supplementary figure 1 a b d - nature research · supplementary figure 1. alox12 is dramatically...

Supplementary Figure 1. ALOX12 is dramatically upregulated during hepatic IR injury. (a,b) Cluster

images showing global sample distribution profiles and relationships analyzed by the hierarchical

clustering (a) and by t-distributed stochastic neighbor embedding (b) based on data from digital gene

Sham Isch 1 h

GAPDH

CPLA2

ALOX5

CYP4A

ALOX15

Alox12 Alox5 Alox15 Gpx1 Ggt1 Cbr2 Ptges

Rel

ativ

e m

RN

A ex

pres

sion

(nor

mal

ized

toAc

tb)

a

c

f

h

g*

**

* *n.s.

n.s.ALOX12

Rep

3 h

-2R

ep 3

h-1

Rep

3 h

-3R

ep 3

h-4

Rep

3 h

-5Is

ch 1

h-2

Isch

1 h

-5Is

ch 1

h-3

Isch

1 h

-1Is

ch 1

h-4

Sham

-3Sh

am-4

Sham

-5Sh

am-1

Sham

-2R

ep 2

4 h-

3R

ep 2

4 h-

5R

ep 2

4 h-

1R

ep 2

4 h-

2R

ep 2

4 h-

4R

ep 6

h-2

Rep

6 h

-3R

ep 6

h-4

Rep

6 h

-1R

ep 6

h-5

Rep

12

h-4

Rep

12

h-5

Rep

12

h-3

Rep

12

h-1

Rep

12

h-2

Supplementary Figure 1

0

25

50

75

100

C1 C2 C3 C4 C5 C6

Per

cent

Lipid metabolism

Amino acid metabolism

Carbohydrate metabolism

Energy metabolism

Glycan biosynthesis and metabolism

Metabolism of cofactors and vitamins

Metabolism of terpenoids and polyketides

Overview of metabolism

Xenobiotics biodegradation and metabolism

Folding, sorting and degradation

Replication and repair

Transcription

Translation

Cell growth and death

Cellular community

Transport and catabolism

Development

Digestive system

Endocrine system

Immune system

Other

Signaling molecules and interaction

Signal transduction

Aging

C3: TNF / MAPK / FoxO / Hippo / cAMP signalingC4: TGF-beta / Hippo / ErbB / MAPK / Sphingolipid signalingC5: RAP1 / cGMP-PKG / Phospholipase D / Notch / Ras singlaing

ShamIsch 1 h

0

1

2

3

4

5

b

−40

0

40

−40 0 40 80

Dimension 1

Dim

ensi

on 2

d

-log 1

0(F

DR

)

log2 (FC)

0

1

2

3

−5.0 −2.5 0.0 2.5 5.0

e

-0.4

0.4

0.2

0

-0.2

Relative proteinabundance

(log2 fold change)

CYP2C38GPX3ALOX12CYP2C68GPX1CYP4A12BPLA2G12BPTGS1CYP2C70CYP4A12ACYP2C55CYP2U1CYP4F14CBR1PTGES2CBR3CYP2C23LTA4HCYP2C50GGT5CYP2C37EPHX2CYP4F13PLA2G6CYP2E1FAM213BCYP4A10CYP2J5PLA2G12APTGES3CYP2C54CYP2C29CYP4A14CYP2B10

Sham Isch 1 h Rep 3 h Rep 6 h Rep 12 h Rep 24 h

ALO

X12

prot

ein

expr

essi

on(n

orm

aliz

ed to

GAP

DH

) BaselineIschemiaReperfusion

n.s.

*

*

Nature Medicine: doi:10.1038/nm.4451

expression (DGE). (c) The corresponding molecular events of each k-means clustering in Fig. 1b. (d)

Volcano plots indicating the differentially expressed genes (DEGs; red, upregulated genes; green,

downregulated genes) in ischemia group relative to sham controls in DGE assay. The vertical dashed

gray lines in the plot represent log2 normalized fold change equal to 1 and -1. The horizontal dashed

gray line represents false discovery rate (FDR) equal to 0.05. For a-d, n = 5 mice per group. (e) Heat

maps generated using the protein abundance data (normalized to the mean value of sham group) of

members in arachidonic acid metabolism pathway detected by proteomics analyses. n = 15 mice in

each group with samples from 5 mice pooled for proteomics analysis; n = 3 pools in each group for

proteomics analysis. (f) The relative mRNA expressions of genes encoding indicated Alox members

and highlighted factors by DGE in livers of mice subjected to hepatic ischemia for 1 h and of sham

controls. n = 5 mice per group. Data are shown as the mean ± s.e.m. * P < 0.05, n.s., not significant;

by two-tailed Student’s t-test. (g) The protein expression levels of ALOX12, ALOX5, ALOX15,

CPLA2, and CYP4A in the livers of mice at 1 h after ischemia challenge or in the sham group. n = 4

mice per group with 3 technical replicates. GAPDH was the loading control. (h) The quantification of

ALOX12 protein expression in the livers of individuals subjected to hepatic IR surgery. n = 19

individuals in each group. * P < 0.05, n.s., not significant; by one-way ANOVA with Tamhane’s T2(M)

post hoc analysis.


Supplementary Figure 2. ALOX12 directly promotes 12-HETE accumulation in hepatic IR injury.

(a) Schematic image showing the major function of ALOX12 as an enzyme that promotes 12-

hydroxyeicosatetraenoic acid (12-HETE) production from arachidonic acid (AA). (b) Statistical

results of Fig. 2a showing major AA metabolites contents in the livers of mice at indicated time points

of hepatic IR process. n = 10 mice in sham group, n = 15 mice at each reperfusion group. * P < 0.05,

** P < 0.01, *** P < 0.001, **** P < 0.0001 compared to corresponding sham group; #### P < 0.0001

compared to corresponding Isch 1 h group; by one-way ANOVA with Tamhane’s T2(M) post hoc

analysis. (c) The contents of 5-, 12-, 15-, and 20-HETE in the serum of mice after 6 h of reperfusion

compared to those in the sham group (n = 6 mice in each group). * P < 0.05, ** P < 0.01, *** P <

0.001, n.s., no significance; by two-tailed Student’s t-test. For b,c, data are shown as the mean ± s.e.m.

(d,e) The protein expression level of ALOX12 in the indicated tissues of Alox12-KO (d), Alox12-HTG

(e; HTG-3 line was used in this study), and their corresponding control mice. For d,e, n = 2 mice in

each group for western blot. n = 3 technical replicates. GAPDH was the loading control.

a b

e

c

d

0

2

4

6 ShamRep 6 h AA

met

abol

ite c

onte

nts

inliv

er (f

old

chan

ge)

HET

E co

nten

ts in

seru

m (f

old

chan

ge)

12-H

ETE

5-HETE

15-H

ETE

20-H

ETE

Liver

Serum

Sham

Rep24

h

Rep 12

h

Rep 6

h

Rep 3

h

Isch 1

h

***

* **

**

**** **** ****

******** **********

*****

*****

*** ****

n.s.


