INFECTION AND IMMUNITY, July 2009, p. 2919–2924
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 77, No. 7
Novel Subtilase Cytotoxin Produced by Shiga-Toxigenic Escherichia coli
Induces Apoptosis in Vero Cells via Mitochondrial
Gen Matsuura,1,2Naoko Morinaga,1* Kinnosuke Yahiro,1,3Reiko Komine,1Joel Moss,3
Hideo Yoshida,2and Masatoshi Noda1
Departments of Molecular Infectiology1and Pediatric Surgery,2Graduate School of Medicine, Chiba University, Chiba, Japan, and
Translational Medicine Branch, NHLBI, NIH, Bethesda, Maryland3
Received 11 December 2008/Returned for modification 19 January 2009/Accepted 15 April 2009
Subtilase cytotoxin (SubAB) is an AB5cytotoxin produced by some strains of Shiga-toxigenic Escherichia coli.
The A subunit is a subtilase-like serine protease and cleaves an endoplasmic reticulum chaperone, BiP, leading
to transient inhibition of protein synthesis and cell cycle arrest at G1phase. Here we show that SubAB, but not
the catalytically inactive mutant SubAB(S272A), induced apoptosis in Vero cells, as detected by DNA frag-
mentation and annexin V binding. SubAB induced activation of caspase-3, -7, and -8. Caspase-3 appeared
earlier than caspase-8, and by use of specific caspase inhibitors, it was determined that caspase-3 may be
upstream of caspase-8. A general caspase inhibitor blocked SubAB-induced apoptosis, detected by annexin V
binding. SubAB also stimulated cytochrome c release from mitochondria, which was not suppressed by caspase
inhibitors. In HeLa cells, Apaf-1 small interfering RNA inhibited caspase-3 activation, suggesting that cyto-
chrome c might form an apoptosome, leading to activation of caspase-3. These data suggested that SubAB
induced caspase-dependent apoptosis in Vero cells through mitochondrial membrane damage.
Shiga-toxigenic Escherichia coli (STEC) is an etiologic agent
of hemorrhagic colitis. Gastrointestinal disease caused by STEC
may progress to systemic complications, including hemolytic ure-
mic syndrome (HUS), which is characterized by thrombocytope-
nia, microangiopathic hemolytic anemia, and renal failure (13,
23). Shiga toxin 1 (Stx1) and Stx2 are both produced by STEC.
However, whether Shiga toxins are the only factors responsible
for these devastating diseases is still not clear.
A new member of the AB5toxin family, named subtilase
cytotoxin (SubAB), was identified (22, 23) in E. coli O113:H21
strain 98NK2, which produced Stx2 and was responsible for an
outbreak of HUS. SubAB consists of one A subunit and five B
subunits, which form a pentamer, similar to the case for Stx.
The SubAB A subunit, with a molecular size of 35 kDa, shares
sequence homology with a subtilase-like serine protease of
Bacillus anthracis, and the toxin was named “subtilase cyto-
toxin.” The A subunit cleaves at a specific single site of endo-
plasmic reticulum (ER) chaperone BiP (21). The B subunits
bind to some N-glycosylated membrane proteins, and ?2?1
integrin has been shown to one of the receptors for vacuolating
activity of B subunits (18, 30). Recently, it was reported that B
subunits specifically bound to glycans terminating in the sialic
acid N-glycolylneuraminic acid (3). SubAB is lethal for mice,
causing extensive microvascular thrombosis as well as necrosis
in the brain, kidney, and liver and apoptosis in the spleen,
kidney, and liver. These findings are similar to the histopatho-
logic, biochemical, and hematologic changes seen in human
HUS (22, 26).
