’).
[3H]-palmitic acid labeling of endogenous lipids and quantification
96 h after transfection, HeLa cells were labeled for 24 h with [3H]-palmitic acid
(4 µCi/ml, Amersham). Cells were then scraped off plates, washed in cold PBS, and
lipids were extracted, as described above. Total radioactivity in each sample was counted
with Betamatic V (Kontron Instruments). Equal amounts of radioactivity were spotted on
silica gel 60 TLC plates (Merck), and developed in CHCl3/MeOH/acetic acid (32.5/14/4).
Spots were visualized with iodine vapor and identified using standards. The plates were
sprayed with EN3HANCE autoradiography enhancer (PerkinElmer Life and Analytical
Sciences, Wellesley, MA, USA) and exposed to SUPER RX films (Fujifilm) for 24 h at
-80 °C. Spots were scraped off plates and counted as above. Radioactivity of CL and PG
spots were normalized with the total radioactivity associated with PE and PC spots. Each
value represents the mean of three independent experiments, analyzed by TLC in
triplicate.
Annexin-V and PI staining of apoptotic cells
After exposure to actinomycin D (3 µM, Sigma) or staurosporine (1 µM, Sigma),
transfected HeLa cells were washed with PBS and trypsinized. All the cells were pooled
(floating, in the wash, trypsinized) and cell survival was quantified by Annexin-V FITC
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(BD Biosciences) and PI (Sigma) staining according to the manufacturer’s instructions
and using a FACScalibur system (Becton Dickinson). Statistical significance was
assessed with a paired Student’s t-test.
Supplementary references
1.
Daum G Lipids of mitochondria. Biochim Biophys Acta 1985; 822: 1-42
2.
Brummelkamp TR, Bernards R and Agami R Stable suppression of
tumorigenicity by virus-mediated RNA interference. Cancer Cell 2002; 2: 243-7
3.
Brummelkamp TR, Bernards R and Agami R A system for stable expression of
short interfering RNAs in mammalian cells. Science 2002; 296: 550-3
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Supplementary Figure 1
Supplementary Figure 2
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Supplementary Figure 3
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Supplementary Figure 4
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Supplementary Figure 5
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