WT KO

ALOX12

GAPDH

NTG HTG NTG HTG NTG HTG NTG HTGALOX12

GAPDH

Liver Heart KidneyBrain

ALOX12

GAPDH

NTG HTG-1 HTG-2 HTG-3

#### ########

0

10

20

30

O

OH

CH3

COOH

OH

O2

ALOX12

Arachidonic acid (AA) 12-HETE


Supplementary Figure 3. ALOX12 aggravates liver dysfunction, cell death and inflammation in

hepatic IR injury. (a) The ALT and AST levels in the serum of mice with Alox12 overexpression or

NTG controls at baseline or at 6 h after reperfusion. n = 10 mice per group. **** P < 0.0001, n.s., not

significant; by one-way ANOVA with Tamhane’s T2(M) post hoc analysis. (b,c) The representative

H&E (b; NA, necrotic area) and TUNEL (c) staining images (green, positive cells; blue, nuclei labeled

Sham Rep 6 h

c-CASP3

GAPDH

BCL2

BAX

HTGNTG HTGNTG

NTG

HTG

HTGNTG HTGNTGSham Rep 6 h

GAPDH

p-p65

p65

p-IκBα

IκBα

Sham Rep 6 h

NTG

HTG

Ly6G

Sham Rep 6 h

CD11b

Sham Rep 6 h

AST

(U/L

)

Sham Rep 6 h

NTG

HTG

02,0004,0006,0008,000

10 000

Sham Rep 6 hSham Rep 6 h CC

L2 c

onte

nt in

ser

um (p

g/m

l)

TNF

cont

ent i

n se

rum

(ng/

ml)

Tnf Il6 Cxcl2Rel

ativ

e m

RN

A ex

pres

sion

(nor

mal

ized

toAc

tb)

NTG, rep 6 hHTG, rep 6 h

NTG, shamHTG, sham

c d

e f

g h

020406080

100

NTGHTG

** ** ** **

** ** ** **

** ** ** **** *

*n.s.

*

*n.s.

**

n.s.

n.s.

n.s.

n.s.

n.s.


050

10010,00015,00020,00025,00030,00035,000

090

18010,00015,00020,00025,00030,00035,000

0

5

10

20

40

60


by DAPI) of liver sections from NTG and Alox12-HTG mice in sham or Rep 6 h groups. n = 4 mice

in each group with 24 images for each mouse. Scale bar, 200 Pm for H&E staining (10u); 50 Pm for

H&E staining (40u); 20 Pm for TUNEL images. (d) Representative western blot (of 3 western blots)

showing protein expression of BCL2, BAX, and cleaved caspase-3 (c-CASP3) in the livers of mice in

indicated groups. GAPDH was the loading control. n = 6 mice in each group. (e) The contents of TNF

and CCL2 in the serum of Alox12-HTG mice and NTG controls at 6 h after reperfusion or without IR

insult. n = 4 mice for sham-NTG group; n = 6 mice for other groups. * P < 0.05, ** P < 0.01, **** P

< 0.0001, n.s., no significance; by one-way ANOVA with Tamhane’s T2(M) (TNF) or with

Bonferroni’s post hoc analysis (CCL2). (f) The relative mRNA expression (relative to Actb) of

cytokines and chemokines in livers of Alox12-HTG and NTG groups after 6 h of hepatic IR injury or

in the sham group. n = 4 mice for each group. * P < 0.05, *** P < 0.001, n.s., no significance; by one-

way ANOVA with Tamhane’s T2(M) post hoc analysis (Il6 and Cxcl2) or with Bonferroni’s post hoc

analysis (Tnf). (g) The representative images of immunofluorescence staining of CD11b+ (left) and

Ly6G+ (right) inflammatory cells (red) in the liver sections of mice in the indicated groups. The nuclei

were labeled with DAPI (blue). n = 4 mice in each group with 24 images for each mouse. Scale bar,

20 Pm. (h) The activation of NF-NB signaling in the livers of Alox12-HTG and NTG mice at sham and

Rep 6 h groups. n = 6 mice in each group; n = 3 western blots for each band. GAPDH served as a

loading control. For statistical plots, data are shown as the mean ± s.e.m.


Supplementary Figure 4. 12-HETE promotes hepatic IR injury via inducing a burst of inflammation.

Sham Rep 6 hHTGNTGHTGNTG

Sham Rep 6 hKOWTKOWT

GAPDH

p-JNK

p-p38

p-ERK

ERK

JNK

p38

GAPDH

p-JNK

p-p38

p-ERK

ERK

JNK

p38

Il1bTnfCcl212-HETE

Il1bTnfCcl212-HETE

Relative m

RN

A expression(norm

alized toActb)

12-H

ETE

cont

ent i

n liv

er(fo

ld c

hang

e)

Relative m

RN

A expression(norm

alized toActb)

12-H

ETE

cont

ent i

n ce

ll ly

sate

(fol

d ch

ange

)

Hyp 5

min

Normox

ia

Hyp 10

min

Hyp 30

min

Hyp 60

min

Reoxy

1 h

Reoxy

6 h

Isch 1

5 min

Sham

Isch 3

0 min

Isch 6

0 min

Rep 1

h

Rep 3

h

Rep 6

h

Rep 12

h

Rep 24

h

a b

g h

** *

** ** **

***

*****

***

******** ****

******


0

10

20

30

40

0

5

10

15

0.0

0.5

1.0

1.5

2.0

2.5

0

2

4

6

NormoxiaAlox12

HypoxiaGFP Alox12GFP

NormoxiashAlox12

HypoxiashGFP shAlox12shGFP

Flag

GAPDH

p-p65

p-JNK

p-p38

p-ERK

ERK

p65

JNK

p38

GAPDH

p-p65

p-JNK

p-p38

p-ERK

ERK

p65

JNK

p38

ALOX12

Rel

ativ

e m

RN

A ex

pres

sion

(nor

mal

ized

toAc

tb)

Rel

ativ

e m

RN

A ex

pres

sion

(nor

mal

ized

toAc

tb)

GFP-HypAlox12-Hyp

GFP-NorAlox12-Nor

sh GFP-Hypsh Alox12-Hyp

sh GFP-Norsh Alox12-Nor

c

d

e

f

TnfIl6 Ccl2 TnfIl6 Ccl2

**** **** **** ******** ****

**** **** * ** **

0

2

4

6

8

10

0

1

2

3

4

5


(a,b) The time-line relationships between 12-HETE with inflammatory mediators in the liver (a) and

primary hepatocytes (b) at indicated time points after hepatic IR injury (a) or HR challenge (b) . For

12-HETE contents in (a), n = 7 in sham, Isch, and Rep 1 h groups, n = 15 in other groups; For qPCR

in (a), n = 5 mice in each group. n = 3 independent experiments in (b). Data are shown as the mean ±

s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 compared to the corresponding sham

(a) or normoxia (b) group by two-tailed Student’s t-test. (c,d) Protein expression of phosphorylated

and total p65, JNK, p38 and ERK (c) and relative mRNA expression of inflammatory mediators (d) in

cell lysates of primary hepatocytes with Alox12 overexpression under normoxia (Nor) or hypoxia (Hyp)

conditions for 1 h. * P < 0.05, ** P < 0.01, **** P < 0.0001; by one-way ANOVA with Bonferroni’s

post hoc analysis (Tnf in d) or with Tamhane’s T2(M) post hoc analysis (Il6 and Ccl2 in d). (e,f) Protein

expression levels of phosphorylated and total p65, JNK, p38 and ERK (e) and relative mRNA

expression of inflammation-related genes (f) in cell lysates of primary hepatocytes with Alox12

knockdown under normoxia or hypoxia conditions for 1 h. ** P < 0.01, **** P < 0.0001; by one-way

ANOVA with Bonferroni’s post hoc analysis. In c-f, n = 3 independent experiments with 2 replicates

each time. (g,h) The protein expression of phosphorylated and total JNK, p38 and ERK in the livers

of Alox12-HTG, NTG (g), Alox12-KO and WT (h) mice at 6 h after reperfusion or in the sham group.

n = 6 mice for each group; n = 3 western blots for each band. For all western blots, GAPDH was the

loading control.