SubAB is cytotoxic to Vero cells. BiP cleavage by the A
subunit is necessary for Vero cell death (17, 18, 21, 22). BiP is
known as a master regulator of ER function and homeostasis
(11). SubAB induces ER stress (17, 27), as shown by activation
of double-stranded RNA-activated protein kinase-like ER ki-
nase (PERK) and eukaryotic initiation factor 2? (eIF2?), lead-
ing to transient protein synthesis inhibition and stress-induc-
ible C/EBP-homologous protein (CHOP) induction, with cell
cycle arrest in G1phase as a result of downregulation of cyclin
Apoptosis, or programmed cell death, is a physiological event
important in a diverse array of biological processes ranging from
embryo development to bacterial infection (7, 31, 33). Morpho-
logically, cells undergoing apoptosis demonstrate nuclear/cyto-
plasmic condensation and membrane protrusions. Biochemically,
apoptotic cells are characterized by reduction in the mitochon-
tion of reactive oxygen species, externalization of phosphatidyl-
serine residues in membrane bilayers, selective proteolysis of a
subset of cellular proteins, and internucleosomal degradation of
DNA, resulting in a typical fragmentation pattern (28). There are
multiple potential participants described for ER stress-induced
apoptosis; however, the precise mechanisms of ER stress-induced
apoptosis have not been fully elucidated (29). Recently, SubAB-
induced apoptosis was partially described (27). We report here
that SubAB triggers apoptosis in Vero cells initiated via mito-
chondrial membrane damage, followed by activation of a caspase-
dependent cell death pathway.
MATERIALS AND METHODS
Cells and reagents. Vero (Vero-C1) cells were cultured at 37°C in a humidified
5% CO2atmosphere in Eagle’s minimum essential medium (EMEM) containing
10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, and 0.1
mg/ml streptomycin. General caspase inhibitor (Z-VAD-FMK) was obtained
* Corresponding author. Mailing address: 1-8-1 Inohana, Chuo-ku,
Chiba 260-8670, Japan. Phone: (81) 43 226 2048. Fax: (81) 43 226 2049.
?Published ahead of print on 20 April 2009.
from BD Biosciences Pharmingen, caspase-8 inhibitor (Z-IETD-FMK) from
R&D Systems, and caspase-3 inhibitor (Z-DQMD-FMK) from Calbiochem.
Antibodies against cytochrome c were obtained from R&D Systems; antibodies
against caspase-3, -7, and -8 and cleaved capase-3 and -7 were from Cell Signal-
ing; and antibodies against GAPDH (glyceraldehyde-3-phosphate dehydroge-
nase) were from Santa Cruz Biotechnology. Apaf-1 small interfering RNA
(siRNA) and control siRNA were obtained from Santa Cruz Biotechnology.
Apaf-1 antibody was purchased from Assay Designs.
Preparation of SubAB. Recombinant His-tagged SubAB and SubAB(S272A)
were purified as previously reported (18).
Apoptosis assays. (i) Detection of apoptotic cells by DNA fragmentation. Cells
(2 ? 106) were grown overnight and incubated with toxin for the indicated times.
After incubation, cells were washed with phosphate-buffered saline (PBS) and
lysed with lysis buffer (company supplied), and then DNA was isolated using the
Apoptotic DNA Ladder kit (Roche Diagnostics). Isolated DNA was incubated
with RNase (2 ?g/ml) for 20 min at room temperature, and the amount was
determined. DNA samples (2 ?g) were loaded onto a 1% agarose gel, separated
by electrophoresis, and then stained with ethidium bromide and visualized with
(ii) Detection of apoptotic cells by TUNEL assay. Terminal deoxynucleotidyl-
transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay was per-
formed using an in situ apoptosis detection kit (Wako Pure Chemical Industries,
(iii) Detection of apoptotic cells by annexin V binding. An annexin V-Fluos
staining kit (Roche Applied Science) was used. Briefly, 1 ? 106cells were
incubated with SubAB for indicated times, and cells were collected with
trypsinization, washed once with PBS, and then incubated with annexin V-Fluos
plus propidium iodide (PI) reagent for 15 min at room temperature. After
treatment, cells were washed with PBS and cell fluorescence was measured using
CellQuest software on a flow cytometer (Becton Dickinson). Fluorescence pa-
rameters were gated using unstained and single-stained (annexin V or PI) un-
treated and treated cells. Apoptosis was expressed as the percentages of annexin
V-positive cells in 10,000 cells. Parameters were as follows: FSC voltage, E00;
amp gain, 1.49; SSC voltage, 474; FL1 voltage, 474; FL2 voltage, 474; FL1, 59.9%
FL2; FL2, 45.4% FL1.
