12
Genes Forward primer (5’-3’) Reverse primer (5’-3’) 18s ACGGAAGGGCACCACCAGGA CACCACCACCCACGGAATCG Ly6g TGCGTTGCTCTGGAGATAGA CAGAGTAGTGGGGCAGATGG F4/80 CTTTGGCTATGGGCTTCCAGTC GCAAGGAGGACAGAGTTTATCGTG Bak AGACCTCCTCTGTGTCCTGG AAAATGGCATCTGGACAAGG Bax GATCAGCTCGGGCACTTTAG TTGCTGATGGCAACTTCAAC Bim GCTCCTGTGCAATCCGTATC GCCCCTACCTCCCTACAGAC Chop GACCAGGTTCTGCTTTCAGG CAGCGACAGAGCCAGAATAA Gadd34 GGAGATAGAAGTTGTGGGCG TTTTGGCAACCAGAACCG Ero1a CACAGGTACAGTCGTCCAGGT CTTGCTCGTTGGACTCCTG Ero1b TGACAAAAAGGGGGCCAAGT TATCGCACCCAACACAGTCC Pdi CCCCGTGTGTGGAAAAGAGA AGCCACAGAGTAATGTGCCC Il1b TCGCTCAGGGTCACAAGAAA CATCAGAGGCAAGGAGGAAAAC Il6 TCCATCCAGTTGCCTTCTTG TTCCACGATTTCCCAGAGAAC Tnfa AGGCTGCCCCGACTACGT GACTTTCTCCTGGTATGAGATAGCAAA Vcam1 TGAACCCAAACAGAGGCAGAGT GGTATCCCATCACTTGAGCAGG Icam1 CAATTTCTCATGCCGCACAG AGCTGGAAGATCGAAAGTCCG Mcp1 TCTGGACCCATTCCTTCTTGG TCAGCCAGATGCAGTTAACGC Mip1a TGAGAGTCTTGGAGGCAGCGA TGTGGCTACTTGGCAGCAAACA Mip1b AACACCATGAAGCTCTGCGT AGAAACAGCAGGAAGTGGGA p47 phox TCCTCTTCAACAGCAGCGTA CTATCTGGAGCCCCTTGACA p67 phox TCTATCAGCTGGTTCCCACG CTATCTGGAGCCCCTTGACA p40 phox ATCGTCTGGAAGCTGCTCAA CCCATCCATCTGCTTTTCTG p22 phox ATGGAGCGATGTGGACAGAAG TAGATCACACTGGCAATGGCC gp91 phox GACCATTGCAAGTGAACACCC AAATGAAGTGGACTCCACGCG Mt1 AAGAGTGAGTTGGGACACCTT CGAGACAATACAATGGCCTCC Mt2 GCCTGCAAATGCAAACAATGC AGCTGCACTTGTCGGAAGC Rab11a TGGGAAAACAATAAAGGCACAGA ATGTGAGATGCTTAGCAATGTCA Rab11b ATTCAAAGTGGTGCTTATTGGGG TCCGATGGTACTCTTGCTCTC Rab35 CCACAATCGGAGTGGATTTCA CGTCGTAAACCACAATGACCC Rab5b TGGGTAAAGGAACTACAGCGG GGCCACCTTACTTCATACTCCA Rab27a TCGGATGGAGATTACGATTACCT TTTTCCCTGAAATCAATGCCCA Rab27b CGTCAGGAAAAGCGTTTAAGGT AGAAGCTCTGTTGACTGGTGA Vamp7 GACAACTTACGGTTCAAGAGCA TCTCCACGTTGAGCAACTAAATC Vamp8 AGTGGGAGTGCCGGAAATG TGAAGTGTTCAGACGTGGCTT Hgs TTCGAGCGTCTCCTAGACAAA GCTTGTGTGTCCCCCTGAC Pdcd61p TAGTGTTTGCACGGAAGACAG GGGAGGACTGATAGGCTGGA Tsg101 TCTAACCGTCCGTCAAACTGT TTGTACCAGTGAGGTTCACCA Stam1 ACCCCTTCGACCAGGATGTT CCACAGTTTGATACACATGCTCC Vta1 AAGAGCATACAGCACCATTTGA GCTTCATTATCCCCCAACTGTTT Ykt6 AGTCAACTGATTGTGGAACGC TCTGGAAGGGTATTCGCTGTC nSmase2 ACACGACCCCCTTTCCTAATA GGCGCTTCTCATAGGTGGTG Bcl2 GTCGCTACCGTCGTGACTTC CAGACATGCACCTACCCAGC Bclxl GACAAGGAGATGCAGGTATTGG TCCCGTAGAGATCCACAAAAGT Primer sequences for real-time PCR Supporting Table 1