Vehicle-Hyp12-HETE-Hyp

Vehicle-Nor12-HETE-Nor

TnfIl6Il1b Cxcl2Ccl2

Rel

ativ

e m

RN

A ex

pres

sion

(nor

mal

ized

toAc

tb)

b

********

*** ********

***

***

*****

*****

** * *

fWT

GPR31B

Gpr31b-KO

GAPDH


d

30100 (min)

GPR31-res

30100

GPR31-KO

30100

WT

12-HETE

GAPDH

p-PKC

JNK

PKC

p-JNK

p-p65

p65

p-p38

p38

p-ERK

ERK

GPR31

GPR31-KO, 12-HETEGPR31-res, 12-HETE

WT, vehicleWT, 12-HETE

TNFIL6 CXCL10CCL2

Rel

ativ

e m

RN

A ex

pres

sion

(nor

mal

ized

to)

e

* * **

* ** *

*

0

2

4

6

8

0

1

2

3

4

a

Low High

Vehicle 12-HETE

Normoxia

Mmp13Ccl3Traf1Tlr2Mmp9Il1bMefvCcl7Ccl4Socs3Icam1Ifi47Bcl2a1dIl1aCreb3l4FasCasp4Mb21d1ItgalNfkb2Sh2d1b1Nlrp3Ccl2Cxcl2IkbkeTnfCcr7Ticam2Il1r1Ccl9Irf5Cd40Nlrx1Cxcl11Cxcl1Il23aZbp1Tlr3PrlrDdx58Il13ra1GsdmdRasgrp2Casp7Bcl2a1bRelbCd48Tlr1H2−M3Ifngr2Rac2Cxcl3Lcn2Casp3SykTnfrsf21Il2rgCtskPidd1OsmrNfkb1Nfatc2Ccl22Jak3Arrb1Tlr6Ptgs2Prex1Bcl2a1aCd14CflarCxcl5Ripk2Mapk11Isg15NfkbiaTnfaip3Il18Raet1dFlt3lTgfb3Dhx58Ncf1Raet1aBirc3Gnb4Tnfsf10Ccl28HckCxcl16PrkcbMyd88Bmp7Tnfsf13bCxcl10Ppp3ccTrex1Csf1Il33Il18rapH2−T22Tnfrsf11b

Vehicle 12-HETE

Hypoxia

Low HighZ-score

cTNFRSF1BPTGS2VAV3THPOBMP2IFI16ROCK1MAPK13ATMFASROCK2IL6STITPR2MAPK4JAK3EDARTRPM7AKT3IL1RAPTNFRSF10CAMHKITLGSOS1PLCB1LCN2IFIH1PLAUTLR3BMPR2RPS6KA5RAET1ECREB1OAS1PIK3CACXCL3INHBETANKSHC3GBP2ATF2CCR10LEPRCXCL8CASP4TLR5LIFRCXCL2SOS2IRAK4MUC5ACTNFRSF25RNF125IL1R1TNFSF12MAP2K6TBKBP1IRF5FYNDNM1LITGB2EPORTRAF1NEK7PIK3CDPDGFRBJAK2CNTFRMAPK15GRK5TBK1CARD6BIRC3SH3BP2OSMRTNFRSF9MUC5BCREB3L3ULBP1CD14SOCS3PRKCZIL23ATAB3MAPK6GNG11BCL3ITPR1NLRC4TGFB3GNG7PDGFBIL4RGBP3SIKE1GNG3MPLAZI2BIRC2CHUKADCY5PRKACBADCY6BMPR1AFLT3LGUSP25BRCC3PRKCBDDX3XXIAPMAPK3NAMPTISG15MAP3K7TAB2SHC2MFN1HSP90B1CYLDCCR7TGFBR1TNFSF14TLR4MAP3K5PIK3R3ITCHMALT1MAP3K1ATG5INHBBTLR6PPP3CACSF1PTK2BPTPN6HSP90AA1TNFSF13CCL28GABARAPL1CASP1IFNGR2MAVSTNFRSF8KRASIL17RCFOSSTAT2IL22RA1CEBPBRAP1ADDX58GNAI3NRASTNFSF4JUNDMAP2K4CTSBWASLBRAFPIK3CBIFNLR1GNAI1POLR3ECREB5CARD16TNFAIP3ACVR2BCARD8MAP3K8TMEM173STAT5BINHBCMETCD70CXCL16ARRB1TNFRSF1ANGFRMEFVGDF5TSLPCSF1RGRK7TLR1CREB3L4MMP13EDN1IL2RBLow

HighZ-score


Supplementary Figure 5. GPR31 mediates the exacerbation of 12-HETE on hepatic IR injury. (a)

The leading genes contributing to 12-HETE function based on a gene set enrichment analysis (GSEA)

of RNA-seq results from primary hepatocytes treated with or without 12-HETE (100 nM) under

normoxia or hypoxia conditions. Data were analyzed based on n = 4 samples in each group. (b) The

relative mRNA expression of cytokines and chemokines in primary hepatocytes treated with 12-HETE

after hypoxia challenge or under normoxia condition. The gene expression was normalized to that of

Actb. n = 3 independent experiments with 2 replicates each time. * P < 0.05, ** P < 0.01, *** P <

0.001, **** P < 0.0001 by one-way ANOVA with Tamhane’s T2(M) post hoc analysis. (c) The heat

maps (n = 3 in each group) of leading genes based on a gene set enrichment analysis of RNA-seq in

Fig. 5e. (d,e) Representative western blot (of 2 western blots) showing protein expression of

phosphorylated and total PKC, p65, JNK, p38 and ERK (d) and relative mRNA expression of

inflammatory mediators (e) in GPR31-KO cells, GPR31-overexpressed cells in the background of


10× 40× 10× 40×

g

Gpr

31b-

KOW

T

Sham Rep 6 h

NA

NA

NA

h Ly6G

Sham

CD11bSham

WT

Gpr

31b-

KO

Rep 6 hRep 6 h


GPR31-KO (GPR31-res), and WT control hepatocytes after 12-HETE treatment. n = 2 independent

experiments with 2 technical replicates. For western blot, GAPDH served as a loading control. For

qPCR, gene expression was normalized to that of ACTB. * P < 0.05, ** P < 0.01, *** P < 0.001, ****

P < 0.0001 by one-way ANOVA with Bonferroni’s post hoc analysis (CCL2) or with Tamhane’s T2(M)

post hoc analysis (IL6, TNF, and CXCL10). (f) Representative western blot (of 3 technical replicates)

of GPR31B expression in the livers of Gpr31b-KO mice and WT controls. n = 4 mice in each group.

GAPDH was the loading control. (g) Representative images of H&E (NA, necrotic area) staining of

liver sections at 6 h after reperfusion of mice with Gpr31b knockout or WT controls. n = 4 mice for

each group with 24 images for each mouse. Scale bar, 200 �Pm for H&E staining (10u); 50 Pm for

H&E staining (40u). (h) The representative staining images of CD11b+ (left) and Ly6G+ (right)

inflammatory cell (red) infiltration in the livers of mice in the indicated groups. The nuclei were labeled

with DAPI (blue). n = 3 mice in each group with 24 images for each mouse. Scale bar, 20 Pm. In all

statistical plots, data are shown as mean ± s.e.m.


c

Rep

6 h

Vehicle ML355 CDC

Vehicle

Rep

6 h

h

Vehi

cle

ML3

55

Sham Isch 1 h Rep 6 h

ShamML355

Rep 6 h

GAPDH

c-CASP3

BCL2

BAX

Vehicle ML355Vehicle

i

Vehicle ML355, 1mg/kg ML355, 2mg/kg ML355, 3mg/kg

Vehicle ML355, 3mg/kgML355, 1mg/kg ML355, 2mg/kg

VehicleML355

Rep 6 h, CDCRep 6 h, vehicleSham, vehicle Rep 6 h, ML355

Rep 6 h, CDCRep 6 h, vehicleSham, vehicle Rep 6 h, ML355

12-H

ETE

cont

ent i

nliv

er (f

old

chan

ge)

