Serum amyloid alpha 1-2 are not required for liver inflammation in the 4T1 murine breast cancer model

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Frontiers in Immunology

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Figure S1: The basic characteristics of Saa1-2

(A) A DNA sequence alignment of Saa1 (accession number: CCDS21284.1) and Saa2 (accession

number: CCDS21285.1). The differences in their sequences are highlighted in red.

(B) The correlations of the mRNA abundances between Saa1-2 and representative neutrophil

related genes. n = 4.

10

20

30

40

50

60

70

Saa1 ATGAAGCTAC TCACCAGCCT GGTCTTCTGC TCCCTGCTCC TGGGAGTCTG CCATGGAGGG TTTTTTTCAT

Saa2 ATGAAGCTAC TCACCAGCCT GGTCTTCTGC TCCCTGCTCC TGGGAGTCTG CCATGGAGGG TTTTTTTCAT

71

80

90

100

110

120

130

140

Saa1 TTGTTCACGA GGCTTTCCAA GGGGCTGGGG ACATGTGGCG AGCCTACACT GACATGAAGG AAGCTAACTG

Saa2 TTATTGGGGA GGCTTTCCAA GGGGCTGGAG ACATGTGGCG AGCCTACACT GACATGAAGG AAGCTGGCTG

141

150

160

170

180

190

200

210

Saa1 GAAAAACTCA GACAAATACT TCCATGCTCG GGGGAACTAT GATGCTGCTC AAAGGGGTCC CGGGGGAGTC

Saa2 GAAAGATGGA GACAAATACT TCCATGCTCG GGGGAACTAT GATGCTGCCC AAAGGGGTCC CGGGGGAGTC

211

220

230

240

250

260

270

280

Saa1 TGGGCTGCTG AGAAAATCAG TGATGGAAGA GAGGCCTTTC AGGAATTCTT CGGCAGAGGA CATGAGGACA

Saa2 TGGGCTGCTG AGAAAATCAG TGATGCAAGA GAGAGCTTTC AGGAATTCTT CGGCAGAGGA CACGAGGACA

281

290

300

310

320

330

340

350

Saa1 CCATTGCTGA CCAGGAAGCC AACAGACATG GCCGCAGTGG CAAAGACCCC AATTACTACA GACCTCCTGG

Saa2 CCATGGCTGA CCAGGAAGCC AACAGACATG GCCGCAGTGG CAAAGACCCC AATTACTACA GACCTCCTGG

351

360

Saa1 ACTGCCTGAC AAATACTGA 369

Saa2 ACTGCCTGCC AAATACTGA 369

Lcn2 vs Saa1

Ifitm1 vs Saa1

RPKM(x102)

12

1.5

20

1.0

10

R2=0.943

p=2.91x10-2

20

40

60

RPKM(x102)

0.5

R2=0.907

p=4.76x10-2

80

20

40

60

RPKM(x102)

80

R2=0.907

p=4.77x10-2

Ifitm1 vs Saa2

Lcn2 vs Saa2

40

60

RPKM(x102)

80

12

1.5

20

20

Lrg1 vs Saa2

2.0

30

RPKM(x102)

Lrg1 vs Saa1

2.0

30

1.0

10

R2=0.907

p=4.75x10-2

20

40

RPKM(x102)

60

0.5

R2=0.825

p=9.17x10-2

20

40

RPKM(x102)

60

R2=0.962

p=1.93x10-2

20

40

RPKM(x102)

60

Figure S2: Western blot analysis of SAA1-2 proteins in the liver

Western blot analysis for SAA1-2 in the livers of sham and 4T1-bearing mice in WT and Saa1-2

KO. Recombinant SAA1 protein (2948-SA: R&D systems, MN, USA) is included as a positive

control.

(kDa)

15

10

WT

Sham

4T1

KO

Sham 4T1

nt

A1 bin

SA com

re

Figure S3: qPCR validations of the RNA-seq experiments in the liver

(A) This figure supports Fig. 3A. Top10 differentially expressed gene names are labelled.

(B) Venn diagrams showing the number of differentially expressed genes in the livers of 4T1bearing WT and Saa1-2 KO mice.

(C) qPCR analysis of S100a8, Ly6g, Mpo, and Itgam (Cd11b) in the livers of sham and 4T1bearing mice in WT and Saa1-2 KO (pooled from the four independent 4T1 transplantation

experiments). Averaged fold change data normalized to the sham group in each genotype are

presented as the mean ± SEM. n.s., not significant, unpaired two-tailed Student’s t-test. n =

11 for the sham groups, n = 13 for 4T1-bearing WT mice, and n = 14 for 4T1-bearing Saa12 KO mice.