Supporting Table 1 Primer sequences for real -time PCR · 2020. 7. 22. · Mt2 GCCTGCAAATGCAAACAATGC. AGCTGCACTTGTCGGAAGC Rab11a TGGGAAAACAATAAAGGCACAGA: ATGTGAGATGCTTAGCAATGTCA Rab11b

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Page 1: Supporting Table 1 Primer sequences for real -time PCR · 2020. 7. 22. · Mt2 GCCTGCAAATGCAAACAATGC. AGCTGCACTTGTCGGAAGC Rab11a TGGGAAAACAATAAAGGCACAGA: ATGTGAGATGCTTAGCAATGTCA Rab11b

Genes Forward primer (5’-3’) Reverse primer (5’-3’)18s ACGGAAGGGCACCACCAGGA CACCACCACCCACGGAATCGLy6g TGCGTTGCTCTGGAGATAGA CAGAGTAGTGGGGCAGATGGF4/80 CTTTGGCTATGGGCTTCCAGTC GCAAGGAGGACAGAGTTTATCGTGBak AGACCTCCTCTGTGTCCTGG AAAATGGCATCTGGACAAGGBax GATCAGCTCGGGCACTTTAG TTGCTGATGGCAACTTCAACBim GCTCCTGTGCAATCCGTATC GCCCCTACCTCCCTACAGACChop GACCAGGTTCTGCTTTCAGG CAGCGACAGAGCCAGAATAAGadd34 GGAGATAGAAGTTGTGGGCG TTTTGGCAACCAGAACCGEro1a CACAGGTACAGTCGTCCAGGT CTTGCTCGTTGGACTCCTGEro1b TGACAAAAAGGGGGCCAAGT TATCGCACCCAACACAGTCCPdi CCCCGTGTGTGGAAAAGAGA AGCCACAGAGTAATGTGCCCIl1b TCGCTCAGGGTCACAAGAAA CATCAGAGGCAAGGAGGAAAACIl6 TCCATCCAGTTGCCTTCTTG TTCCACGATTTCCCAGAGAACTnfa AGGCTGCCCCGACTACGT GACTTTCTCCTGGTATGAGATAGCAAAVcam1 TGAACCCAAACAGAGGCAGAGT GGTATCCCATCACTTGAGCAGGIcam1 CAATTTCTCATGCCGCACAG AGCTGGAAGATCGAAAGTCCGMcp1 TCTGGACCCATTCCTTCTTGG TCAGCCAGATGCAGTTAACGCMip1a TGAGAGTCTTGGAGGCAGCGA TGTGGCTACTTGGCAGCAAACAMip1b AACACCATGAAGCTCTGCGT AGAAACAGCAGGAAGTGGGA

