Supplementary Information
Linoleic acid metabolite drives severe asthma by inducing mitochondrial dysfunction and
airway epithelial injury
Ulaganathan Mabalirajan1*, Rakhshinda Rehman1, Tanveer Ahmad1, Sarvesh Kumar2, Sucitha
Singh1, Geeta D. Leishangthem3, Jyotirmoi Aich1, Manish Kumar1, Kritika Khanna1, Vijay P.
Singh1, Amit K. Dinda3, Shyam Biswal2, Anurag Agrawal1, and Balaram Ghosh1
1Molecular
Immunogenetics Laboratory and Centre of Excellence for Translational Research in
Asthma and Lung Disease, CSIR-Institute of Genomics and Integrative Biology, India.
2Department
of Environmental Health Sciences, Bloomberg School of Public Health, Johns
Hopkins University, USA.
3Division
of Renal Pathology, Department of Pathology, All India Institute of Medical Sciences,
India.
*Corresponding Author:
Ulaganathan Mabalirajan, Molecular Immunogenetics Laboratory, CSIR-Institute of Genomics
and Integrative Biology, Mall Road, Delhi-110 007. Phone: 91-11-27662580. Fax: 91-1127667471. E-mail address: um.rajan@igib.res.in
Supplementary Figure S1. Murine models used in this study. a) Different concentrations of
13-S-hydroxyoctadecadienoic acid (13-S-HODE) or vehicle (0-0.6–mg dose per mouse in
BALB/c mice or 0.6-mg dose per mouse in C57BL/6 mice) were instilled intranasally on days 1
to 3 in isoflurane-anesthetized mice. Respiratory mechanics was measured, and euthanasia was
performed 6 hours after the last dose. Intraperitoneal administration of 0.05 mg/kg calpeptin
(CALP) or 2.5 mg/kg capsazepine (CPZ) or vehicle or intranasal administration of Transient
Receptor Potential Cation Channel, Vanilloid-type 1 (TRPV1) siRNA or scrambled siRNA was
performed in mice in which 13-S-HODE had been administered. b) BALB/c mice were
sensitized on days 0, 7, and 14, followed by seven challenges with OVA, and AHR was
measured and euthanasia was performed 24 hours after the last challenge. 13-S-HODE antibody
or isotype control was administered intraperitoneally on days 25 to 27 in BALB/c mice. TRPV1
siRNA, or scrambled siRNA was administered intranasally on days 25 and 27 in BALB/c mice.
CPZ (2.5 mg/kg) or VEH twice a day was administered intraperitoneally on days 21 to 27 in
BALB/c mice. AHR, airway hyperresponsiveness; VEH, vehicle.
Supplementary Figure S2. Effect of ethanol on normal airways. To determine the possible
toxic effects of vehicle in which 13-S-HODE was dissolved (50 % ethanol), histopathology of
alveolar regions and baseline lung compliance of vehicle administered BALB/c mice were
estimated and compared with normal control mice. a) Photomicrographs of hematoxylin and
eosin stained alveolar regions of normal mice and mice which were administered 50 % ethanol
intranasally once a day for 3 consecutive days followed by sacrifice 6 hours after the last
administration (n = 5-6 mice per group). All images are at 20X magnification. Invasive
measurements to determine baseline lung compliance (b) and lung elastance in different groups
of BALB/c mice (c). Control, naïve mice which were not given either vehicle or 13-S-HODE.
Supplementary Figure S3. 13-S-HODE–induced mitochondrial dysfunction and epithelial
injury seem to be mediated by TRPV1. a) Inflammation scoring was performed in lung
sections stained with hematoxylin and eosin from mice in which 13-S-HODE was administered
and that were treated with CPZ (a pharmacological antagonist of TRPV1) n = 6 mice per group).
