ATF6 Mediates a Pro-Inflammatory Synergy Between ER Stress and TLR Activation in the Pathogenesis of Liver Ischemia-Reperfusion Injury
Article first published online: 5 JUN 2014
© Copyright 2014 The American Society of Transplantation and the American Society of Transplant Surgeons
American Journal of Transplantation
Volume 14, Issue 7, pages 1552–1561, July 2014
How to Cite
Rao, J., Yue, S., Fu, Y., Zhu, J., Wang, X., Busuttil, R. W., Kupiec-Weglinski, J. W., Lu, L. and Zhai, Y. (2014), ATF6 Mediates a Pro-Inflammatory Synergy Between ER Stress and TLR Activation in the Pathogenesis of Liver Ischemia-Reperfusion Injury. American Journal of Transplantation, 14: 1552–1561. doi: 10.1111/ajt.12711
- Issue published online: 20 JUN 2014
- Article first published online: 5 JUN 2014
- Manuscript Accepted: 15 FEB 2014
- Manuscript Revised: 7 FEB 2014
- Manuscript Received: 16 DEC 2013
- NIH. Grant Number: RO1 DK083408
- The Dumont Research Foundations
- National Nature Science Foundation of China. Grant Numbers: 81100270, 1310108001, 81210108017
Additional Supporting Information may be found in the online version of this article.
Figure S1: Effects of ATF6 siRNA. (A) ATF6 siRNA selectively inhibited the ATF6 expression in ER-stressed macrophages. The ATF6- or NS-siRNA transfected BMMs were pretreated with Tm, followed by stimulations with LPS, as described in the Materials and Methods section. Cells were harvested at 4 h to measure gene expressions of ATF6, XBP1, CHOP and Grp78 by qRT-PCR. (B) ER stress did not alter IP-10 expressions. BMMs were pretreated with Tm or Tg, followed by LPS stimulations for 4 h, as described in the Materials and Methods section. IP-10 gene expressions were measured by qRT-PCR. (C) ATF6 siRNA did not regulate macrophage IP-10 gene expression. As described in (A), IP-10 expression in siRNA transfected macrophages under ER stress was measured by qRT-PCR. Results are representative of at least two separate experiments, n = 3 replicates/group/expt., t-test, *p < 0.05.
Figure S2: Targeted delivery of siRNA in vivo by mannose-conjugated polymers. AlexaFluor488-labeled siRNA (green) were mixed with mannose-conjugated polymers and injected via tail vein, as described in the Materials and Methods section. Spleen and livers were collected at 4 h postinjection. Tissue specimens were fixed with 10% neutral formaldehyde, embedded and frozen in O.C.T (Tissue-Tek® optimum cutting temperature compound, VWR). Liver sections (4 µm) were stained with anti-CD68 antibody (Santa Cruz Biotechnology, Inc., San Diego, CA) and Cy3-conjugated AffiniPure goat anti-mouse IgG antibody (red; Jackson Immunoresearch, West Grove, PA). DAPI (blue) was used for nuclear counterstaining. The biodistribution of green fluoresence positive cells in spleen were indicative of phagocytes (expressing mannose receptors) being transfected. In livers, these positive cells merged with red immunofluorescence-stained CD68 positive cells.
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