p47phox TCCTCTTCAACAGCAGCGTA CTATCTGGAGCCCCTTGACA

p67phox TCTATCAGCTGGTTCCCACG CTATCTGGAGCCCCTTGACA

p40phox ATCGTCTGGAAGCTGCTCAA CCCATCCATCTGCTTTTCTG

p22phox ATGGAGCGATGTGGACAGAAG TAGATCACACTGGCAATGGCC

gp91phox GACCATTGCAAGTGAACACCC AAATGAAGTGGACTCCACGCGMt1 AAGAGTGAGTTGGGACACCTT CGAGACAATACAATGGCCTCCMt2 GCCTGCAAATGCAAACAATGC AGCTGCACTTGTCGGAAGCRab11a TGGGAAAACAATAAAGGCACAGA ATGTGAGATGCTTAGCAATGTCARab11b ATTCAAAGTGGTGCTTATTGGGG TCCGATGGTACTCTTGCTCTCRab35 CCACAATCGGAGTGGATTTCA CGTCGTAAACCACAATGACCCRab5b TGGGTAAAGGAACTACAGCGG GGCCACCTTACTTCATACTCCARab27a TCGGATGGAGATTACGATTACCT TTTTCCCTGAAATCAATGCCCARab27b CGTCAGGAAAAGCGTTTAAGGT AGAAGCTCTGTTGACTGGTGAVamp7 GACAACTTACGGTTCAAGAGCA TCTCCACGTTGAGCAACTAAATCVamp8 AGTGGGAGTGCCGGAAATG TGAAGTGTTCAGACGTGGCTTHgs TTCGAGCGTCTCCTAGACAAA GCTTGTGTGTCCCCCTGACPdcd61p TAGTGTTTGCACGGAAGACAG GGGAGGACTGATAGGCTGGATsg101 TCTAACCGTCCGTCAAACTGT TTGTACCAGTGAGGTTCACCAStam1 ACCCCTTCGACCAGGATGTT CCACAGTTTGATACACATGCTCCVta1 AAGAGCATACAGCACCATTTGA GCTTCATTATCCCCCAACTGTTTYkt6 AGTCAACTGATTGTGGAACGC TCTGGAAGGGTATTCGCTGTCnSmase2 ACACGACCCCCTTTCCTAATA GGCGCTTCTCATAGGTGGTGBcl2 GTCGCTACCGTCGTGACTTC CAGACATGCACCTACCCAGCBclxl GACAAGGAGATGCAGGTATTGG TCCCGTAGAGATCCACAAAAGT

Primer sequences for real-time PCRSupporting Table 1

Page 2: Supporting Table 1 Primer sequences for real -time PCR · 2020. 7. 22. · Mt2 GCCTGCAAATGCAAACAATGC. AGCTGCACTTGTCGGAAGC Rab11a TGGGAAAACAATAAAGGCACAGA: ATGTGAGATGCTTAGCAATGTCA Rab11b

0

20

40

60

Rel

ativ

e m

RN

A Le

vels

(fol

d)

**

**

****

Mt1 Mt2

ChowChow+Binge

HFDHFD+Binge

0

5

10

15

Rel

ativ

e m

RN

A Le

vels

(fol

d)

Mt1 Mt2

****

****

Pair-fed E10d 1B

0

5

10

15

20

Rel

ativ

eM

t1m

RN

A Le

vels

(fol

d)

****

ControlMaltose1B

Liver Kidney Heart Spleen Pancreas0

5

10

15**** Control

Maltose1B

Liver Kidney Heart Spleen PancreasRel

ativ

e M

t2m

RN

A Le

vels

(fol

d)

BA

C D

Supporting Fig. S1: Mt expression in different alcohol injury models. (A) C57BL6 N micewere pair-fed or fed an ethanol diet for 10 days or one binge (1B). Mt1 and Mt2 levels inliver tissue were examined by RT-qPCR. (B) C57BL6 N mice were fed chow diet for 3months, chow diet for 3 months plus one binge, high fat diet (HFD) for 3 months or HFDfor 3 months plus binge. Mt1 and Mt2 levels in liver tissue were examined by RT-qPCR.(C) Different tissues from control, maltose or one binge groups were subjected to RT-qPCR for Mt1 expression. (D) Different tissues from control, maltose or one binge groupswere subjected to RT-qPCR for Mt2 expression. Values represent mean±SEM. Statisticalevaluation was performed by Student’s t-test or one-way ANOVA with Tukey’s post hoctest for multiple comparisons. (**p<0.01)

Page 3: Supporting Table 1 Primer sequences for real -time PCR · 2020. 7. 22. · Mt2 GCCTGCAAATGCAAACAATGC. AGCTGCACTTGTCGGAAGC Rab11a TGGGAAAACAATAAAGGCACAGA: ATGTGAGATGCTTAGCAATGTCA Rab11b

MPO F4/80

Pair-fed

WT

E10+1B

Mt1

/2-/-

Pair-fed E10+1B

Supporting Fig. S2: Infiltration of neutrophils and macrophages in the liver ofMt1/2-/- mice subjected to E10d+1B treatment. Mice were subjected to chronic-plus-binge ethanol feeding or pair-feeding. Mice were euthanatized 9 hours postgavage. Liver tissues from WT and Mt1/2-/- were subjected to immunostaining withan anti-MPO or anti-F4/80 antibody. Representative photographs are shown (scalebar:100μm).

