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Version of Record online: 6 DEC 2012
Copyright © 2012 American Association for the Study of Liver Diseases
Volume 57, Issue 2, pages 786–796, February 2013
How to Cite
Ghiassi-Nejad, Z., Hernandez-Gea, V., Woodrell, C., Lang, U. E., Dumic, K., Kwong, A. and Friedman, S. L. (2013), Reduced hepatic stellate cell expression of kruppel-like factor 6 tumor suppressor isoforms amplifies fibrosis during acute and chronic rodent liver injury. Hepatology, 57: 786–796. doi: 10.1002/hep.26056
Potential conflict of interest: Nothing to report.
Supported by MSTP grant (to Z.G.N.), DK56621, DK47340, and P20AA017067 (to S.L.F.), Summer Student support from MSSM.
- Issue online: 5 FEB 2013
- Version of Record online: 6 DEC 2012
- Accepted manuscript online: 7 SEP 2012 09:01AM EST
- Manuscript Accepted: 27 AUG 2012
- Manuscript Received: 12 APR 2012
Additional Supporting Information may be found in the online version of this article.
|HEP_26056_sm_SuppFig1.tif||4454K||Supporting Information Figure 1. All rat KLF6 isoforms can undergo proteasomal degradation; and as predicted, only rKLF6top, but not the shorter isoforms, can localize to the nucleus. A. 293FT cells were transfected with pcDNA3.1/rKlf6top-V5, pcDNA3.1/rKlf6mid-V5, pcDNA3.1/rKlf6bot-V5, or pcDNA3.1/V5. 24 hours later cells were either treated with proteasomal inhibitor MG132 (10μM) or an equal volume of DMSO. Protein was harvested 5 hours after addition of MG132, and SDS-PAGE was performed followed by a western blot for V5. All 3 isoforms show degradation by the proteasomal pathway, as indicated by increasing protein in presence of MG132; B. 293FT cells were transfected as before with each of the three rat Klf6 expression vectors, and V5 immunofluorescence was performed 24h later. Confocal microscopy failed to show nuclear staining of rKLF6mid and rKLF6bot.|
|HEP_26056_sm_SuppFig2.tif||6662K||Supporting Information Figure 2. Validation of constructs employed in generating stellate cell specific transgenic mice, and efficient expression of transgenes following CCl4 administration to these transgenic mice. A. Mouse stellate cell line (JS1) was transfected with Cola2(I)-GFP, Cola2(I)-KLF6WT , Cola2(I)-KLF6SV1 construct and protein was harvested 48h later for SDS-PAGE followed by KLF6 immunoblotting. The constructs expressed the appropriate KLF6 isoforms; B. Efficient induction (∼40 fold) of transgene 24h after a single injection of CCl4 in 2 transgenic lines from each of the Cola2(I)-KLF6WT mice Cola2(I)-KLF6SV1 mice (2 mice per line per treatment, data was normalized to wild type littermates receiving oil injection) (p-value<0.01).|
|HEP_26056_sm_SuppFig3.tif||6893K||Supporting Information Figure 3. Stellate cell specific KLF6WT and KLF6SV1 transgenic mice have decreased number of ASMA positive cells following CCl4 liver injury. A. Livers from Cola2(I)-KLF6WT, Cola2(I)-KLF6SV1 and wild type mice following 8 weeks of chronic CCl4 were immunostained with ASMA as described. Transgenic mice over-expressing either isoform show reduced number of ASMA positive cells. B. Quantification of ASMA positive cells is shown. For each animal 30 images taken at high magnification (40X objective lens) were employed (p-value<0.01).|
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