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Additional Supporting Information may be found in the online version of this article.

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HEP_26007_sm_SuppFig1to1.tif6029KSupporting Information Figure 1-1. (A) DEN-treated rats were sacrificed at indicated time intervals and the gross appearance of their livers were shown. (B) Tumor nodules of rats in the 22nd week were assessed by H&E staining. Representative images of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC) like regions were shown as indicated. (C) Liver sections of rats treated with DEN were subjected to immnuohistochemistry against OV-6 and proliferation of oval cells was determined based on the percentage of positively stained cells. (D) Liver sections of DEN-treated rats were stained with OV-6 (green), Ki67 (red) and Hochest 33342 (blue); white arrow indicated Ki67+OV-6+ cells; scale bar, 25 μm. (E) Liver sections of DEN-treated rats were stained with OV-6 (green), p-Smad2 (red) and Hochest 33342 (blue); white arrow indicated p-Smad2+OV-6+ cells; scale bar, 25 μm. (F) The mRNA expression of PAI-1, CDKN1A, CDKN2B and TGFB1I1 in the liver tissue of DEN-treated rats.
HEP_26007_sm_SuppFig1to2.tif2323KSupporting Information Figure Part 1-2.
HEP_26007_sm_SuppFig2.tif2892KSupporting Information Figure 2. Rats following 2-acetylaminofluorene/partial hepatectomy (2-AAF/PHx) approach were sacrificed seven days after the partial hepatectomy and their liver sections were subjected to H&&E staining, immnuohistochemistry against α-SMA, OV-6 and TGF-β. Rats treated with the partial hepatectomy only were used as control; scale bar, 100 μm.
HEP_26007_sm_SuppFig3.tif3393KSupporting Information Figure 3. Liver sections of mice fed with diethyldithiocarbamate (DDC) or saline for 4 months were subjected to H&E staining, immnuohistochemistry of TGF-β and immnuofluorescence of A6 (green) and DAPI (blue); scale bar, 100 μm.
HEP_26007_sm_SuppFig4to1.tif1787KSupporting Information Figure 4-1. (A) WB-F344 cells treated with saline or TGF-β for indicated time intervals were subjected to immnuoblotting assay. (B) Relative expression analysis of TGF-β response genes in WB-CON and WB-TβLT cells. Data are presented in a heatmap format in which rows and columns represent genes and samples at indicated time intervals, respectively. Proliferation (C) and apoptosis (D) of TGF-β-treated WB-F344 and control cells were determined by Cell Counting Kit 8 and FACS assay. (E) WB-CON and WB-TβLT cells were subjected to immunofluorescent staining of E-cadherin and Vimentin (red). DAPI were used to show the location of nucleus (blue); scale bar, 50 μm. (F) WB-CON and WB-TβLT cells were seeded into 12-well plates at 70% confluence and then subjected to wound healing assay. Representative images were taken at indicated time point and the relative migration rate was shown. (G) 1×105 of WB-CON or WB-TβLT cells were seeded into the upper chamber of Corning 3422 transwell plates with serum-free DMEM. The lower chamber was filled with DMEM with 10% FBS. The cells were fixed and stained with crystal violet.
HEP_26007_sm_SuppFig4to2.tif3073KSupporting Information Figure 4-2.
HEP_26007_sm_SuppFig5.tif331KSupporting Information Figure 5. WB-CON and WB-TβLT cells were cultured in spheroid condition for seven days and the spheroids were collected. The mRNA level of CD90 and CD133 were measured using Realtime PCR analysis.
HEP_26007_sm_SuppFig6.tif316KSupporting Information Figure 6. WB-CON and WB-TβLT cells were treated with 5'-fluorouracil (5-FU) at 200 ?μM and etoposide (ETO) at 50 μM for 24hrs and their relative proliferation rates were analyzed by CCK-8.
HEP_26007_sm_SuppFig7.tif854KSupporting Information Figure 7. (A) LEPCs were treated with saline or TGF-β at 0.25 ng/mL for 6 weeks and subjected to spheroid assay at 10000 cells per well. Spheroids were counted 7 days later and representative images were shown; scale bar, 100 μm. (B) TGF-β-treated LEPCs and control cells were subjected to limiting dilution assay at doses of 25, 100, 200 and 400 cells per well. The results were shown as natural logarithm of the proportion of stem cells. (C) TGF-β-treated LEPCs and control cells were subjected to colony formation assay at 3000 cells per well and colonies formed were counted 7 days later. (D) TGF-β-treated LEPCs and control cells were subjected to immnuoblotting analysis as indicated. (E) The microRNA levels of TGF-β-treated LEPCs and control cells were determined by Realtime-PCR assay. *, p<0.05.
HEP_26007_sm_SuppFig8.tif840KSupporting Information Figure 8. WB-CON and WB-TβLT cells were treated with SIS3 (Merck KGaA, Germany) at 10 μM and then subjected to spheroid assay (A) and limiting dilution assay (B), DMSO was used as solvent control. WB-CON and WB-TβLT cells were transfected with small interference RNA against Smad2 (RiboBio Biotechnology, Guangzhou, China) followed by spheroid assay (C) and limiting dilution assay (D). (E) Realtime-PCR analysis of microRNA-216a expression in siSmad2-transfected WB-CON and WB-TβLT cells. Scramble siRNA was used as negative control. WB-CON and WB-TβLT cells were treated with SIS3 at indicated concentration and then subjected to immnuoblotting analysis of PTEN (F) and phosphorylation of Akt (G), GAPDH was used as loading control.
HEP_26007_sm_SuppInfo.doc94KSupporting Information

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