Western blotting to detect caspases. Cells (5 ? 105cells/well) were cultured
overnight in 24-well plates with EMEM containing 10% FBS. Prior to treatment
with SubAB, the medium was changed to EMEM containing 1% FBS. Cells were
then treated with SubAB (100 ng/ml) for the indicated times. After treatment,
cells were lysed with sodium dodecyl sulfate (SDS) sample buffer (0.0625 M Tris
[pH 6.8], 1% SDS, 10% glycerol, 2.5% mercaptoethanol, 0.001% bromophenol
blue) and heated at 100°C for 5 min before proteins were separated by SDS-
polyacrylamide gel electrophoresis. After electrophoresis at room temperature,
separated proteins were transferred onto polyvinylidene difluoride membranes
at 100 V for 1 h. Membranes were blocked with 5% nonfat milk in TTBS (20 mM
Tris [pH 7.6], 137 mM NaCl, 0.1% Tween 20) for 30 min and then incubated with
primary antibodies overnight at 4°C. After the membranes were washed three
times for 5 min with TTBS, they were incubated with horseradish peroxidase-
labeled secondary antibodies for 1 h at room temperature. Bands were visualized
using the Las 1000 (Fuji film). To investigate the effect of caspase inhibitors, cells
were treated with inhibitors for 30 min prior to treatment with toxin and then
incubated for 36 h with SubAB.
Detection of cytochrome c release from mitochondria. To evaluate the cyto-
chrome c release from mitochondria into cytosol, cytosol was fractionated fol-
lowing the method described by Chen et al. (5) with slight modifications. Briefly,
cells (1 ? 106cells) were treated with SubAB (100 ng/ml) in EMEM with 1%
FBS for the indicated times, collected with a cell scraper, and homogenized for
5 min in buffer (75 mM KCl, 1 mM Na2PO4, 8 mM Na2HPO4, 250 mM sucrose,
1 mM EDTA) containing 50 ?g/ml digitonin and protease inhibitor cocktail
(Roche Diagnostics). Following centrifugation at 10, 000 ? g for 10 min, the
supernatant was collected and then stored as cytosolic fractions. Mitochondrial
contamination was detected using antibody against Tom20 (Santa Cruz), a mi-
tochondrial marker. Cytochrome c was detected by Western blotting.
Apaf-1 gene silencing in Vero cells. Vero cells (1 ? 105cells) in a 12-well plate
were cultured overnight (50 to 60% confluent) and were transfected with control
siRNA (38 pmol) or siRNA for Apaf-1 (38 or 50 pmol) in Lipofectamine 2000
transfection reagent (Invitrogen) for 48 h following the company’s instructions.
After incubation, cells were washed with PBS and dissolved with SDS sample
buffer. Transfection efficiency was evaluated by Western blotting using Apaf-1
SubAB induces Vero cell apoptosis. SubAB cleaves BiP and
induces ER stress, as shown by activation of PERK and eIF2?,
leading to transient protein synthesis inhibition and cell cycle
arrest in G1phase as a result of downregulation of cyclin D1,
resulting in cell death (17, 27). Treatment of Vero cells with
SubAB, but notwitha catalytically
SubAB(S272A), in which the critical A subunit serine was
replaced by alanine, induced membrane blebbing followed by
loss of adhesion and retraction from the matrix, leading to
detachment (Fig. 1). The appearance was similar to that in-
duced by tunicamycin (TM), an N-glycosylation inhibitor which
causes ER stress, leading to apoptotic death in many cells. Stx1
also induces apoptosis (6, 10, 14), although bleb formation was
not observed at this time point (Fig. 1). To investigate whether
the cell death was due to apoptosis, DNA fragmentation was
evaluated (Fig. 2). DNA fragmentation was detected in
SubAB-, TM-, or Stx1-treated cells but not untreated control
cells or SubAB(S272A)-treated cells (Fig. 2). A typical DNA
ladder was more clearly evident in cells detached from the
matrix by treatment with SubAB than in cells still attached to
the matrix (data not shown). Similarly, cells treated with
SubAB, but not with SubAB(S272A), were TUNEL positive
(data not shown). Further, flow cytometric analysis revealed
that SubAB increased annexin V binding to Vero cells in a
time-dependent manner, consistent with exposure of phospha-
tidylserine residues in the outer leaflet of the plasma mem-
brane, which is an early event during apoptosis (Fig. 3A and
B). In contrast, SubAB(S272A)-treated cells showed only a
minor increase in apoptosis at 48 h.