b) Graph shows data from ELISA of 8-isoprostane, a marker of oxidative stress. c) Graph shows
data from photomicrographs of TUNEL-stained lung sections that were assessed for the
percentage of TUNEL-positive bronchial epithelium. d) Inflammation scoring was performed in
lung sections stained with hematoxylin and eosin from mice in which 13-S-HODE was
administered and that were treated with either TRPV1 siRNA or scrambled siRNA (n = 4-6 mice
per group). e) Graph shows data from ELISA of 8-isoprostane, a marker of oxidative stress in
lung cytosol. f) Differential cell count was performed in BAL fluid and expressed as the
percentage of each cell type. Results are shown as mean ± s.e.m., significance is determined with
unpaired Student t test or Mann-Whitney test (*P ˂ 0.0001; ***P ˂ 0.05). Macro, macrophage;
Mono, mononuclear agranulocytes such as monocytes and lymphocytes; and Neutro, neutrophil.
Supplementary Figure S4. Pharmacological antagonism of TRPV1 attenuates asthma
features, restores mitochondrial dysfunction, and reduces epithelial injury in allergic mice.
a) Graph of inflammation score derived from photomicrographs of bronchovascular regions
stained with hematoxylin and eosin from mice that were sensitized and challenged with alum or
OVA for 7 consecutive days and administered either CPZ or vehicle (n = 6 mice per group). b)
Differential cell count was performed in BAL fluid and expressed as the percentage of each cell
type. Macro, macrophage; Mono, mononuclear agranulocytes such as monocytes and
lymphocytes; Eosino, eosinophil; and Neutro, neutrophil. c) Activity of mitochondrial complex I
(n = 4-6 mice per group). (d, e) Graphs of data from ELISA and fluorescence-based activity
assays showed cytochrome c and caspase 3 activity in lung cytosol. Results shown as mean ±
s.e.m., significance determined with unpaired Student t test or Mann-Whitney test (***P ˂ 0.05).
SHAM/PBS/VEH, PBS–sensitized, and PBS-challenged mice treated with vehicle;
OVA/OVA/VEH, OVA–sensitized, and OVA-challenged mice treated with vehicle; and
OVA/OVA/CPZ, OVA–sensitized, and OVA-challenged mice treated with capsazepine.
Supplementary Table 1.
Symbol
Mitochondrial functions
Mitochondrial dynamics
Mfn1
Mfn2
Msto1
Dnm1l
Fis1
Opa1
Fold change vs. HODE
Fold change vs. OVA
Gene name
1.5227
-1.3044
2.4061
1.8747
1.4406
1.5547
1.1514
-1.3074
-1.1947
-1.0994
1.1837
1.2002
Uncoupling
Ucp2
Ucp3
3.6723
-1.2086
2.3349 Uncoupling protein 2 (mitochondrial, proton carrier)
-1.0257 Uncoupling protein 3 (mitochondrial, proton carrier)
Chaperones and antioxidants
Hsp90aa1
Hspd1
Sod1
Sod2
-1.651
-1.5298
1.4109
1.1701
-1.0842
1.217
-2.214
1.3692
Heat shock
Heat shock
Superoxide
Superoxide
Solute carriers
Slc25a1
Slc25a10
Slc25a20
Slc25a21
Slc25a22
Slc25a15
Slc25a2
Slc25a12
Slc25a13
Slc25a3
Slc25a23
Slc25a24
Slc25a19
Slc25a25
Slc25a4
Slc25a5
Slc25a14
Slc25a16
Slc25a17
Slc25a27
Slc25a30
Slc25a31
Slc25a37
1.4607
1.3074
2.4566
-1.7451
1.2114
2.0515
1.3819
2.3403
2.4228
1.8747
1.1865
3.2641
-1.4273
1.2198
-1.1355
1.4709