12-H

ETE

cont

ent i

nliv

er (f

old

chan

ge)

12-H

ETE

cont

ent i

nse

rum

(fol

d ch

ange

)

12-H

ETE

cont

ent i

nse

rum

(fol

d ch

ange

)

ALT

(U/L

)

AST

(U/L

)

ALT

(U/L

)

AST

(U/L

)

ALT

(U/L

)

AST

(U/L

)

Sham

Isch 1

h

Rep 1

h

Rep 3

h

Rep 12

h

Rep 6

h

Rep 24

hSha

m

Isch 1

h

Rep 1

h

Rep 3

h

Rep 12

h

Rep 6

h

Rep 24

h0

5,000

10,000

15,000

20,000

0

5,000

10,000

15,000

20,000

25,000

VehicleML355

Sham Rep 6 h Sham Rep 6 hSham Rep 6 h

CC

L2 c

onte

nt in

seru

m (p

g/m

l)

CX

CL1

0 co

nten

t in

seru

m (p

g/m

l)

TNF

cont

ent i

nse

rum

(ng/

ml)

a b

d

e f

g

j

10×20×

20× 20×10×10×

NA

NA

NA

NA

NA NA

NA

NA

NA

NA

NA

NA

NA

NANA

******* **** ****

***

***

***** ****

*** ****

**** ****

****

****

** **

***** **

***

**** *** ***

* *

*

**

**

* **

*

**

*

** *

n.s.

n.s. n.s.

* *

*


0200

400

600

8001,000

0

2,000

4,000

6,000

8,000

0

20

40

60

0

5

10

15

0

2

4

6

8

0.0

0.5

1.0

1.5

0.0

0.5

1.0

1.5

080

4,00010,00016,00022,00028,000

030

5,000

15,000

25,000

35,000

Liver Serum


Supplementary Figure 6. Blocking 12-HETE production protects mice against IR-induced liver

damage. (a,b) The liver and serum 12-HETE contents (a) and serum ALT and AST levels (b) in mice

treated with ML355, CDC or vehicle controls in the sham group or at 6 h after reperfusion. n = 6 mice

in each group. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; by one-way ANOVA with

Bonferroni’s post hoc analysis. (c) Representative H&E staining images (NA, necrotic area) of mice

in the indicated groups at 6 h after reperfusion. n = 4 mice for each group with 24 images for each

mouse. Scale bar, 200 Pm for H&E staining (10u); 100 Pm for H&E staining (20u). (d,e) The liver

and serum 12-HETE contents (d, n = 6) and serum ALT and AST levels (e, n = 7) of mice treated with

ML355 at a dose of 1, 2, or 3 mg per kg body weight. For d, * P < 0.05, *** P < 0.001, **** P <

0.0001; by one-way ANOVA with Bonferroni’s post hoc analysis. For e, * P < 0.05, ** P < 0.01; by

one-way ANOVA with Bonferroni’s post hoc analysis (ALT) or with Tamhane’s T2(M) post hoc

analysis (AST). (f) Representative H&E staining images (NA, necrotic area) of liver sections from

mice subjected to 1 h of ischemia followed by 6 h of reperfusion and treated with 1, 2, or 3 mg per kg

body weight of ML355. n = 4 mice for each group with 24 images for each mouse. Scale bar, 200 Pm

for H&E staining (10u); 100 Pm for H&E staining (20u). (g) The ALT and AST levels in the serum of

m Sham

ML355

Rep 6 h

Vehicle ML355Vehicle

GAPDH

p-p65

p-JNK

p-p38

p-ERK

ERK

p65

JNK

p38

Ly6GSham Isch 1 h Rep 6 h


Vehi

cle

ML3

55

Sham Isch 1 h Rep 6 hCD11b

Rel

ativ

e m

RN

A ex

pres

sion

(nor

mal

ized

toAc

tb)

Tnf Il1b Il6 Cxcl2Ccl2

k l

*** * * ***

VehicleML355

0.0

0.5

1.0

1.5

2.0


mice treated with ML355 or vehicle controls at different time points during hepatic IR injury. n = 10

mice in each group. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 compared to

corresponding time point of vehicle controls by two-tailed Student’s t-test. (h) Representative TUNEL

images (green) of liver sections from mice with ML355 administration or vehicle at 6 h after

reperfusion. The nuclei were labeled with DAPI (blue). n = 4 for each group with 24 images for each

mouse. Scale bar, 20 Pm. (i) Protein expression of apoptotic factors in the livers of mice from indicated

groups. GAPDH was the loading control. n = 6 mice for each group; n = 3 western blots for each band.

(j,k) The serum level (j, n = 4 in vehicle-sham group; n = 6 in other groups) and hepatic relative mRNA

expression (k, n = 4; gene expression was normalized to that of Actb) of inflammatory cytokines and

chemokines in mice treated with ML355 or vehicle control. For j, * P < 0.05, ** P < 0.01, **** P <

0.0001, n.s., not significant; by one-way ANOVA with Bonferroni’s post hoc analysis (CXCL10) or

with Tamhane’s T2(M) post hoc analysis (TNF and CCL2). For k, * P < 0.05, ** P < 0.01, *** P <

0.001; by two-tailed Student’s t-test. (l) The CD11b+ (left) and Ly6G+ (right) inflammatory cell (red)

infiltration in the livers of mice in the indicated groups after 6 h of reperfusion. The nuclei were labeled

with DAPI (blue). n = 4 mice for each group with 24 images for each mouse. Scale bar, 20 Pm. (m)

The protein expression of key factors activated by 12-HETE in the livers of mice with ML355 or

vehicle treatment after 6 h of reperfusion. GAPDH was the loading control. n = 6 mice in 3 western

blots. Data are shown as mean ± s.e.m.


Supplementary Figure 7. Blocking 12-HETE production protects pigs against IR-induced liver

damage. (a) Schematic images of experimental protocols of pig hepatic IR injury model with ML355

a

Time pointof hepatic IR

1-2-2.5 0 3 6 12 24 36 48 (h)

Ischemia ReperfusionSerum collection at each time point Tissue collection

ML355 (0.4mg/kg)or Vehicle

ML355 (0.4mg/kg)or Vehicle

d H&E(10×)

H&E(40×)

Vehi

cle

ML3

55

TUNEL(40×)

e

g

Vehi

cle

ML3

55

CD68Ly6GCD11b

Vehicle ML355

GAPDH

p-p65

p-JNK

p-p38

p-ERK

ERK

p65

JNK

p38

12-H

ETE

cont

ent i

nse

rum

(fol

d ch

ange

)

12-H

ETE

cont

ent i

nliv

er (f

old

chan

ge)

Baseline Rep 12 hRep 48 h

Baseline Rep 12 hBaseline Rep 12 h

Vehicle ML355

Vehicle ML355

Vehicle ML355

Vehicle ML355

Baseli

ne

Isch 1

h

Rep 1

h

Rep 3

h

Rep 12

h

Rep 6

h

Rep 24

h

Rep 36

h

Rep 48

h

Isch 2

h

Baseli

ne

Isch 1

h

Rep 1

h

Rep 3

h

Rep 12

h

Rep 6

h

Rep 24

h

Rep 36

h

Rep 48

h

Isch 2

h

Baseli

ne

Isch 1

h

Rep 1

h

Rep 3

h

Rep 12

h

Rep 6

h

Rep 24

h

Rep 36

h

Rep 48

h

Isch 2

h

Baseli

ne

Isch 1

h

Rep 1

h

Rep 3

h

Rep 12

h

Rep 6

h

Rep 24

h

Rep 36

h

Rep 48

h

Isch 2

h

Rel

ativ

e m

RN

A ex

pres

sion

(nor

mal

ized

toAC

TB)

TNF IL1B CXCL2CCL2

CC

L2 c

onte

nt in

ser

um(p

g/m

l)

IL6

cont

ent i

n se

rum

(pg/

ml)

ALT

(U/L

)

AST

(U/L

)

ALP

(U/L

)

LDH

(U/L

)

b

c

f

h

0

20

40

60

0

2

4

6

*** ***

*** *

* ***

**

**

* * ** ** ****

**** *******

***

**** * * * *

n.s.

n.s.n.s.