100um

100um

100um

100um

Page 4: Supporting Table 1 Primer sequences for real -time PCR · 2020. 7. 22. · Mt2 GCCTGCAAATGCAAACAATGC. AGCTGCACTTGTCGGAAGC Rab11a TGGGAAAACAATAAAGGCACAGA: ATGTGAGATGCTTAGCAATGTCA Rab11b

B

A

4-H

NE

MD

A

Pair-fed E10d+1B

p-AS

K1

p-p3

8

Supporting Fig. S3: Representative images of oxidative stress and stress kinaseactivation staining in the livers of E10+1B model (scale bar:200μm). C57BL6N micewere pair-fed or ethanol diet for 10 day (E10d) or 10 day plus one binge (E10d+1B)and were euthanized 9 hours after gavage. (A) Liver tissues were subjected toimmunostaining with anti-MDA and anti-4-HNE antibodies. (B) Liver tissues weresubjected to immunostaining with anti-p-ASK1 and p-p38 antibodies.

Page 5: Supporting Table 1 Primer sequences for real -time PCR · 2020. 7. 22. · Mt2 GCCTGCAAATGCAAACAATGC. AGCTGCACTTGTCGGAAGC Rab11a TGGGAAAACAATAAAGGCACAGA: ATGTGAGATGCTTAGCAATGTCA Rab11b

p-p38

JNK

p-STAT3

STAT3

p38

p-JNK

β-Actin

Ask1-/-WTA B

F4/8

0

MPO

C WT Ask1-/-

0.0

0.5

1.0

1.5

2.0*

F4/8

0(fo

ld)

67891011 *

F4/8

0+ar

ea%

0

10

20

30 *

MPO

+ce

lls/fi

eld

0.00.51.01.52.02.5

Ly6g

(fold

)

WT Ask1-/-WT Ask1-/-

0.0

0.5

1.0

1.5

2.0

Rel

ativ

e m

RN

A Le

vels

(fol

d)

Il6 Il1b Tnfa Icam1 Vcam1 Mip1a Mip2

**

*

**

*

0.0

0.5

1.0

1.5

2.0

Rel

ativ

e m

RN

A Le

vels

(fol

d)

Chop Ero1a Ero1b Bim Bak Gadd34 Pdi1 Bax

**

***

* ***

WT

Ask1-/-

0.0

0.5

1.0

1.5

2.0

Rel

ativ

e m

RN

A Le

vels

(fol

d)

p47phox p40phoxp67phox gp91phox p22phox

* * * *

D E

F

0

2

4

6

8

Neu

trop

hils

(109

/L)

Ask1-/-WT

WT

Ask1-/-

Page 6: Supporting Table 1 Primer sequences for real -time PCR · 2020. 7. 22. · Mt2 GCCTGCAAATGCAAACAATGC. AGCTGCACTTGTCGGAAGC Rab11a TGGGAAAACAATAAAGGCACAGA: ATGTGAGATGCTTAGCAATGTCA Rab11b

WT Ask1-/-

TUN

EL s

tain

ing

G

Supporting Fig. S4: Deletion of Ask1 ameliorates chronic-plus-binge ethanol-induced liverinjury. WT and Ask1-/- mice were subjected to E10d+1B feeding and were euthanized 9 hoursafter gavage. (A) Liver tissues were subjected to western blot analysis of p-p38, p38, p-JNK,JNK, p-STAT3 and STAT3 and β-actin; (B) Circulating neutrophil numbers were counted. (C)Liver tissues were subjected to immunostaining of MPO and F4/80. Representativephotographs are shown on the left (scale bar:100μm). The number of MPO+ cells and thepercentage of F4/80+ area were quantified on the right. Hepatic Ly6g and F4/80 weredetected by real-time PCR and were shown on the right; (D, E, F) Liver tissues were subjectedto RT-qPCR analyses of inflammatory cytokine genes (panel D), ROS-associated genes (panelE), ER-stress-associated genes (panel F). (G) Liver tissues were subjected to TUNEL staining.Representative images are shown. Values represent mean ± SEM. Statistical evaluation wasperformed by Student’s t-test. (*p<0.05; **p<0.01; ***p<0.001)