1.0619
2.3729
1.9274
2.3403
1.8747
2.3894
2.0373
1.488
-1.7132
1.1837
-3.1529
1.5404
1.3503
-1.4406
1.9498
1.5948
1.2953
-1.9141
1.8965
-1.0046
-1.6896
-1.2541
1.5298
-1.2114
1.0163
1.0817
-1.2983
1.0521
-1.6434
-1.2283
Solute
Solute
Solute
Solute
Solute
Solute
Solute
Solute
Solute
Solute
Solute
Solute
Solute
Solute
Solute
Solute
Solute
Solute
Solute
Solute
Solute
Solute
Solute
Mitochondrial import machinery
Fxc1
Timm10
Timm17a
Timm17b
Timm22
Timm23
Timm44
Timm50
Timm8a1
Timm8b
Timm9
Grpel1
Dnajc19
Tomm20
Tomm22
Tomm34
Tomm40
Tomm40l
Tomm70a
Mtx2
Sh3glb1
Cox18
Aip
Immp1l
Immp2l
Mipep
1.0619
1.0994
1.3441
1.0767
1.7613
-1.1045
1.6208
1.9141
-1.5619
1.0546
-1.234
2.0515
1.7492
1.7736
-1.3787
-1.3044
1.5122
1.146
1.1147
-1.2953
-1.1674
1.7251
1.154
-1.0668
-1.674
-1.1755
1.6283
-1.2894
-1.2894
1.0163
-1.1381
1.0817
-1.1147
1.8575
1.0817
-1.4208
-1.4208
2.4852
1.1199
-1.3915
-1.8234
-2.2294
-1.0918
1.7695
1.1199
1.0668
-1.2198
2.434
1.2086
-1.2894
-1.0046
-1.3629
Fractured callus expressed transcript 1
Translocase of inner mitochondrial membrane 10 homolog (yeast)
Translocase of inner mitochondrial membrane 17a
Translocase of inner mitochondrial membrane 17b
Translocase of inner mitochondrial membrane 22 homolog (yeast)
Translocase of inner mitochondrial membrane 23 homolog (yeast)
Translocase of inner mitochondrial membrane 44
Translocase of inner mitochondrial membrane 50 homolog (yeast)
Translocase of inner mitochondrial membrane 8 homolog a1 (yeast)
Translocase of inner mitochondrial membrane 8 homolog b (yeast)
Translocase of inner mitochondrial membrane 9 homolog (yeast)
GrpE-like 1, mitochondrial
DnaJ (Hsp40) homolog, subfamily C, member 19
Translocase of outer mitochondrial membrane 20 homolog (yeast)
Translocase of outer mitochondrial membrane 22 homolog (yeast)
Translocase of outer mitochondrial membrane 34
Translocase of outer mitochondrial membrane 40 homolog (yeast)
Translocase of outer mitochondrial membrane 40 homolog-like (yeast)
Translocase of outer mitochondrial membrane 70 homolog A (yeast)
Metaxin 2
SH3-domain GRB2-like B1 (endophilin)
COX18 cytochrome c oxidase assembly homolog (S. cerevisiae)
Aryl-hydrocarbon receptor-interacting protein
IMP1 inner mitochondrial membrane peptidase-like (S. cerevisiae)
IMP2 inner mitochondrial membrane peptidase-like (S. cerevisiae)
Mitochondrial intermediate peptidase
Mitochondrial trafficking
Rhot1
Rhot2
Lipid and heme metabolism
Stard3
Tspo
Cpt2
Cpt1b
Taz
Cox10
2.4737
1.3256
1.1865
1.2198
1.5984
7.1437
1.9954
-1.8067
RNA stabiity
Lrpprc
Uxt
1.0767
1.3074
Cell survival
Cdkn2a
Sfn
Pmaip1
Trp53
Aifm2
Bid
Bnip3
Bak1
Bbc3
Bcl2
Bcl2l1
-1.0892
-1.341
2.0946
-1.398
1.0329
1.154
4.3974
1.1947
1.7132
1.2541
3.7494
Nefl
2.5082
Mitofusin 1
Mitofusin 2
Misato homolog 1 (Drosophila)
Dynamin 1-like
Fission 1 (mitochondrial outer membrane) homolog (yeast)
Optic atrophy 1 homolog (human)
protein 90, alpha (cytosolic), class A member 1
protein 1 (chaperonin)
dismutase 1, soluble
dismutase 2, mitochondrial
carrier
carrier
carrier
carrier