NA NA

NA

NA

NA

NA

NA

NA

0

1

2

3

4

0.0

0.5

1.0

1.5

2.0

0

1,000

2,000

3,000

0

200

400

600

800

0

50

100

150

0

300

600

900

1,200

0.0

0.5

1.0

1.5

2.0


administration. n = 6 pigs per group at each time point. (b,c) The liver and serum 12-HETE contents

(b, n = 5 pigs in each group) and serum enzyme levels (c, n = 6 per group) of pigs treated with ML355

at indicated time points after hepatic IR injury. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P <

0.0001, n.s., not significant; compared to corresponding time points of vehicle control by two-tailed

Student’s t-test (left b,c) or by one-way ANOVA with Bonferroni’s post hoc analysis (right b). (d)

Representative images of H&E (NA, necrotic area) and TUNEL staining (green, positive cells; blue,

nuclei labeled by DAPI) of liver sections from ML355- or vehicle-treated pigs at 48 h after reperfusion.

n = 5 pigs in each group with 36 images for each pig. Scale bar, 200 Pm for H&E staining (10u); 20

Pm for H&E staining and TUNEL (40u). (e,f) The serum contents (e, n = 3 in vehicle group; n = 4 in

ML355 group) and hepatic relative mRNA expression (f, n = 4 for vehicle group; n = 6 for ML355

group) of inflammatory mediators of pigs treated with ML355. * P < 0.05, ** P < 0.01, *** P < 0.001,

n.s., no significance; by one-way ANOVA with Bonferroni’s post hoc analysis (e) or by two-tailed

Student’s t-test (f). (g,h) The representative images of inflammatory cell infiltration (g; n = 4 for each

group with 10 images for each pig) and the protein expression of key inflammation-related factors

influenced by 12-HETE (h; n = 4 pigs in each group with 3 technical replicates; GAPDH served as the

loading control) in the livers of pigs treated with ML355 or vehicle controls at 48 h after reperfusion.

Scale bar, 20 Pm. For statistical blots, data are shown as mean ± s.e.m.



Ischemia 30 minIschemia 120 min Ischemia 60 min

ALP

in s

erum

(U

/L)

a

AST

in s

erum

(U

/L)

Baseli

ne

Ische

mia

Rep 0

h

Rep 1

h

Rep 3

h

Rep 6

h

Rep 12

h

Rep 24

h

Rep 36

h

Rep 48

h

Rep 72

h

Rep 1

w

Rep 2

w

Rep 3

w

Rep 4

w

Baseli

ne

Ische

mia

Rep 0

h

Rep 1

h

Rep 3

h

Rep 6

h

Rep 12

h

Rep 24

h

Rep 36

h

Rep 48

h

Rep 72

h

Rep 1

w

Rep 2

w

Rep 3

w

Rep 4

w

Baseli

ne

Ische

mia

Rep 0

h

Rep 1

h

Rep 3

h

Rep 6

h

Rep 12

h

Rep 24

h

Rep 36

h

Rep 48

h

Rep 72

h

Rep 1

w

Rep 2

w

Rep 3

w

Rep 4

w

ALT

in s

erum

(U

/L) ** ** **

** **

* *** **** **

*

** ** *

**

*

Sham

HAP HPP

HTP HPI

HAP HPP

HTP HPI

HAP HPP

HTP HPI

HAP HPP

HTP HPI

HAP HPP

HTP HPI

HAP HPP

HTP HPI

Rep 2 weeks Rep 3 weeks Rep 4 weeks

Rep 48 h Rep 1 weekIschemia - 30 minb

Rep 48 h Rep 1 week

Rep 2 weeks Rep 3 weeks Rep 4 weeks

Sham

HAP HPP

HTP HPI

HAP HPP

HTP HPI

HAP HPP

HTP HPI

HAP HPP

HTP HPI

HAP HPP

HTP HPI

HAP HPP

HTP HPI

Ischemia - 120 minc

0

500

1,000

1,500

2 000

0

500

1,000

1,500

2,000

0

200

400

600

800

1,000



Control Rep 48 h

ALOX12

GAPDH

Control Rep 48 hRel

ativ

eAL

OX1

2 ex

pres

sion

(nor

mal

ized

to18

S rR

NA)

e

j

f

*

d

ML3

55 c

onte

nt in

ser

um (u

g/m

l)

012345

10203040

0.0

0.5

1.0

1.5

2.0

2.5

Ischemia - 60 min

12-H

ETE

cont

ent i

nse

rum

(fol

d ch

ange

)

12-H

ETE

cont

ent i

nliv

er (f

old

chan

ge)

Baseline Rep 12 hRep 48 h

Vehicle ML355 Vehicle ML355iLiver Serum

** *n.s.

0369

12

0.00.51.01.52.0

g

3 w 4 w10 3 6 12 48 72 (h)

Ischemia Reperfusion

24 36

Serum collection at each time pointLiver biopsy at Rep 48 h

Liver CT at each time point(except 72 h)

ML355 (3mg/kg)or Vehicle

ML355 (3mg/kg)or Vehicle

1 w 2 wTime points of hepatic IR

-1.5 -1

Rep

48

h

Vehicle ML355

Baseline Rep 12 h

Vehicle ML355

GAPDH

p-p65

p-JNK

p-p38

p-ERK

ERK

p65

JNK

p38

k

l

IL6

cont

ent i

n se

rum

(pg/

ml)

** * *

n.s.

0

5

10

15

20

Baseline Rep 12 hCC

L2 c

onte

nt in

ser

um(p

g/m

l)

* *

n.s.

0

2

4

6

8


Supplementary Figure 8. ML355 inhibits liver damage in a monkey hepatic IR model. (a) The ALT,

AST and ALP levels in the serum of monkeys at indicated time points after a 30 min-, 60 min-, or 120

min-ischemia in the left lateral lobe of liver. n = 5 in each group. * P < 0.05, ** P < 0.01 compared to

corresponding time points in ischemia 60 min group by two-tailed Student’s t-test. (b-d)

Representative perfusion CT images of monkeys exposed to a 30 min-(b), 120 min-(c) or 60 min-

ischemia (d) followed by reperfusion for indicated periods. HAP, hepatic arterial perfusion; HPP,

hepatic portal perfusion; HTP, hepatic total perfusion; HPI, hepatic perfusion index. n = 5 monkeys in

each group; each image was generated from 39 scans in each monkey. (e,f) Relative ALOX12 mRNA

(e, n = 5 monkeys per group; normalized to 18S rRNA) and protein expression (f, n = 4 monkeys in

each group with 3 technical replicates) in the IR-challenged lobes and control lobes of monkey livers

at 48 h after reperfusion. * P < 0.05 by two-tailed Student’s t-test. (g) Schematic illustration of protocol

used to evaluate ML355 effects on the monkey hepatic IR injury. (h) The serum ML355 concentration

of monkeys at each indicated time points before or after hepatic IR surgery. n = 5 monkeys in each

time point. (i) The hepatic and serum 12-HETE contents (n = 5 in each group) in monkeys treated by

ML355 (3 mg per kg body weight) or vehicle controls after 60 min of hepatic ischemia followed by

indicated periods of reperfusion. * P < 0.05, n.s., not significant; by two-tailed Student’s t-test (left) or

by one-way ANOVA with Bonferroni’s post hoc analysis (right). (j) Representative images of TUNEL

staining (green) on the monkey liver sections in ML355 and vehicle groups after 48 h of reperfusion.