WT Ask1-/-0

5

10

15 ***

TUN

EL+

hepa

tocy

tes/

field

Page 7: Supporting Table 1 Primer sequences for real -time PCR · 2020. 7. 22. · Mt2 GCCTGCAAATGCAAACAATGC. AGCTGCACTTGTCGGAAGC Rab11a TGGGAAAACAATAAAGGCACAGA: ATGTGAGATGCTTAGCAATGTCA Rab11b

Veh+E NQ+E

β-Actin

ASK1

p-p38

p-ASK1

p38

A GS+E

Veh+E

NQ+E

GS+E

B

C

D

0

1

2

3 *

***

*

Tnfa Il1b Il6 Vcam1 Icam1 Mip1a Mip2 Mcp1

Rel

ativ

e m

RN

A Le

vels

(fol

d)

0

2

4

6

Rel

ativ

e m

RN

A Le

vels

(fol

d)

p47phox p40phoxp67phox gp91phox p22phox Mt1 Mt2

**

** *

*

**

**

0.0

0.5

1.0

1.5

2.0

2.5**

p=0.06

*

* ***

***

***

Rel

ativ

e m

RN

A Le

vels

(fol

d)

Chop Ero1a Ero1b BimBakGadd34 Pdi1 Bax

Veh+E

NQ+E

GS+E

Veh+E

NQ+E

GS+E

Page 8: Supporting Table 1 Primer sequences for real -time PCR · 2020. 7. 22. · Mt2 GCCTGCAAATGCAAACAATGC. AGCTGCACTTGTCGGAAGC Rab11a TGGGAAAACAATAAAGGCACAGA: ATGTGAGATGCTTAGCAATGTCA Rab11b

Veh+E NQ+E GS+ETU

NEL

sta

inin

g

E

Supporting Fig. S5: Inhibition of ASK1 ameliorates chronic-plus-binge ethanol-inducedliver injury. C57BL/6J mice were subjected to E10d+1B feeding and received i.p. injectionof ASK1 inhibitors (GS-4997 at 3mg/kg; NQDI-1 at 4mg/kg) 30min before gavage. Micewere euthanized 9 hours after gavage. (A) Western blot analysis of p-ASK1, ASK1, p-p38,p38 and β-Actin. (B, C, D) Liver tissues were subjected to RT-qPCR analyses ofinflammatory genes (panel B), reactive oxygen speices (ROS)-associated genes (panel C),and ER-stress-associated genes (panel D). (E) Liver tissues were subjected to TUNELstaining. Representative images are shown. Values represent mean ± SEM. Statisticalevaluation was performed by Student’s t-test or one-way ANOVA with Tukey’s post hoctest for multiple comparisons. (*p<0.05; **p<0.01; ***p<0.001).

0

5

10

15

20 ******

TUN

EL+

hepa

tocy

tes/

field

Veh+E NQ+E GS+E

Page 9: Supporting Table 1 Primer sequences for real -time PCR · 2020. 7. 22. · Mt2 GCCTGCAAATGCAAACAATGC. AGCTGCACTTGTCGGAAGC Rab11a TGGGAAAACAATAAAGGCACAGA: ATGTGAGATGCTTAGCAATGTCA Rab11b

0.0

0.5

1.0

1.5

2.0

2.5

p=0.06

Chop Ero1a Ero1b Bax Bak Bim Pdi Gadd34

Rel

ativ

e m

RN

A Le

vels

(fol

d)

0.0

0.5

1.0

1.5

2.0

2.5

Rel

ativ

e m

RN

A Le

vels

(fol

d)

p47phox p67phox p40phox gp91phox p22phox

WT

p38aHep-/-

A

B

WT

p38aHep-/-

**

Supporting Fig. S6: Hepatocyte-specific deletion of p38a gene ameliorates chronic-plus-binge ethanol-induced liver injury. WT and p38aHep-/- mice were subjected toE10d+1B feeding and were euthanized 9 hours after gavage. Liver tissues were subjectedto RT-qPCR analyses of ROS-associated genes (panel A) and ER-stress-associated genes(panel B). Values represent mean ± SEM. Statistical evaluation was performed byStudent’s t-test. (**p<0.01).