carrier
carrier
carrier
carrier
carrier
carrier
carrier
carrier
carrier
carrier
carrier
carrier
carrier
carrier
carrier
carrier
carrier
carrier
carrier
family
family
family
family
family
family
family
family
family
family
family
family
family
family
family
family
family
family
family
family
family
family
family
25 (mitochondrial
25 (mitochondrial
25 (mitochondrial
25 (mitochondrial
25 (mitochondrial
25 (mitochondrial
25 (mitochondrial
25 (mitochondrial
25 (mitochondrial
25 (mitochondrial
25 (mitochondrial
25 (mitochondrial
25 (mitochondrial
25 (mitochondrial
25 (mitochondrial
25 (mitochondrial
25 (mitochondrial
25 (mitochondrial
25 (mitochondrial
25, member 27
25, member 30
25 (mitochondrial
25, member 37
carrier, citrate transporter), member 1
carrier, dicarboxylate transporter), member 10
carnitine/acylcarnitine translocase), member 20
oxodicarboxylate carrier), member 21
carrier, glutamate), member 22
carrier ornithine transporter), member 15
carrier, ornithine transporter) member 2
carrier, Aralar), member 12
carrier, adenine nucleotide translocator), member 13
carrier, phosphate carrier), member 3
carrier; phosphate carrier), member 23
carrier, phosphate carrier), member 24
thiamine pyrophosphate carrier), member 19
carrier, phosphate carrier), member 25
carrier, adenine nucleotide translocator), member 4
carrier, adenine nucleotide translocator), member 5
carrier, brain), member 14
carrier, Graves disease autoantigen), member 16
carrier, peroxisomal membrane protein), member 17
carrier; adenine nucleotide translocator), member 31
1.5087 Ras homolog gene family, member T1
-1.0767 Ras homolog gene family, member T2
1.3044
1.4473
1.1594
2.835
-1.4406
1.0023
START domain containing 3
Translocator protein
Carnitine palmitoyltransferase 2
Carnitine palmitoyltransferase 1b, muscle
Tafazzin
COX10 homolog, cytochrome c oxidase assembly protein, heme A: farnesyltransferase (yeast)
-1.0994 Leucine-rich PPR-motif containing
1.674 Ubiquitously expressed transcript
1.8965
-3.3096
1.1199
1.0595
-1.6096
1.923
-1.5547
1.3692
-1.0693
2.2868
2.2243
Cyclin-dependent kinase inhibitor 2A
Stratifin
Phorbol-12-myristate-13-acetate-induced protein 1
Transformation related protein 53
Apoptosis-inducing factor, mitochondrion-associated 2
BH3 interacting domain death agonist
BCL2/adenovirus E1B interacting protein 3
BCL2-antagonist/killer 1
BCL2 binding component 3
B-cell leukemia/lymphoma 2
Bcl2-like 1
5.7491 Neurofilament, light polypeptide
Suppl. Table 1. Quantitative real-time PCR array profiling of key genes of mitochondrial functions
shows the differentially expressed genes in murine lung samples in which 13-S-HODE was
administered and or allergic mice. OVA-induced asthmatic mice had common upregulated genes such
as Ucp2, GrPel1, Cpt1b, Bcl2l, and Nefl compared with findings in 13-S-HODE–induced asthmatic mice.
More than or less than twofold increase or decrease indicates differentially expressed genes compared to
either SHAM control mice or vehicle administered mice.
Supplementary Table 2.