The nuclei were labeled by DAPI (blue). n = 5 monkeys per group with 24 images for each monkey.

Scale bar, 20 Pm. (k) The serum contents of inflammatory cytokines and chemokines of ML355- or

vehicle-treated monkeys at 12 h after reperfusion. n = 4 monkeys in vehicle group; n = 5 monkeys in

ML355 group. * P < 0.05, *** P < 0.001, n.s., no significance; by one-way ANOVA with Bonferroni’s

post hoc analysis (IL6) or with Tamhane’s T2(M) post hoc analysis (CCL2). (l) The protein expression

of phosphorylated and total p65, JNK, p38 and ERK in the livers of monkeys treated with ML355 or

vehicle after 60 min of ischemia followed by 48 h of reperfusion. n = 4 monkeys in each group; n = 3

technical replicates. GAPDH served as a control. For all statistical analysis, data are shown as mean ±

s.e.m.


Supplementary Figure 9. ML355 exerts negligible impacts on myocardial infarction. The

concentrations of CK (left) and LDH (middle) in the serum and EF% (right) of mice subjected to a

myocardial infarction insult. n = 6 mice in each group. * P < 0.05, n.s., not significant; by two-tailed

Student’s t-test. Date are shown as mean ± s.e.m.


Sham

CK

(U/L

)

LDH

(U/L

)

EF (%

)

1 d 3 d 7 d Sham 1 d 3 d 7 d Sham 1 d 3 d 7 d

Vehicle ML355

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

*

n.s.n.s. n.s. n.s.

0

500

1,000

1,500

0

500

1,000

1,500

2,000

0

50

100

150


170130100

7055

40

35

25

15

GAPDH

170130100

70554035

25

15

ALOX12

170130100

7055403525

15

GAPDH

Case1 Case2 Case3 Case4

B I R B I R B I R B I R

Western blots for Figure 1

1i

ALOX12

170130100

705540

35

25

15

1h

Supplementary Figure 10. Original uncropped gels for western blot analyses shown in Figure 1.


170130100

705540

3525

15

2d Normoxia Hypoxia

shGFP shAlox12 shGFP shAlox12

GAPDH

ALOX12

2e

170130100

7055403525

GAPDH

170130100

705540

35

25

15

ALOX12

Normoxia Hypoxia

GFP Alox12 GFP Alox12


170130100

7055

40

3525

15



3d

170130100

7055

403525

15

WT KO WT KO

Sham Rep 6 h

BCL2

170130100

7055403525

15BAX

170130100

70554035

25

15

170130100

7055403525

15

c-CASP3

GAPDH

3h

170130100

7055403525

p-IκBα

170130100

7055

40

35

25

15

IκBα

170130100

705540

35

25

15

p-p65

170130100

705540

3525

15

GAPDH

170130100

705540

35

25

15

p65

WT KO WT KOSham Rep 6 h


15



4c

p-p65

p65

p-p38

p38

GAPDH

130100

70554035

25

15

170

130100

70554035

25

15

170

130100

705540

35

25

15

170

130100

705540

35

25

15

170

130100

705540

35

25

15

170

130100

705540

3525

170

15

130100

705540

3525

170

15

130100

7055

4035

25

170

15

130100

70554035

25

170

15

Time(min) 10 20 40 60012-HETE

Time(min) 10 20 40 60012-HETE

p-JNK

JNK

p-ERK

ERK

4c



4f

p-p38

p38

GAPDH

p-p65

p65

Vehicle ML355Normoxia Hypoxia

Vehicle ML355

1301007055403525

15

130100

70554035

25

15

170

130100

7055403525

15

170

130100

705540

35

25

15

170

130100

705540

3525

15

170

130100

705540

3525

170

15

130100

705540

35

25

170

15

130100

7055

40

35

25

170

15

1301007055403525

170

15

p-JNK

JNK

p-ERK

ERK

Vehicle ML355Normoxia Hypoxia

Vehicle ML3554f

170




5c

GPR40

GPR75

GPR120

p-PKC

p-JNK

GAPDH

12-HETE (100 nM) - + - + - + - +

130100

705540

35

25

170

15

130100

705540

35

25

170

15

130100

7055

40

3525

170

130100

7055

4035

25

170

15

130100

705540

35

25

15

170

130100

70554035

25

15

170

130100

70554035

25

15

170

- +

GPR31

PKC

JNK

12-HETE (100 nM) - + - + - + - + - +

130100

7055

40

35

25

170

5c


130100

7055403525

170

15



ALOX12

ALOX5

ALOX15

CYP4A

CPLA2

GAPDH

Sham Isch 1 h

130100

705540

3525

170

130100

70554035

25

15

170

130100

705540

3525

15

170

130100

7055403525

15

170

130100

7055403525

15

170

15

Western blots for Supplementary Figure1

130100

7055403525

170

1g

Supplementary Figure 15. Original uncropped gels for western blot analyses shown in Supplementary Figure 1.


