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FilenameFormatSizeDescription
hep26585-sup-0001-supptable1.doc51KTable SI : Primary antibodies used for immunodetection M, mouse; R, rabbit; WB, western blot; IF, immunofluorescence; IHC, immunohistochemistry; FC, flow cytometry.
hep26585-sup-0002-supptable2.doc32KTable SII : sequence of primers used
hep26585-sup-0003-suppfig1.tif2638KFigure S1: Characterization of HLMF and expression of EGFR in CCA cells (A) Morphology of HLMF in primary culture one week after isolation (P0) using a phase contrast microscope. Arrows indicate vitamin A-storing lipid droplets demonstrating that HLMF derives in part from hepatic stellate cells. (B) Detection of α-SMA, HepPar and CD31 in HLMF was performed using flow cytometry. Representative histograms of mean fluorescence indicating the density of the specific markers are shown. (C) EGFR expression in CCA cells was analyzed by Western blot using an anti-EGFR antibody. β−actin is used as loading control.
hep26585-sup-0004-suppfig2.tif976KFigure S2: HLMF induce CCA cell proliferation through EGFR (A) Mz-ChA-1 cells were pretreated or not with gefitinib (1 μM) for 30 min prior to HLMF-CM (n=3) (A, B), FBS (A, B) and HB-EGF (50 ng/ml) (B) stimulation. Quantification of Ki67 positive cells by immunofluorescence is diplayed for in each group. Mean ±SEM. * p<0.01; control without gefitinib vs. stimulated conditions without gefitinib. # p<0.01; conditions without gefitinib vs. conditions with gefitinib.
hep26585-sup-0005-suppfig3.tif6483KFigure S3: HLMF promote EGFR activation and disruption of adherens junctions in SK-ChA-1 cells (A) Cells were pretreated with neutralizing antibodies against HB-EGF (2 μg/μl) or EGFR (4 μg/μl) 30 min prior to incubation for 10 min with HLMF-CM (n=4). Activated and total EGFR levels were examined by Western blot. Representative blots of 2 experiments are shown. (B-C) Cells were pretreated with geftinib (1 μM) or with neutralizing antibodies against EGFR (4 μg/μl) 30 min prior to incubation for 24 h with HLMF-CM (n=3). Cell dispersion was analyzed using phase contrast microscope (B) and localization of E-cadherin was assessed by immunofluorescence using confocal microscopy (C). Scale bar, 20 μm.
hep26585-sup-0006-suppfig4.tif5917KFigure S4: HLMF promote EGFR activation and disruption of adherens junctions in EGI-1 cells (A) Cells were pretreated with neutralizing antibodies against HB-EGF (2 μg/μl) or EGFR (4 μg/μl) 30 min prior to incubation for 10 min with HLMF-CM (n=4). Activated and total EGFR levels were examined by Western blot. Representative blots of 2 experiments are shown. (B-C) Cells were pretreated with geftinib (1 μM) or with neutralizing antibodies against EGFR (4 μg/μl) 30 min prior to incubation for 24h with HLMFs-CM (n=3). Cell dispersion was analyzed using phase contrast microscope (B) and localization of E-cadherin was assessed by immunofluorescence using confocal microscopy (C). Scale bar, 20 μm.
hep26585-sup-0007-suppfig5.tif2833KFigure S5: Activation of HLMF by TGF-β1 (A) Representative immunohistochemical stainings of TGF-β1 and TGF-β1 RII in human CCA cell components. Arrowheads indicate CCA cells and arrows indicate MF. Magnification: x200. (B) HLMF were stimulated with TGF-β1 (5 ng/ml) for 6 h (left panel) and 24 h (right panel). mRNA levels of α-SMA were evaluated by RT-qPCR. Quantitative data are means ± SEM for 3 experiments performed in duplicate. * p<0.01 vs control medium. Cell morphology was analyzed using phase contrast microscope. Scale bar, 35 μm.
hep26585-sup-0008-suppinfo01.tif2272KSupporting Information
hep26585-sup-0009-suppinfo02.doc59KSupporting Information

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