SUPPLEMENTARY MATERIALS
Lung Tissue Bioenergetics and Caspase Activity in Rodents
Ahmed R. Alsuwaidi1; Mohammed T. Alsamri1; Ali S. Alfazari2;
Saeeda Almarzooqi3; Alia Albawardi3; Aws R. Othman1; Thachillath Pramathan1;
Stacey M. Hartwig4; Steven M. Varga4; Abdul-Kader Souid1,*
Departments of Pediatrics1, Medicine2 and Pathology3, United Arab Emirates University, P.O.
Box 17666, Al Ain, UAE
Department of Microbiology4, Department of Pathology and Interdisciplinary Graduate Program
in Immunology5, University of Iowa, Iowa City, IA 52242
(ARA): alsuwaidia@uaeu.ac.ae
(MTA): mohammed.alsamri@uaeu.ac.ae
(ASA): a.almelaih@uaeu.ac.ae
(SA): saeeda.almarzooqi@uaeu.ac.ae
(AA): alia.albawardi@uaeu.ac.ae
(ARO): aws.rashad@uaeu.ac.ae
(TP): pramathant@uaeu.ac.ae
(SMH): stacey-hartwig@uiowa.edu
(SMV): steven-varga@uiowa.edu
(AKS): asouid@uaeu.ac.ae
* To whom correspondence should be addressed: (AKS) e-mail: asouid@uaeu.ac.ae; Tel. +9713-713-7429, fax +971-3-767-2022
1
Figure S1
A
B
250
-1
kc (with dactinomycin) = 0.14 + 0.02 M O2 min mg (p = 0.400)
-1
-1
Ac-DEVD-AMC
peak
1600
U
0.17
T
U
0.12
0.16
T
0.16
1400
fluorescent intensity (arbitrary units)
U
0.16
300
T
0.14
U
0.13
250
[O2], M
200
150
100
1200
1000
fluorescent intensity (arbitrary units)
kc (without dactinomycin) = 0.16 + 0.02 M O2 min mg
-1
2 hr with 8 M dactinomycin
200
150
2 hr without dactinomycin
100
0 hr
50
800
0
3.5
600
4
4.5
5
5.5
6
6.5
min
400
AMC peak
200
50
0
0
2
4
6
8
10
min
0
0
1
2
3
4
5
6
7
hr
C
D
0.25
kc-untreated
AMC-untreated
kc-dactinomycin
AMC-dactinomycin
20
1.5
0.1
Fluorescent intensity
(arbitrary units)
6
AMC peak area (÷10 )
2
2
k
c
-1
-1
(M O min mg )
0.15
with 8 M dactinomycin
15
3
0.2
2.5
AMC peak
4 hr incubation
3.5
10
Ac-DEVD-AMC peak
5
without treatment
1
0.05
0.5
0
0
0
0
0
1
2
3
4
5
10
20
30
40
min
6
hr
Figure S1. Lung tissue respiration and caspase activity with and without 8 M
dactinomycin. Panels A-C: Lung fragments from a C57Bl/6 mouse were incubated at 37oC
with and without 8 µM dactinomycin in 50 mL KH buffer gassed with 95% O2:5% CO2 for 60
sec every hour. At indicated time points, samples were removed from the incubation medium
and processed for measurements of O2 consumption and caspase activity. U (untreated), without
dactinomycin; T (treated), with dactinomycin. Panel A: Runs of cellular mitochondrial O2
consumption are shown; t = 0 corresponds to animal sacrifice. The rate of respiration (k, M O2
2
min-1) was set as the negative of the slope of [O2] vs. t. The values of kc (M O2 min-1 mg-1) are
shown at the top of the runs. Panel B: HPLC runs of caspase activity at 0 h, at 2 h without
dactinomycin and at 2 h with dactinomycin. The running solvent was HPLC-grade
methanol:dH2O 1:1 (isocratic). The retention time (Rt) for Ac-DEVD-AMC was ~2.5 min and
AMC ~4.8 min (insert, the AMC peaks, reflecting caspase activity). Panel C: The values of kc
and AMC peak areas (÷106) are plotted as a function of time of incubation. Panel D: Lung
fragments from a C57Bl/6 mouse were incubated at 37oC with and without 8 µM dactinomycin
in 50 mL KH buffer continuously gassed with 95% O2:5% CO2 for 4 h. Solvent A was HPLCgrade CH3CN:H2O [1:3, v/v] and Solvent B was dH2O (isocratic). The Rt for Ac-DEVD-AMC
was ~2.5 min and AMC ~29.0 min.