KO

WT

60

-log10(p_value)

150

100

40

50

20

-10

-5

10

-10

-5

10

log2(fold change (4T1/Sham))

Up-regulated genes

Down-regulated genes

WT

WT

356

1323

211

36

101

43

KO

KO

S100a8

Fold change

8000

Ly6g

n.s.

6000

400

Mpo

n.s.

Itgam (Cd11b)

n.s.

150

150

n.s.

300

4000

200

2000

100

Sham 4T1 Sham 4T1

WT

KO

100

100

50

50

Sham 4T1 Sham 4T1

WT

KO

Sham 4T1 Sham 4T1

WT

KO

Sham 4T1 Sham 4T1

WT

KO

Figure S4: Gating strategies in the flow cytometry experiments

250K

250K

200K

P1

150K

100K

150K

P2

100K

P2

50K

50K

Comp-APC-Cy7-A :: LiveDead

P1

FSC-W

SSC-A

200K

105

104

103

-103

50K

100K

150K

200K

250K

50K

100K

150K

200K

CD45+

-103

250K

105

104

105

105

Monocytes+Macrophages

104

CD11b+

10

CD11b

104

P3

103

P3

-103

-103

-103

103

104

Comp-PE-Cy7-A :: CD11b

105

Comp-APC-A :: F4_80

Neutrophils

Comp-FITC-A :: Ly6G

Comp-APC-Cy7-A :: LiveDead

CD45+

103

Comp-AmCyan-A :: CD45

FSC-H

105

104

103

-103

-103

103

104

Comp-PE-Cy7-A :: CD11b

105

-103

103

104

Comp-PE-Cy7-A :: CD11b

105

Figure S5: qPCR validations of the RNA-seq experiments in the bone marrow

(A) Venn diagrams showing the number of differentially expressed genes in the bone marrows of

4T1-bearing WT and Saa1-2 KO mice.

(B) qPCR analysis of Wfdc17, Lrg1, Ifitm1, and Saa3 in the bone marrows of sham and 4T1bearing mice in WT and Saa1-2 KO (pooled from the four independent 4T1 transplantation

experiments).

Data are normalized with 18s rRNA and are presented as the mean ± SEM. n.s., not significant,

unpaired two-tailed Student’s t-test. n = 11 for sham-treated WT mice, n = 14 for 4T1-bearing

WT mice, n = 9 for sham-treated WT mice, and n = 12 for 4T1-bearing Saa1-2 KO mice.

Up-regulated genes

WT

Down-regulated genes

WT

803

191

325

296

1082

436

KO

KO

Wfdc17

Fold change

500

Lrg1

n.s.

400

40

n.s.

30

300

20

200

10

100

Sham 4T1 Sham 4T1

Sham 4T1 Sham 4T1

WT

WT

KO

Ifitm1

Saa3

n.s.

Fold change

300

KO

8000

n.s.

6000

200

4000

100

2000

Sham 4T1 Sham 4T1

WT

KO

Sham 4T1 Sham 4T1

WT

KO

Figure S6: Saa3 is induced in the livers of 4T1-bearing mice even in the absence of Saa1-2

(A) qPCR analysis of Saa3 in the livers of sham and 4T1-bearing mice in WT and Saa1-2 KO

(pooled from the four independent 4T1 transplantation experiments). Averaged fold change

data normalized to the sham group in each genotype are presented as the mean ± SEM. n.s.,

not significant, unpaired two-tailed Student’s t-test. n = 11 for the sham groups, n = 13 for

4T1-bearing WT mice, and n = 14 for 4T1-bearing Saa1-2 KO mice.

(B) An amino acid sequence alignment of SAA1 (accession number: CCDS21284.1), SAA2

(accession number: CCDS21285.1) and SAA3 (accession number: CCDS21282.1).

Saa3

80

n.s.

Fold change

60

40

20

Sham

4T1

WT

Sham

4T1

KO

10

20

30

40

50

60

SAA1 MKLLTSLVFC SLLLGVCHGG FFSFVHEAFQ GAGDMWRAYT DMKEANWKNS DKYFHARGNY

SAA2 MKLLTSLVFC SLLLGVCHGG FFSF I GEAFQ GAGDMWRAYT DMKEAGWKDG DKYFHARGNY

SAA3 MKPSI AI I LC I L I LGVDSQR WVQFMKEAGQ GSRDMWRAYS DMKKANWKNS DKYFHARGNY

61

70

80

90

100

110

120

SAA1 DAAQRGPGGV WAAEKISDGR EAFQEFFGRG HEDTI ADQEA NRHGRSGKDP NYYRPPGLPD KY

SAA2 DAAQRGPGGV WAAEKISDAR ESFQEFFGRG HEDTMADQEA NRHGRSGKDP NYYRPPGLPA KY

SAA3 DAARRGPGGA WAAKVISDAR EAVQKFTGHG AEDSRADQFA NEWGRSGKDP NHFRPAGLPK ZY

...