SubAB activates caspase-3, -7, and -8. To determine
whether SubAB-induced apoptosis was caspase dependent, we
investigated caspase activation, as measured by cleavage of
procaspase. Activated caspase-3, -7, and -8 were detected fol-
lowing incubation with SubAB at a concentration of 0.1 ng/ml
or higher for 36 h (data not shown). Activated caspases fol-
lowing incubation with SubAB (100 ng/ml) occurred in a time-
dependent manner (Fig. 4). Caspase-3 and -7 were detected at
FIG. 1. SubAB-induced morphological changes in Vero cells. Cells
were treated with 100 ng/ml of SubAB or SubAB(S272A), 1 ?g/ml of
TM, or 100 ng/ml of Stx1 for 48 h. Phase-contrast microscopy pictures
are shown. Arrowheads indicate membrane blebbing, and arrows show
cells detached from the matrix. Bars, 50 ?m. The data shown are
characteristic of most of independent fields in two independent exper-
2920 MATSUURA ET AL.INFECT. IMMUN.
27 h, while caspase-8 was seen first at 33 h. The difference in
the time-dependent appearance of caspase-8 and caspase-3
A caspase-3 inhibitor inhibits cleavage of procaspase-8. To
confirm the order of caspase activation, we blocked caspase
activities with caspase inhibitors and examined the activation
of caspase-3, -7, and -8 (Fig. 5). A general caspase inhibitor
(Z-VAD-FMK) inhibited cleavage of procaspase-3, -7, and -8.
A caspase-3 inhibitor (Z-DQMD-FMK) inhibited not only
cleavage of procaspase-3 but also cleavage of procaspase-7 and
-8. A caspase-8 inhibitor (Z-IETD-FMK) inhibited cleavage of
procaspase-8 but did not have a significant effect on cleavage of
procaspase-3. It partially inhibited cleavage of procaspase-7 at
100 ?M. These results suggested that caspase-3 was upstream
of caspase-8 and -7.
SubAB-induced apoptosis is suppressed by the caspase in-
hibitor Z-VAD-FMK. We next evaluated whether a caspase
inhibitor suppressed SubAB-induced apoptosis in Vero cells.
Apoptosis was evaluated by annexin V binding as shown in Fig.
3A. We investigated at 36 h of incubation. The percentage of
annexin V-positive early apoptotic cells was suppressed signif-
icantly by incubation with Z-VAD-FMK (Fig. 6).
SubAB induces cytochrome c release from mitochondria. To
investigate whether SubAB-induced apoptosis was induced via
a mitochondrion-dependent pathway, cytochrome c release
from mitochondria into the cytosol was investigated. The pre-
pared cytosolic fractions were not contaminated by mitochon-
dria, which was verified using mitochondrial marker, Tom20
(data not shown). Stx1, which is known to induce cytochrome
c release (10, 14), was used as a positive control. SubAB in-
duced cytochrome c release in a time-dependent manner (Fig.
7A). Caspase inhibitors did not affect cytochrome c release by
SubAB (Fig. 7B), suggesting that changes in mitochondrial
membrane permeability might be upstream of caspase activa-
tion by SubAB. In the intrinsic pathway, cytochrome c released
from mitochondria triggers the formation of an apoptosome
composed of Apaf-1, procaspase-9, and cytochrome c, leading
to activation of caspase-9 and subsequent activation of caspase-3
(15, 32). We were unable to detect either procaspase-9 or
FIG. 2. SubAB-induced DNA fragmentation. Cells were treated
with 100 ng/ml of SubAB, SubAB(S272A), or Stx1 or with 1 ?g/ml of
TM for 48 h. DNA was isolated, and 2 ?g of each sample was analyzed
by 1% agarose gel electrophoresis. Lane 1, molecular size marker; lane
2, control without toxin; lane 3, SubAB; lane 4, Stx1; lane 5, TM; lane
6, SubAB(S272A). Data shown are representative of three separate
FIG. 3. Cells were incubated with 100 ng/ml of SubAB for the indicated times and then stained with annexin V and PI and analyzed by flow
cytometry. (A) Representative dot blots showing percentages of cells staining annexin V positive and PI negative (referred to as early apoptotic
cells; lower right quadrants), annexin V/PI double positive (referred to as late apoptosis including other dead cells; upper right quadrants), annexin
V/PI double negative (lower left quadrants), and annexin V negative and PI positive (upper left quadrants). Numbers represent the percentage
of cells in each quadrant out of a total of 10,000 cells. (B) Early apoptosis was expressed as percentages of annexin V-positive and PI-negative cells.