Symbol
Fold change
Description
Complex I
Core
Core
Core
Core
Core
Core
Core
Regulation of complex I activity
Supernumerary
Supernumerary
Supernumerary
Supernumerary
Supernumerary
Supernumerary
Supernumerary
Accessory subunit
Accessory subunit
Accessory subunit
Accessory subunit
Accessory subunit
Accessory subunit
Accessory subunit
Accessory subunit
Accessory subunit
Accessory subunit
Accessory subunit
Accessory subunit
Accessory subunit
Accessory subunit
Accessory subunit
Accessory subunit
Accessory subunit
Accessory subunit
Ndufs7
Ndufs8
Ndufv1
Ndufv2
Ndufs1
Ndufs2
Ndufs3
Ndufs4
Ndufa1
Ndufa10
Ndufa11
Ndufa2
Ndufab1
Ndufb9
Ndufs6
Ndufa3
Ndufa4
Ndufa5
Ndufa6
Ndufa7
Ndufa8
Ndufb10
Ndufb2
Ndufb3
Ndufb4
Ndufb5
Ndufb6
Ndufb7
Ndufb8
Ndufc1
Ndufc2
Ndufs5
Ndufv3
-1.3379
-1.4743
-1.2924
-1.4641
1.0497
-1.2397
1.2311
1.0353
1.5476
1.8277
1.5911
-1.8404
-1.5369
1.4044
-1.257
-1.6818
-1.2658
-1.1408
1.2311
-1.2058
1.007
1.1096
-1.0718
-1.1019
-1.1892
-1.2834
-1.1975
-1.2483
-1.0867
-1.1251
1.0425
-1.1251
-1.257
NADH
NADH
NADH
NADH
NADH
NADH
NADH
NADH
NADH
NADH
NADH
NADH
NADH
NADH
NADH
NADH
NADH
NADH
NADH
NADH
NADH
NADH
NADH
NADH
NADH
NADH
NADH
NADH
NADH
NADH
NADH
NADH
NADH
dehydrogenase
dehydrogenase
dehydrogenase
dehydrogenase
dehydrogenase
dehydrogenase
dehydrogenase
dehydrogenase
dehydrogenase
dehydrogenase
dehydrogenase
dehydrogenase
dehydrogenase
dehydrogenase
dehydrogenase
dehydrogenase
dehydrogenase
dehydrogenase
dehydrogenase
dehydrogenase
dehydrogenase
dehydrogenase
dehydrogenase
dehydrogenase
dehydrogenase
dehydrogenase
dehydrogenase
dehydrogenase
dehydrogenase
dehydrogenase
dehydrogenase
dehydrogenase
dehydrogenase
(ubiquinone)
(ubiquinone)
(ubiquinone)
(ubiquinone)
(ubiquinone)
(ubiquinone)
(ubiquinone)
(ubiquinone)
(ubiquinone)
(ubiquinone)
(ubiquinone)
(ubiquinone)
(ubiquinone)
(ubiquinone)
(ubiquinone)
(ubiquinone)
(ubiquinone)
(ubiquinone)
(ubiquinone)
(ubiquinone)
(ubiquinone)
(ubiquinone)
(ubiquinone)
(ubiquinone)
(ubiquinone)
(ubiquinone)
(ubiquinone)
(ubiquinone)
(ubiquinone)
(ubiquinone)
(ubiquinone)
(ubiquinone)
(ubiquinone)
Fe-S protein 7
Fe-S protein 8
flavoprotein 1
flavoprotein 2
Fe-S protein 1
Fe-S protein 2
Fe-S protein 3
Fe-S protein 4
1 alpha subcomplex, 1
1 alpha subcomplex 10
1 alpha subcomplex 11
1 alpha subcomplex, 2
1, alpha/beta subcomplex, 1
1 beta subcomplex, 9
Fe-S protein 6
1 alpha subcomplex, 3
1 alpha subcomplex, 4
1 alpha subcomplex, 5
1 alpha subcomplex, 6 (B14)
1 alpha subcomplex, 7 (B14.5a)
1 alpha subcomplex, 8
1 beta subcomplex, 10
1 beta subcomplex, 2
1 beta subcomplex 3
1 beta subcomplex 4
1 beta subcomplex, 5
1 beta subcomplex, 6
1 beta subcomplex, 7
1 beta subcomplex 8
1, subcomplex unknown, 1
1, subcomplex unknown, 2
Fe-S protein 5
flavoprotein 3
Sdha
Sdhb
Sdhc
Sdhd
1.5052
1.6358
-1.3379
1.2658
Succinate
Succinate
Succinate
Succinate
Uqcrc1
Uqcrc2
Cyc1
Uqcrfs1
Uqcr11
Uqcrh
Uqcrq
Bcs1l
1.0644
-1.1647
-1.3566
1.2658
-1.5583
-1.1728
-1.2746
1.0644
Ubiquinol-cytochrome
Ubiquinol cytochrome
Cytochrome c-1
Ubiquinol-cytochrome
Ubiquinol-cytochrome
Ubiquinol-cytochrome
Ubiquinol-cytochrome
BCS1-like (yeast)
Cox4i1
Cox4i2
Cox5a
Cox5b
Cox6a1
Cox6a2
Cox6b1
Cox6b2
Cox6c
Cox7a2
Cox7a2l
Cox7b
Cox8a
Cox8c
Oxa1l