3
Figure S2
A
B
200
k = 0.14  M O min mg
-1
c
300
-1
2
AMC peak
without zVAD
-1
ATP = 17 + 0.7 pmol mg
k = 0.18 M O min mg
-1
c
-1
2
-1
ATP = 10 + 1.4 pmol mg
150
Fluorescent Intensity
(arbitrary units)
250
k = 0.08 M O min mg
-1
c
-1
2
-1
ATP = 186 + 6 pmol mg
[O2], M
200
150
0hr-AMC
3hr-AMC
6hr-AMC
100
50
100
0
50
-50
2
2.5
3
3.5
4
4.5
5
5.5
6
0
0
1
2
3
4
5
6
min
7
hr
C
D
1
200
without zVAD
with zVAD
AMC peak area (arbitrary unit mg -1 ÷10 6)
with zVAD
Fluorescent Intensity
(arbitrary units)
150
0hr+zVAD
3hr+zVAD
6hr+zVAD
AMC peak
100
50
0
0.8
0.6
0.4
0.2
-50
0
2
2.5
3
3.5
4
4.5
5
5.5
6
-1
0
1
2
3
4
5
6
7
min
Hr
Figure S2 Lung tissue respiration, ATP content and caspase activity at 5% CO2. Lung
fragments from a Wistar rat were incubated at 37oC in 50 mL MEM in room air saturated with
5% CO2 (in a CO2 tissue culture incubator). At indicated time points, samples were removed
from the incubation medium and processed for measurements of O2 consumption, ATP content
and caspase activity. Panel A: Runs of cellular mitochondrial O2 consumption are shown; t = 0
corresponds to animal sacrifice. The values of kc (M O2 min-1 mg-1) and ATP content (pmol
mg-1) are shown at the top of the runs. Panels B-C: HPLC runs of caspase activity at 0, 3, and 6
h with (Panel C) and without (Panel B) the addition of zVAD. The running solvent was HPLC4
grade methanol:dH2O 1:1 (isocratic). The Rt for AMC peak was ~4.8 min. Panel D: The values
of AMC peak areas corrected by sample weight (arbitrary unit mg-1 ÷106) are plotted as a
function of time of incubation.
5
Figure S3
A
B
no addition
250
0.2
IAV
no addition
200
k (M O min mg )
no addition
-1
IAV
-1
2
2
[O ], M
IAV
no addition
150
0.15
c
100
0.1
no addition
with IAV
0.05
50
0.11
0.12
0.12
0.17
0
0
1
2
3
0.12
4
0.14
5
0.13
6
0
7
0
8
2
4
6
8
10
hr
hr
C
D
AMC peak
1200
AMC peak
1200
influenza A virus (IAV)
no addition
1000
Fluorescent Intensity
(arbitrary units)
Fluorescent Intensity
(arbitrary units)
1000
0hr-A
3hr-A
6hr-A
9hr-A
800
600
800
0hr-B
3hr-B
6hr-B
9hr-B
600
400
400
200
200
0
0
2
2.5
3
3.5
4
4.5
5
5.5
6
2
2.5
3
3.5
min
E
10
no addition
IAV
-1
6
AMC peak area (arbitrary unit mg ÷10 )
4
min
8
6
4
2
0
0
2
4
6
hr
6
8
10
4.5
5
5.5
6
Figure S3 Respiration and caspase activity in lung tissue exposed in vitro to influenza A
virus (IAV). Lung fragments from a Wistar rat were incubated at 37oC in 50 mL MEM
continuously gassed with 95% O2:5% CO2 with and without added 10 L of the influenza A
virus (IAV) suspension (IAV/PR8/34 108.1 TCIU50). At indicated time points, samples were
removed from the incubation medium and processed for measurements of O2 consumption and
caspase activity. Panel A: Runs of cellular mitochondrial O2 consumption are shown; t = 0
corresponds to animal sacrifice. The values of kc (M O2 min-1 mg-1) are shown at the top of the
runs. Panel B: The values are kc are plotted as a function of time. Panel C-D: HPLC runs of
caspase activity at 0, 3, 6, and 9 h without addition (Panel C) and with the addition of IAV (Panel
D). The running solvent was HPLC-grade methanol:dH2O 1:1 (isocratic). The retention time
(Rt) for AMC was ~4.8 min. Panel E: The values of AMC peak areas (arbitrary unit mg-1 ÷106)
are plotted as a function of time of incubation.
7
Table S1. Lung tissue respiration and ATP content in Wistar rats – Impact of continuous
oxygenation
Conditions
Oxygenated buffer
Sevoflurane inhalation
Unoxygenated buffer
Sevoflurane inhalation
kc
(M O2 min-1 mg-1)
0.15 ± 0.04 (7)
CV = 26%
0.12 ± 0.04 (6)
CV = 33%
ATP content
(pmol mg-1)
91.0 ± 25.3 (6)
CV = 28%
2.9 ± 1.4 (8) *
CV = 48%
Values are mean ± SD (n) for t <8 h. CV, coefficient of variation; * p<0.05
8
Table S2. Lung tissue caspase activity in Wistar rats – Impact of anesthesia
Conditions
Experiments
Urethane inhalation
I
II
Sevoflurane anesthesia
III
IV
AMC peak area
(arbitrary units÷106
mg-1)
46.5 ± 0.7 (5)
CV = 2%
2.1 ± 1.5 (4) *
CV = 71%
2.7 ± 1.6 (4) *
CV = 59%
2.6 ± 2.1 (8) *
CV = 81%
Values are mean ± SD (n) for t <8 h. CV, coefficient of variation; * p<0.05
9
Table S3 Lung tissue caspase activity in C57Bl/6 and BALB/c mice (oxygenated buffer sevoflurane inhalation)
Experiments
I
II
8 independent
experiments
kc
(M O2 min-1 mg-1)
C57Bl/6 mice
0.16 ± 0.02 (4)
CV = 13%
0.15 ± 0.05 (4)
CV = 33%
BALB/c mice
0.08 ± 0.03 (11)
CV = 38%
AMC peak area
(arbitrary units÷106 mg-1)
0.13 ± 1.3 (5)
3.3 ± 2.5 (4)
CV = 76%
-
Values are mean ± SD (n = number of runs for t <8 h). CV, coefficient of variation
10