Data are means ? standard deviations for three samples.*, P ? 0.01 versus control without SubAB.
VOL. 77, 2009APOPTOSIS INDUCED BY SUBTILASE CYTOTOXIN 2921
caspase-9 in Vero cells using human caspase-9 antibody (data not
shown). Human anti-caspase-9 antibodies may not react with
monkey caspase-9. HeLa cells, derived from human cervical can-
cer, exhibit almost the same sensitivity as Vero cells to SubAB,
and SubAB induced cell cycle arrest at G1in HeLa cells as well
(17). In contrast to the case for Vero cells, cleavage of pro-
caspase-9 into 39- and 37-kDa caspase-9 was observed in SubAB-
treated HeLa cells. To confirm that apoptosome formation is
necessary for caspase-3 activation, we transfected HeLa cells with
siRNA against Apaf-1 (Fig. 8). The cleaved form of procaspase-9,
especially the appearance of a 37-kDa band, was suppressed in
cells transfected with Apaf-1 compared to cells treated with con-
trol siRNA, and similarly, procaspase-3 cleavage was suppressed
in cells transfected with Apaf-1 siRNA. In contrast, cytochrome c
SubAB induced caspase-3 activation via cytochrome c release,
followed by apoptosome formation and caspase-9 activation.
There are two well-described caspase-dependent pathways
that induce apoptotic cell death. One is the extrinsic pathway,
in which binding of death receptors by death ligands is fol-
lowed by recruitment of adaptor molecules and activation of
caspase-8. The other is the intrinsic pathway, in which cyto-
chrome c release from mitochondria triggers the formation of
the apoptosome composed of Apaf-1, procaspase-9, and cyto-
chrome c, which results in the activation of caspase-3. SubAB
FIG. 4. Kinetics of SubAB-induced caspase activation. Cells were incubated with SubAB (100 ng/ml) for the indicated times. After incubation,
cell were lysed and analyzed by Western blotting with specific anticaspase antibodies. The left panel shows a representative blot from three
experiments. After quantification of caspase-3, -7, and -8 by densitometry of the blot, the percentage of caspase was calculated as caspase/(caspase ?
procaspase) ? 100 (right panel). ?, caspase-3; ‚, caspase-7; E, caspase-8. Data are means ? standard deviations for three samples.*, P ? 0.01;
**, P ? 0.02; and***, P ? 0.03 (versus caspase-8 at each time point).
FIG. 5. Effects of caspase inhibitors on cleavage of procaspase-3,
-7, and -8. Cells were treated with a specific caspase-3 inhibitor
(DQMD), a caspase-8 inhibitor (IETD), or general caspase inhibitor
(VAD) (10 or 100 ?M) for 30 min before incubation with SubAB (100
ng/ml) for 36 h. After incubation, cell were lysed and analyzed by
Western blotting with specific anticaspase antibodies. Quantification of
caspase-3, -7, and -8 was performed by densitometry, and percentages
of control values were calculated as caspase in the presence of inhib-
itor/caspase of control cells without inhibitor ? 100. Data are means ?
standard deviations for three samples.*, P ? 0.03 versus control
FIG. 6. Effect of caspase inhibitor on SubAB-induced apoptosis.
Cells were treated with a general caspase inhibitor (VAD) (50 or 100
?M) for 30 min before incubation with SubAB (100 ng/ml) for 36 h,
and apoptotic cells were analyzed by annexin V and PI staining as
shown in Fig. 3A. Early apoptosis was expressed as the percentage of
annexin V-positive and PI-negative cells. Data are means ? standard
deviations for three samples. White bars, inhibitor alone; gray bars,
inhibitor with SubAB. Data are means ? standard deviations for three
samples.*, P ? 0.02;**, P ? 0.05 (versus SubAB without inhibitor).
2922MATSUURA ET AL.INFECT. IMMUN.
is bound to cells, internalized by endocytosis, and transported
from Golgi apparatus to ER in a retrograde manner. In the ER
lumen, it cleaved BiP, leading to ER stress, which was demon-
strated by activation of PERK and eIF2?, leading to transient
protein synthesis inhibition and stress-inducible CHOP induction
(17, 27). A catalytically inactive mutant, SubAB(S272A), did not
cleave BiP and did not induce apoptosis. Therefore, SubAB-
induced apoptosis is believed to be initiated not by cell recep-
tor recognition but by ER stress resulting from BiP cleavage.