170130100

7055

40

35

25

15

WT KO

130100

705540

35

25

15

170

2d

Western blots for Supplementary Figure 2

GAPDH

ALOX12

NTG HTG NTG HTG NTG HTG NTG HTG

Liver Heart KidneyBrain

GAPDH

170130100

7055

40

3525

15

ALOX12

2e-1

170130100

705540

3525

170130100

7055

40

3525

15

GAPDH

ALOX12170130100

705540

3525

15

NTG HTG-1 HTG-2 HTG-32e-2



3d

BCL2

GAPDH

BAX

c-CASP3

17013010070

55403525

170130100

705540

3525

170130100

705540

3525

NTG HTG NTG HTG

Sham Rep 6 h3h

170130100

705540

35 GAPDH

17013010070554035

25

15

p65

p-p65

17013010070

55403525

15

170130100

705540

352515

IκBα

170130100

705540

3525

p-IκBα

NTG HTG NTG HTG

Sham Rep 6 h

1007055

40

35

25

15


15


15

15


4c

p-p65

p65

p-JNK

JNK

p-p38

p38

p-ERK

ERK

GAPDH

170130100

705540

35

25

15

130100

705540

3525

15

170

130100

70554035

25

15

170

130100

7055403525

15

170

130100

705540

3525

15

170

130100

7055403525

170

13010070

55403525

15

170

130100

705540

35

25

15

170

13010070

554035

170

GFP Alox12 GFP Alox12Normoxia Hypoxia

Flag

GFP Alox12 GFP Alox12

Normoxia Hypoxia4c

170130

100

70

55

40

35

2515

15



4e

p-p65

ALOX12

p65

p-p38

p38

GAPDH

shGFP shAlox12 shGFP shAlox12Normoxia Hypoxia

170130100

70554035

25

15

130100

70

5540

35

25

15

170

130100

7055403525

15

170

130100

7055403525

15

170

130100

705540

3525

15

170

130100

7055403525

15

170

130100

705540

3525

15

170

130100

705540

35

25

15

170

130100

705540

35

25

15

170

130100

7055403525

15

170

shGFP shAlox12 shGFP shAlox12

Normoxia Hypoxia

p-JNK

JNK

p-ERK

ERK

4e




p-JNK

JNK

p-p38

p38

p-ERK

ERK

GAPDH

Sham

NTG HTG NTG HTG

Rep 6 h

Sham

WT KO WT KO

Rep 6 h

p-JNK

JNK

p-p38

p38

p-ERK

ERK

GAPDH

130100

705540

3525

170

15

130100

7055403525

170

15

130100

705540

35

25

170

15

130100

7055403525

170

15

130100

705540

3525

170

15

130100

705540

3525

170

15

130100

7055403525

170

15

130100

7055403525

170

15

130100

705540

3525

170

15

130100

705540

3525

170

15

130100

7055

40

35

25

170

15

130100

7055

403525

170

15

130100

7055

40

35

25

15

170130100

705540

35

25

15

170

4g 4h



5d

p-PKC

PKC

p-p65

p65

p-p38

p38

GAPDH

GPR31

12-HETE (100 nM) 0 10 30 0 10 30 0 10 30

WT GPR31-KO GPR31-res

130100

7055403525

15

170

130100

7055403525

15

170

130100

705540

35

25

15

170

130100

70554035

25

15

170

130100

70

55

40

35

25

170

13010070

5540352515

170

130100

7055

4035

25

15

170

130100

7055

4035

25

15

170

130100

70554035

25

15

170

130100

705540

3525

15

170

130100

70554035

25

15

170

0 10 30 0 10 30 0 10 30WT GPR31-KO GPR31-res12-HETE

(100 nM)

p-JNK

JNK

p-ERK

ERK

130

1007055

40

35

25

15

170

5d


(min) (min)


5f

GAPDH

130100

7055403525

15

170

GPR31B

WT Gpr31b-KO

130100

70

5540

3525

15

170




6i

BCL2

BAX

c-CASP3

GAPDH

130100

7055403525

15

170

130100

7055403525

15

170

130100

7055403525

15

170

130100

7055403525

15

170

Vehicle ML355Sham

Vehicle ML355Rep 6 h


6m

p-p65

p65

Vehicle ML355Sham

Vehicle ML355

Rep 6 h

130100

705540

3525

15

170

130100

705540

3525

15

170

130100

70554035

25

15

170

130100

7055

40

3525

15

170

p-JNK

JNK

p-p38

p38

130100

7055403525

15

170

130100

7055

4035

25

15

170

1301007055403525

15

170

p-ERK


GAPDH

130100

7055403525

15

170

130100

705540

3525

15

170

ERK

Vehicle ML355

Sham

Vehicle ML355

Rep 6 h6m


p-p65

p65

Vehicle ML355

130100

70554035

25

15

170

7055403525

15

705540

3525

15

130100

705540

3525

15

170

130170100

130170100

p38

GAPDH

130100

70554035

25

15

170

705540

35

25

15

7055

40

3525

15

130100

7055403525

15

170

170100130

100130170

Vehicle ML355

p-JNK

JNK

p-ERK

ERK

7h


p-p38

130100

705540

3525

15

170


7h



8f

GAPDH

ALOX12

Control Rep 48 h

130100

7055403525

15

170

1301007055403525

15

170


8l

p-p65

p65

Vehicle ML355

130100

705540

3525

170

1301007055403525

15

170

130100

7055403525

15

170

p-JNK

JNK

130100

7055403525

15

170

p-p38

p38

130100

7055403525

170

130100

705540

3525

15

170

GAPDH

130100

7055403525

15

170

130100

7055403525

15

170

ERK

8l Vehicle ML355

130100

7055403525

15

170

p-ERK


Supplementary Table 1. Primers for mouse generation.

Primer name/Target sites Sequence 5'---3'

Alb-Flag-Alox12 mouse

Alox12(l)-F CGACGCGTATGGGCCGCTACCGC Alox12(l)-R ATAAGAATGCGGCCGCTCAGATGGT

GATACTGTTCTCTATGCGG Alb-seq-F ACACCCTGGTCATCATCCTG Alb-seq-R GCTCAAGGGGCTTCATGATG

Alox12-KO mouse

Alox12-sgRNA GGAGGGTATAAACACGTTTGAGG pUC57-sgRNA-F GATCCCTAATACGACTCACTATAG pUC57-sgRNA-R AAAAAAAGCACCGACTCGGT Alox12-P1 TGGACTTTGAATGGACGTTG Alox12-P2 GGGAGCACAGAAAGGACAAG

Gpr31b-KO mouse

Gpr31b-sgRNA1 GGTGTGTGCTTTGTGACAGGAAGGG Gpr31b-sgRNA2 GGTCTTCTGGAGGGTCCTGATGAGG Gpr31b-sgRNA1 F TAGGTGTGTGCTTTGTGACAGGAA Gpr31b-sgRNA1 R AAACTTCCTGTCACAAAGCACACA Gpr31b-sgRNA2 F TAGGTCTTCTGGAGGGTCCTGATG Gpr31b-sgRNA2 R AAACCATCAGGACCCTCCAGAAGA Gpr31b-P1 CCCAAACAAATTCTGCTGGCT Gpr31b-P2 TTGAGCACCTTCCGGTATGAG


Supplementary Table 2. Clinical information for individuals included in examination of ALOX12 expression and 12-HETE contents.

Baseline Ischemia Reperfusion ALT (U/L) 24.1 ± 3.0 50.6 ± 5.6 400.5 ± 56.9 AST (U/L) 27.1 ± 3.3 66.8 ± 8.4 400.1 ± 64.2 ALP(U/L) 75.7 ± 10.3 62.8 ± 7.0 73.9 ± 6.6

Period(min) N/A 16.9 ± 1.7 33.1 ± 5.4 Sex Male (n=14, 73.7%) Female (n=5, 26.3%)

Age(years) 55.0 ± 1.9 Data are expressed as mean ± s.e.m. in this table. ALT, alanine amino transferase; AST, aspartate amino transferase; ALP, alkaline phosphatase.


Supplementary Table 3. Primers for qPCR analysis.

Mice Gene Sequence 5'---3' Actb F GTGACGTTGACATCCGTAAAGA R GCCGGACTCATCGTACTCC Alox12 F TCCCTCAACCTAGTGCGTTTG R GTTGCAGCTCCAGTTTCGC Tnf F AGCCGATGGGTTGTACCTTG R ATAGCAAATCGGCTGACGGT Il1b F CCGTGGACCTTCCAGGATGA R GGGAACGTCACACACCAGCA Il6 F AGGATACCACTCCCAACAGACCT R CAAGTGCATCATCGTTGTTCATAC Ccl2 F TTGGCTCAGCCAGATGCA R CCTACTCATTGGGATCATCTTGC Cxcl2 F GCGCCCAGACAGAAGTCATA R CAGTTAGCCTTGCCTTTGTTCA Alox5 F AACGATCACCCACCTTCTGC R TCGCAGATAAGCTGTTCCCG Alox15 F GCTGCCCAATCCTAATCAGTC R TTCCTTATCCAAGGCAGCCAG Gpx1 F TCGGTTTCCCGTGCAATCAG R GTCGGACGTACTTGAGGGAA Ggt1 F CCGCCTACTATGAGCCTGAA R GCGAGCTGAAGTCATCCATC Cbr2 F GGCCCATGTCACCTTTCCTA R TTACCCGGATCTTGTGTGGC Ptges F AGGATGCGCTGAAACGTGGA R ATGAGTACACGAAGCCGAGG