Cox11
1.1019
1.815
-2.395
-1.0867
1.3195
1.1019
1.2311
-1.3755
-1.0943
-1.1892
-1.1487
-1.5692
-1.0644
1.1892
1.6245
-1.434
Cytochrome c oxidase subunit IV isoform 1
Cytochrome c oxidase subunit IV isoform 2
Cytochrome c oxidase, subunit Va
Cytochrome c oxidase, subunit Vb
Cytochrome c oxidase, subunit VI a, polypeptide 1
Cytochrome c oxidase, subunit VI a, polypeptide 2
Cytochrome c oxidase, subunit VIb polypeptide 1
Cytochrome c oxidase subunit VIb polypeptide 2
Cytochrome c oxidase, subunit VIc
Cytochrome c oxidase, subunit VIIa 2
Cytochrome c oxidase subunit VIIa polypeptide 2-like
Cytochrome c oxidase subunit VIIb
Cytochrome c oxidase, subunit VIIIa
Cytochrome c oxidase, subunit VIIIc
Oxidase assembly 1-like
COX11 homolog, cytochrome c oxidase assembly protein (yeast)
1.1892
1.0497
1.1892
-2.0849
1.2397
1.4948
-2.5847
-1.4641
1.3379
1.4241
1.8404
1.815
-1.7171
-1.2142
1.0353
1.5476
1.0425
-1.5583
1.1892
1.1892
ATPase, H+/K+ transporting, nongastric, alpha polypeptide
ATPase, H+/K+ exchanging, gastric, alpha polypeptide
ATPase, H+/K+ exchanging, beta polypeptide
ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit 1
ATP synthase, H+ transporting mitochondrial F1 complex, beta subunit
ATP synthase, H+ transporting, mitochondrial F1 complex, gamma polypeptide 1
ATP synthase, H+ transporting, mitochondrial F1 complex, delta subunit
ATP synthase, H+ transporting, mitochondrial F0 complex, subunit B1
ATP synthase, H+ transporting, mitochondrial F0 complex, subunit c1 (subunit 9)
ATP synthase, H+ transporting, mitochondrial F0 complex, subunit C2 (subunit 9)
ATP synthase, H+ transporting, mitochondrial F0 complex, subunit C3 (subunit 9)
ATP synthase, H+ transporting, mitochondrial F0 complex, subunit d
ATP synthase, H+ transporting, mitochondrial F0 complex, subunit F
ATP synthase, H+ transporting, mitochondrial F0 complex, subunit F2
ATP synthase, H+ transporting, mitochondrial F1 complex, O subunit
ATPase, H+ transporting, lysosomal V0 subunit A2
ATPase, H+ transporting, lysosomal V0 subunit D2
ATPase, H+ transporting, lysosomal V1 subunit C2
ATPase, H+ transporting, lysosomal V1 subunit E2
ATPase, H+ transporting, lysosomal V1 subunit G3
Complex II
Catalytic site
Catalytic site
Membrane-anchoring subunit
Membrane-anchoring subunit
dehydrogenase
dehydrogenase
dehydrogenase
dehydrogenase
complex,
complex,
complex,
complex,
subunit
subunit
subunit
subunit
A,
B,
C,
D,
flavoprotein (Fp)
iron sulfur (Ip)
integral membrane protein
integral membrane protein
Complex III
Core subunit
Core subunit
Respiratory
Respiratory
Low molecular weight
Low molecular weight
Low molecular weight
assembly of complex
subunit
subunit
subunit
III
c reductase core protein 1
c reductase core protein 2
c
c
c
c
reductase, Rieske iron-sulfur polypeptide 1
reductase, complex III subunit XI
reductase hinge protein
reductase, complex III subunit VII
Complex IV
regulation and assembly
regulation and assembly
regulation and assembly
regulation and assembly
regulation and assembly
regulation and assembly
regulation and assembly
Connects the two COX monomers
regulation and assembly
regulation and assembly
regulation and assembly
regulation and assembly
regulation and assembly
