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Supplementary material for this article can be found on the H EPATOLOGY website ( http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html ).

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jws-hep.20966.fig1.tif29KSTAT3&&num;8211;DNA binding activity in cholangiocarcinoma cells is regulated via an IL-6/STAT3 cascade. ELISA-based STAT3&&num;8211;DNA binding assay was performed using nuclear proteins (10 &&num;956;g) extracted from the cancer cells, which were incubated in the absence (vehicle) or presence of AG490 at various concentrations (50-200 &&num;956;mol/L) for 30 minutes. * P< .01. ** P< .001 versus cells treated with vehicle alone. The data represent four independent experiments using TFK-1 cells. Veh, vehicle.
jws-hep.20966.fig2.tif32KMcl-1 mRNA expression is regulated by a STAT3 pathway. TFK-1 cells were treated with IL-6 (10 ng/mL) in the absence (vehicle) or presence of AG490 at a dose of 200 &&num;956;mol/L for 3 hours. Total cellular RNA was isolated and real-time reverse-transcriptase PCR using SYBR green was performed. The relative expression levels of Mcl-1 mRNA were expressed as a ratio of Mcl-1/18S ribosomal RNA (internal control) copies per microliter. The data are representative of four individual experiments. * P< .01 for IL-6&&num;8211;stimulated cells treated with vehicle versus cells treated with IL-6 plus AG490. (B) TFK-1 cells were transfected with DN STAT3 or an empty vector. After 48-hour transfection, total cellular RNA was isolated and was subjected to real-time reverse-transcriptase PCR using SYBR green. The data are representative of six individual experiments. ** P< .05 for IL-6&&num;8211;stimulated cells transfected with DN STAT3 versus cells transfected with empty vector. Mcl-1, myeloid cell leukemia-1; mRNA, messenger RNA; Veh, vehicle; IL-6, interleukin 6; DN STAT3, pRcCMV-dominant negative Y705F STAT3 mutant plasmid.

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