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Supplemental data

Disruption of CXC motif chemokine ligand-14 in mice ameliorates obesity-induced insulin resistance

By Noriko Nara, Yuki Nakayama, Shiki Okamoto, Hiroshi Tamura, Mari Kiyono, Masatoshi Muraoka, Kiyoko

Tanaka, Choji Taya, Hiroshi Shitara, Rie Ishii, Hiromichi Yonekawa, Yasuhiko Minokoshi, and Takahiko Hara

Contents: 6 figures (Fig. S1, S2, S3, S4, S5 and S6), and 1 table (Table S1)

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Fig. S1 Disruption of CXCL14 in mice. (A) Schematic representation of the wild-type murine CXCL14 genomic

locus, the CXCL14 gene-targeting vector, and the mutant CXCL14 allele. Regions corresponding to exons (black

boxes), the probe used in Southern blot analysis (open box), and the PCR primers (P1 and P2) are indicated. (B)

Splenic DNA from CXCL14+/+, CXCL14+/- or CXCL14-/- female mice was analyzed by Southern blot

hybridization using the 3’ probe. (C) Total RNA from the indicated organs from lean CXCL14+/+, CXCL14+/- or

CXCL14-/- female mice was subjected to Northern blot analysis using CXCL14 or β-actin cDNA (loading

control) as a probe. (D) Total brain protein (50 µg) from CXCL14+/+, CXCL14+/- or CXCL14-/- female mice was

analyzed by Western blot using anti-CXCL14 antibody.

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Fig. S2 Histogram of adipocyte size in WAT. Size distribution of cells from periovarian fat pads was determined

using NIH Image software to analyze hematoxylin and eosin-stained paraffin sections from representative RD-

fed CXCL14+/- (A), HFD-fed CXCL14+/- (B), RD-fed CXCL14-/- (C), or HFD-fed CXCL14-/- (D) female mice.

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Fig. S3 Partial protection of CXCL14-/- mice from HFD-induced hepatic steatosis. (A) Photographs of

representative livers of HFD-fed CXCL14+/- and CXCL14-/- female mice. (B) Oil Red O staining of frozen liver

sections from HFD-fed CXCL14+/+, CXCL14+/- and CXCL14-/- female mice. A CXCL14+/+ female mouse was

used as a healthy lean control. Scale bars, 100 µm. (C) Amount of triglyceride in 1g of liver from HFD-fed

CXCL14+/- (n = 3) and CXCL14-/- (n = 4) female mice. Data represents the means ± S.E. **, P < 0.05 compared

to CXCL14+/- mice. (D) Total RNA from the livers of RD-fed CXCL14+/+ or HFD-fed CXCL14+/- mice (two mice

for each group) was subjected to Northern blot analysis using CXCL14 cDNA as a probe. Ctr, WAT RNA from

HFD-fed CXCL14+/- mice. GAPDH was analyzed as a loading control.

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Fig. S4 Responsiveness of HFD-fed male CXCL14-/- mice to insulin or glucose. IITT (A) and IGTT (B) of

CXCL14+/- and CXCL14-/- male mice fed an HFD. Data represents the means ± S.E. In A, number and mean body

weights (g) ± S.E. for each group of mice are shown above the data legend.

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Fig. S5 Slight reduction of serum cholesterol levels in HFD-fed CXCL14-/- mice. Concentrations of cholesterol

(A), triglyceride (B) and FFA (C) in the serum of HFD-fed CXCL14+/- and CXCL14-/- female mice (n = 5 for

each). Data represents the means ± S.E. **, P < 0.05; n.s., not significant compared to CXCL14+/- mice.

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Fig. S6 Expression of the mRNAs of metabolic regulator genes in CXCL14-/- mice. (A-C) Total RNA from BAT

(A), WAT (B) and skeletal muscle (C) of RD-fed or HFD-fed CXCL14+/- and CXCL14-/- female mice were

subjected to Northern blot analysis using a UCP-1, UCP-3, or GLUT4 cDNA as a probe. (D) Total RNA from

the livers of fasted HFD-fed CXCL14+/- and CXCL14-/- female mice (2 mice for each group) were subjected to

Northern blot analysis using a Pepck or G6Pase cDNA as a probe. In A-D, GAPDH was analyzed as a loading

control.

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Table S1 Primers for RT-PCR reactions in this study

Gene Forward primers (5’ to 3’) Reverse primers (5’ to 3’) PCR

product

(bp)

CXCL14 5’-CCAAGATTCGCTATAGCGAC-3’ 5’-CCTGCGCTTCTCGTTCCAGG-3’ 191

UCP-1 5’-ACAGAAGGATTGCCGAAAC-3’ 5’- GTAAATGGCAGGGGACGTCA-3’ 448

UCP-3 5’- CATGGTTGGACTTCAGCCC-3’ 5’-TTCCTCTCTCCTCCAGTTCC-3’ 462

GLUT4 5’-CTCATTGGCATCATTTCTC -3’ 5’-ACACATCAGCCCAGCCGGT -3’ 469

GAPDH 5’-ACCACAGTCCATGCCATCAC-3’ 5’-TCCACCACCCTGTTGCTGTA-3’ 452

Pepck 5’-ATGCCTCCTCAGCTGCATAAC-3’ 5’-CATGCTGGCCACCACATAGGG-3’ 510

G6Pase 5’-CTCCATGACTTTGGGATCCAG-3’ 5’-CAAGGTAGATCCGGGACAGAC-3’ 499

CCL2 5’-AGCCAGCTCTCTCTTCCTCC -3’ 5’-AAGGCATCACAGTCCGAGTC -3’ 496

IL-6 5’-CCGGAGAGGAGACTTCACAG -3’ 5’-TGGTCTTGGTCCTTAGCCAC -3’ 479

IL-1β 5’-CTGGAGAGTGTGGATCCCAAG -3’ 5’-GGAAGACACGGATTCCATGGTG -3’ 213

TNF-α 5’-TCCCCAAAGGGATGAGAAGTTC -3’ 5’- TCATACCAGGGTTTGAGCTCAG-3’ 411

α-tubulin 5’-GCACTCTGATTGTGCCTTCA-3’ 5’-CACAGTGGGAGGCTGGTAAT-3’ 499