The precise mechanisms of how ER stress results in the acti-
vation of caspases have not been fully elucidated (25, 29).
Murine caspase-12 and human caspase-4, the counterpart of
murine caspase-12, are candidates for involvement in the ini-
tial events of ER stress-induced apoptosis (12, 20); however,
recent reports questioned their participation. Further ER
stress-induced apoptosis required mitochondrion-dependent
apoptosome formation by a caspase-12-independent mecha-
nism (8, 24). SubAB induced changes in mitochondrial perme-
ability and released cytochrome c in a caspase-independent
manner, suggesting that caspase activation by SubAB might be
downstream of the changes in mitochondrial membrane per-
meability. Similar to the case for the intrinsic pathway, in HeLa
cells, a decrease in Apaf-1 expression clearly suppressed pro-
caspase-9 and -3 cleavage with no change in cytochrome c
release, suggesting that apoptosome formation was necessary
to induce caspase-3 activation. Treatment with SubAB might
induce a similar pathway for caspase-3 activation in Vero cells.
Caspase inhibitors did not suppress cytochrome c release by
SubAB, also suggesting that there might be caspase-independent
apoptosis, i.e., mitochondrial release of apoptosis-inducing factor
(2, 4). Further studies are necessary to define the potential func-
tion of apoptosis-inducing factor and other factors.
We found that activation of procaspase-8, known as an ini-
tiator caspase, was downstream of caspase-3. We reached this
conclusion because caspase-8 appeared later than caspase-3
and caspase-8-specific inhibitors did not suppress procaspase-3
cleavage, while a caspase-3-specific inhibitor suppressed pro-
caspase-8 cleavage. However, this pharmacological approach
may be compromised by possible off-target effects of the in-
hibitors. The use of a specific caspase siRNA may yield more
conclusive results. Also, there is still a possibility that caspase-8
activation may partially occur independently of caspase-3.
With regard to caspase-8, however, a recent report showed that
unlike its proximal role in receptor signaling, in the mitochon-
drial pathway caspase-8 functions as an amplifying executioner
caspase (9). Similar to the results in that report, cytochrome c
release by SubAB was not suppressed by caspase inhibitors,
and caspase-8 activation was a postmitochondrial event initi-
ated by caspase-3. Therefore, caspase-8 might enhance the
apoptotic signal initiated by mitochondria.
The mechanisms of how ER stress by SubAB induces
mitochondrial damage have not been identified. Bcl-2 family
proteins regulate apoptosis by controlling mitochondrial
permeability. CHOP, a transcription factor, involved in ER
stress-induced apoptosis that reduces expression of Bcl-2 (16),
was activated in SubAB-treated cells. Further, ER stress also
leads to increased cytosolic calcium levels, which activated
m-calpain and resulted in cleavage of Bcl-XL(19). The study of
FIG. 7. Cytochrome c release by SubAB. (A) Vero cells were treated
with SubAB (100 ng/ml) for the indicated times at 37°C, and the cyto-
plasmic fraction was prepared as described in Materials and Methods.
Cytochrome c was detected by Western blotting. As a positive control,
Vero cells were treated with Stx1 (100 ng/ml) for 30 h. (B) Cells were
incubated with 50 ?M caspase inhibitors for 30 min before treatment with
SubAB for 48 h, and cytochrome c release was assayed. The data shown
are representative of three separate experiments.
FIG. 8. Effect of Apaf-1 siRNA treatment on SubAB-induced
caspase activation. HeLa cells which had been transfected with control
siRNA (38 pmol) or Apaf-1 siRNA (38 or 50 pmol) for 48 h were
treated with SubAB (100 ng/ml) for another 48 h. After incubation,
cells were analyzed by Western blotting with antibodies against cyto-
chrome c, caspase-9 and -3, and GAPDH. 1, control siRNA; 2, Apaf-1
siRNA (38 pmol); 3, Apaf-1 siRNA (50 pmol). The left panel shows
representative blots, and the right panel shows the fold increase after
quantification of blots by densitometry. Data are means and standard
deviations for three samples.*, P ? 0.05 versus control siRNA.
VOL. 77, 2009 APOPTOSIS INDUCED BY SUBTILASE CYTOTOXIN2923