regulation and assembly
activity and assembly of cytochrome oxidase
insertion of the copper B into subunit I
Complex V
catalytic subunit of the ouabain-sensitive H+/K+ -ATPase
Atp12a
catalytic alpha subunit of the gastric H+, K+-ATPase
Atp4a
catalytic beta subunit of the gastric H+, K+-ATPase
Atp4b
alpha subunit of the catalytic core
Atp5a1
beta subunit of the catalytic core
Atp5b
gamma subunit of the catalytic core
Atp5c1
delta subunit of the catalytic core
Atp5d
b subunit of the proton channel
Atp5f1
subunit c of the proton channel
Atp5g1
subunit c of the proton channel
Atp5g2
subunit c of the proton channel
Atp5g3
d subunit of the proton channel
Atp5h
F6 subunit of proton channel
Atp5j
f subunit of the proton channel
Atp5j2
part of the connector linking catalytic core and proton channel
Atp5o
component of the V(0) domain vacuolar ATPase
Atp6v0a2
Subunit of the integral membrane V0 complex of vacuolar ATPase
Atp6v0d2
Subunit of the peripheral V1 complex of vacuolar ATPase
Atp6v1c2
Subunit of the peripheral V1 complex of vacuolar ATPase
Atp6v1e2
Catalytic subunit of peripheral V1 vacuolar ATPase
Atp6v1g3
other nuclear coded OXPHOS genes
Lhpp
Ppa1
Ppa2
1.0943 Phospholysine phosphohistidine inorganic pyrophosphate phosphatase
1.0792 Pyrophosphatase (inorganic) 1
-1.5583 Pyrophosphatase (inorganic) 2
Supplementary Table 2. Quantitative real-time PCR array profiling of key genes of the mitochondrial
electron transport chain shows the differentially expressed genes in lungs in which 13-S-HODE was
administered. Downregulated genes are in blue, and unaltered genes are in black; more than or less than twofold
increase or decrease indicates differentially expressed genes compared to either SHAM control mice or vehicle
administered mice.
Legends for supplementary videos:
1. Supplementary Video 1 (MOV 162 K)
Breathing pattern of mouse in which VEH (ethanol) was administered. The breathing pattern
was captured 10 to 15 minutes after administration of 30 µl of ethanol in isofluraneanesthetized BALB/c mice.
2. Supplementary Video 2 (MOV 231 K)
Breathing pattern of mouse in which 13-S-HODE was administered. The breathing pattern
was captured 10 to 15 minutes after administration of 30 µl of 13-S-HODE (0.6 mg/d per
mouse for 3 d) in isoflurane-anesthetized BALB/c mice. The moving mouse with the yellow
label was administered VEH. There was visible difficulty in breathing characterized by
forced abdominal movement with complete focus on breathing in 13-S-HODE–treated mice
compared with no focus on breathing in ethanol-treated mice.
3. Supplementary Video 3 (MOV 110 K)
Breathing pattern of mouse in which 13-S-HODE was administered. The breathing pattern
was captured 10 to 15 minutes after administration of 30 µl of 13-S-HODE (0.6 mg/d per
mouse for 3 d) in isoflurane-anesthetized BALB/c mice that were pretreated with 2.5 mg/kg
CPZ.
4. Supplementary Video 4 (MOV 327 K)
Breathing pattern of mouse in which 13-S-HODE was administered. The breathing pattern
was captured 10 to 15 minutes after administration of 30 µl of 13-S-HODE (0.6 mg/d per
mouse for 3 d) in isoflurane-anesthetized BALB/c mice that were pretreated with 50 µg of
TRPV1 siRNA per day per mouse.