Page 10: Supporting Table 1 Primer sequences for real -time PCR · 2020. 7. 22. · Mt2 GCCTGCAAATGCAAACAATGC. AGCTGCACTTGTCGGAAGC Rab11a TGGGAAAACAATAAAGGCACAGA: ATGTGAGATGCTTAGCAATGTCA Rab11b

ALY+E

β-Actin

ASK1p-p38

p-ASK1

p38

Veh+E PH+E SB+EVeh+E

0

1

2

3

4

**

*

** **

**

***

****

p47phox p67phox p40phox gp91phox p22phox Mt1 Mt2

Rel

ativ

e m

RN

A Le

vels

(fol

d)

B

C

Veh+E

LY+E

PH+E

Veh+E

LY+E

SB+E

0.0

0.5

1.0

1.5

2.0

2.5*

**

**

Rel

ativ

e m

RN

A Le

vels

(fol

d)

Chop Ero1α Ero1b Bax BakBimPdi Gadd34 Bcl2 Bclxl

Supporting Fig. S7: Inhibition of p38 ameliorates chronic-plus-binge ethanol-induced liver injury. C57BL/6J mice were subjected to E10d+1B feeding and receivedi.p. injection of p38 inhibitors (LY2228820 at 3mg/kg, PH797804 at 12mg/kg andSB239063 at 10mg/kg) 30min before gavage. Mice were euthanized 9 hours aftergavage. (A) Liver tissues were subjected to western blot analysis of p-ASK1, ASK1, p-p38, p38 and β-actin; (B, C) Liver tissues were subjected to RT-PCR analyses of ROS-associated genes (panel B) and ER-stress-associated genes (panel C). Values representmean ± SEM. Statistical evaluation was performed by Student’s t-test or one-wayANOVA with Tukey’s post hoc test for multiple comparisons. (*p<0.05; **p<0.01; ***p<0.001).

Page 11: Supporting Table 1 Primer sequences for real -time PCR · 2020. 7. 22. · Mt2 GCCTGCAAATGCAAACAATGC. AGCTGCACTTGTCGGAAGC Rab11a TGGGAAAACAATAAAGGCACAGA: ATGTGAGATGCTTAGCAATGTCA Rab11b

0

200

400

600

800

ALT(

U/L

)

WTEV

Ask1-/-

EV

0

2

4

6

8

p=0.06

***

Neu

trop

hils

(109

/L)

0 3h 6h 9h

WT

Ask1-/-

A

B

0

20

40

60 p=0.07

Neu

trop

hils

%

0 3h 6h 9h

Supporting Fig. S8: EVs derived from E10d+1B treated WT but not Ask1-/- miceinduce neutrophilia in E10d-treated mice. E10d-fed B6J mice were i.v. injected withEVs isolated from either E10d + 1B treated WT or Ask1-/- mice. Mice were euthanized9 hours after injection. (A) Serum ALT levels and (B) the numbers and percentage ofperipheral neutrophils were measured (n = 5 per group). Student’s t test wasperformed. ***p< 0.001.

WT

Ask1-/-

Page 12: Supporting Table 1 Primer sequences for real -time PCR · 2020. 7. 22. · Mt2 GCCTGCAAATGCAAACAATGC. AGCTGCACTTGTCGGAAGC Rab11a TGGGAAAACAATAAAGGCACAGA: ATGTGAGATGCTTAGCAATGTCA Rab11b

NSMase2

Rab27A

ALIX

β-Actin

PBS E+GSEtOH 100mM E+LYN

SMas

e2/β

-Act

in (f

old)

0.0

0.5

1.0

1.5*

*****

0.0

0.5

1.0

1.5*

******

ALIX

/β-A

ctin

(fol

d)

0.0

0.5

1.0

1.5 p=0.07

****

Rab

27A/β-

Actin

(fol

d)

NSMase2

ALIX

Rab27A

β-Actin

Supporting Fig. S9: Inhibition of ASK1 and p38 kinases attenuates the expression ofEV biogenesis induced by ethanol in AML12 cells. AML12 cells were pretreated withGS-4997 (30μM, 1 hr) or LY2228820 (2 μM, 1 hr) and then exposed to ethanol (100mM)for 24h. AML12 cells were collected for western blot analysis of nSMase2, ALIX,Rab27A and β-Actin. Statistical evaluation was performed by Student’s t-test. (*p<0.05;***p<0.001). PBS: phosphate-buffered solution.