Pigs

Gene Sequence 5'---3' ACTB F ACGATATTGCTGCGCTCGTG R TTCTCCATGTCGTCCCAGTTG TNF F TGCACTTCGAGGTTATCGGC R GGCATACCCACTCTGCCATT IL1B F CCCATCATCCTTGAAACGTGC R TCCTTGCACAAAGCTCATGC CCL2 F GAGTCACCAGCAGCAAGTGT R TCAAGGCTTCGGAGTTTGGTT CXCL2 F CCCACTGTGACCAAACGGAA R CAGTTGGCACTGCTCTTGTT


Non-human primates

Gene Sequence 5'---3' 18S rRNA F GAGCCTGCGGCTTAATTTGA R AACTAAGAACGGCCATGCAC ALOX12 F CCATGTGCAGTTGCTTCGTC R CCCAGAGCCGTAAAGCATCA TNF F AAGGGTCCTGCCTCTTACAAT R CAGATGGTGAGTCTGCTCAGT IL1B F TTGGGCCTCAAGGCAAAGAA R GGGAATTGGGCAGACTCGAA CCL2 F GCAGCAAGTGTCCCAAAGAAG R CCATGGAATCCTGAACCCACT CXCL5 F GTTTACAGACCACGCAGGGA R GAGACAAACTTCCGTCCCGT

Human Gene Sequence 5'---3' ACTB F CGGGGACCTGACTGACTACC R AGGAAGGCTGGAAGAGTGC ALOX12 F GATCCCCCATATCCGCTACAC R CCACCTGTGCTCACTGCCTTAT IL6 F TCTGGATTCAATGAGGAGACTTG R GTTGGGTCAGGGGTGGTTAT TNF F TACTCCCAGGTCCTCTTCAAGG R TTGATGGCAGAGAGGAGGTTG CCL2 F GTCTCTGCCGCCCTTCTG R ACTTGCTGCTGGTGATTCTTCT CXCL10 F GTGGCATTCAAGGAGTACCTC R TGATGGCCTTCGATTCTGGATT


Supplementary Table 4. Plasmids and primers for expression constructs.

Plasmids name Reference sequence

Species Sequence5'---3'

pcDNA5-hGPR31-Flag NM_005299.2 Human F:ACACCGGCGGCCACGCGTATGCCATTCCCAAACTGCTC R:GGAGGTACCTCCGGATCCTTACTTATCGTCGTCATCCTTG

pGL3-U6-hGPR31-gRNA NM_005299.2 Human F: ACCGTCCACACCCTGACCCGGAAC R: AAACGTTCCGGGTCAGGGTGTGGA

pLKO.1-sh hGPR31 NM_005299.2 Human

F:CCGGCACTCTCCTGCCTTCAGTTTGCTCGAGCAAAC TGAAGGCAGGA1GAGTGTTTTTG R:AATTCAAAAACACTCTCCTGCCTTCAGTTTGCTCGA GCAAACTGAAGGCAGGAGAGTG


F: CCGGCATCACAGCCTTCTGCTACGTCTCGAGACGTA GCAGAAGGCTGTGATGTTTTTG R:AATTCAAAAACATCACAGCCTTCTGCTACGTCTCGA GACGTAGCAGAAGGCTGTGATG


F:CCGGCCGACCAGGAAATTTCGATTTCTCGAGAAATC GAAATTTCCTGGTCGGTTTTTG R:AATTCAAAAACCGACCAGGAAATTTCGATTTCTCGA GAAATCGAAATTTCCTGGTCGG


F:CCGGCTGATAGTAATGGACTTTATTCTCGAGAATAAA GTCCATTACTATCAGTTTTTG R:AATTCAAAAACTGATAGTAATGGACTTTATTCTCGAG AATAAAGTCCATTACTATCAG


Supplementary Table 5. Parameters of pigs during hepatic IR surgery. Index Baseline Ischemia Reperfusion Body weight (kg) Vehicle 26.7 r 2.5 N/A N/A ML355 21.7 r 0.9 N/A N/A Heart rate / min Vehicle 102.5 r 4.9 117.2 r 13.5 140.0 r 16.3 ML355 109.0 r 5.7 103.7 r 11.4 128.6 r 7.2 Breathing rate / min Vehicle 37.5 r 2.2 43.2 r 1.0 40.0 r 1.3 ML355 36.4 r 0.9 36.4 r 0.9 36.4 r 0.9 Tidal volume Vehicle 146.3 r 13.8 118.0 r 5.2 118.0 r 6.3 ML355 120.0 r 4.9 120.0 r 3.8 118.6 r 4.6 Blood pressure (High) Vehicle 120.1 r 5.3 77.1 r 8.6 98.8 r 5.1 ML355 105.3 r 5.4 63.1 r 7.8 90.6 r 9.0 Blood pressure (Low) Vehicle 81.4 r 5.2 48.4 r 4.9 60.3 r 4.1 ML355 73.7 r 4.5 43.0 r 5.6 60.4 r 5.8 Oxygen saturation Vehicle 97.3 r 0.8 95.5 r 1.9 97.5 r 1.0 ML355 98.4 r 0.7 97.0 r 1.9 99.0 r 0.7 Plasma flow Vehicle 216.7 r 40.6 35.8 r 6.6 160.0 r 10.0 ML355 181.0 r 34.5 35.4 r 6.2 157.0 r 24.6 Data are expressed as mean ± s.e.m. in this table.


Supplementary Table 6. Parameters of monkeys during hepatic IR surgery.

Index Vehicle-120min Vehicle-60min Vehicle-30min ML355-60min Body weight (kg) 4.6 r 0.2 4.9 r 0.2 4.8 r 0.1 4.8 r 0.3 Heart rate-Base (/min) 123.7 r 7.0 121.2 r 7.6 129.2 r 4.5 100.3 r 5.6 Heart rate-Isch (/min) 117.3 r 5.1 107.2 r 11.6 122.2 r 6.3 98.0 r 5.0 Heart rate-Rep (/min) 101.2 r 4.0 116.6 r 8.2 126.4 r 4.3 105.2 r 7.4 Blood pressure (high)-Base (mmHg) 98.8 r 7.0 90.2 r 7.5 120.0 r 5.3 84.7 r 4.3 Blood pressure (low)-Base (mmHg) 67.5 r 4.3 61.0 r 3.4 72.8 r 4.4 57.5 r 3.3 Blood pressure (high)-Isch (mmHg) 83.3 r 5.3 85.8 r 6.8 94.0 r 5.5 81.3 r 4.5 Blood pressure (low)-Isch (mmHg) 55.8 r 3.1 58.6 r 6.2 58.2 r 4.7 58.2 r 5.4 Blood pressure (high)-Rep(mmHg) 85.3 r 7.6 92.0 r 10.1 92.2 r 1.9 77.2 r 2.4 Blood pressure (low)-Rep (mmHg) 57.2 r 6.4 64.2 r 9.4 59.4 r 3.4 53.5 r 2.6 Oxygen saturation 100 100 100 100 Rectal temperature-Base (oC) 37.0 r 0.3 37.2 r 0.1 37.3 r 0.2 37.2 r 0.3 Rectal temperature- Isch (oC) 37.1 r 0.3 37.2 r 0.3 37.6 r 0.3 37.0 r 0.2 Rectal temperature-Rep(oC) 37.4 r 0.2 37.5 r 0.3 37.6 r 0.3 37.3 r 0.3 Plasma flow-Base 205.0 r 28.2 190.0 r 14.8 161.6 r 17.2 160.5 r 19.5 Plasma flow-Isch 36.0 r 4.3 26.0 r 6.2 25.5 r 1.9 35.5 r 2.6 Plasma flow-Rep 152.5 r 13.0 147.5 r 20.6 147.0 r 13.2 166.7 r 36.6

Data are expressed as mean ± s.e.m. in this table.


supplementary figure 1 a b d - nature research · supplementary figure 1. alox12 is dramatically...

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