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SUPPLEMENTARY
Supplementary Methods
Immunoblotting
Lung, aortaes and macrophages obtained from mice were homogenized and subjected to
immunoblotting as described previously1, 2. For experiments involving TNF-α
stimulation, mice were injected with recombinant murine TNF-α (R&D Systems Inc, MN,
USA), and killed 30 minutes later. Antibodies to IB(C-15) (Santa Cruz Biotechnology,
CA, USA) were commercially obtained.
Isolation and culture of endothelial cells
Endothelial cells were isolated from murine lung using a MACS separation unit
(Miltenyi Biotec, Surry, UK) as previously described3. To quantify VCAM-1 expression,
purified endothelial cells were pre-incubated for 9 h, and then stimulated with or without
TNF- for 7h, followed by quantitative RT-PCR analysis.
Isolation of Macrophages
Peritoneal macrophages were harvested from lavage of E-DNIB mice and their
wild-type littermates 4 days after intraperitoneal injection of 4% thioglycollate (3ml) as
previously described4. To quantify IL-6 expression, isolated macrophages were
pre-incubated for 4 h, and then stimulated with or without TNF- for 5h, followed by
quantitative RT-PCR analysis.
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Supplementary Figure Legends
Supplementary Figure 1. Expressions of IB protein and MCP-1 mRNA.
(A) Extracts from lung, aortae and macrophages were immunoblotted with anti-IB
antibody. Exogenous (human) IBα has a slightly higher molecular weight than
endogenous (murine) IBα.
Supplementary Figure 2. (A) Lung extracts of control and E-DNIB mice were
obtained 30 minutes after injection of the indicated amounts of TNF-, followed by
immunoblotting with anti-IB antibody.
(B) Relative amounts of mRNA of VCAM-1 in isolated endothelial cells from control
and E-DNIB mice (n=5 per group). Isolated endothelial cells were stimulated with and
without TNF- at different concentrations as indicated. (C) Relative amounts of mRNA
of IL-6 in isolated macrophages from control and E-DNIB mice (n=5 per group).
Isolated macrophages were stimulated with and without TNF- at different
concentrations as indicated. Data are presented as means  SEM. *P<0.05 compared
with control littermate group by one-way ANOVA.
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Supplementary References
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Imai J, Katagiri H, Yamada T, Ishigaki Y, Suzuki T, Kudo H et al. Regulation of
pancreatic beta cell mass by neuronal signals from the liver. Science.
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2.
Hasegawa Y, Saito T, Ogihara T, Ishigaki Y, Yamada T, Imai J et al. Blockade of
the nuclear factor-kappaB pathway in the endothelium prevents insulin resistance
and prolongs life spans. Circulation. 2012:125:1122-1133.
3.
Marelli-Berg FM, Peek E, Lidington EA, Stauss HJ, Lechler RI. Isolation of
endothelial cells from murine tissue. J Immunol Methods. 2000;244:205-215.
4.
Gao J, Ishigaki Y, Yamada T, Kondo K, Yamaguchi S, Imai J et al. Involvement
of endoplasmic stress protein C/EBP homologous protein in arteriosclerosis
acceleration with augmented biological stress responses.
Circulation.2011:124:830-839.