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

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HEP_24587_sm_SuppFig1.tif9040KSupporting Information Figure 1. Expression of CTGF and YAP in healthy and diseased human liver. (A) CTGF and YAP mRNA levels were measured in healthy livers (HL, n-9), healthy liver tissue from organs harbouring a hepatocellular carcinoma (HL+HCC, n-12), cirrhotic liver tissue from organs harbouring a hepatocellular carcinoma (Cirr+HCC, n-17) and hepatocellular carcinoma (HCC, n-20). “+” indicates the mean value of CTGF or YAP expression for each set of samples. For CTGF expression statistical significance was: HL vs HL+HCC p-0.002; HL vs Cirr+HCC p- 0.0002; HL vs HCC p-0.026. For YAP expression statistical significance was: HL vs HL+HCC p-0.0003; HL vs Cirr+HCC p-0.0001; HL vs HCC p-0.0001. (B) Immunohistochemical detection of CTGF and YAP proteins in sections from HL, Cirr+HCC and HCC liver tissues. Representative photomicrographs are shown (magnification × 200).
HEP_24587_sm_SuppFig2.tif4972KSupporting Information Figure 2. EGFR activation stimulates CTGF and YAP expression in MCF-10A cells. (A) CTGF mRNA levels in cells treated with AR (50 nmol/L) or HB-EGF (50 nmol/L) for 3h. (B) YAP mRNA levels in cells treated with AR (50 nmol/L) or HB-EGF (50 nmol/L) for 3h. (C) YAP protein levels in cells treated with AR (50 nmol/L) or HB-EGF (50 nmol/L) for 12h analyzed by Western blotting. (D) Immunofluorescent detection of YAP protein in control and AR (50 nmol/L, 12h) treated MCF-10A cells.
HEP_24587_sm_SuppFig3.tif85KSupporting Information Figure 3. Effect of AR on miRNA-375 expression. Levels of miRNA-375 in control and AR (50 nmol/L, 3h) stimulated Hep3B cells (A) and primary human hepatocytes (B).
HEP_24587_sm_SuppFig4.tif788KSupporting Information Figure 4. Effect of CTGF knockdown on anchorage-independent cell growth in soft agar and on in vivo growth in nude mice. (A) PLC/PRF/5 cells were transfected with control (siGL) or CTGF specific (siCTGF) siRNAs. After 48h cells were harvested, counted, resuspended in 0.2% soft agar, and seeded onto 0.4% soft agar in DMEM supplemented with 10% fetal calf serum (104 cells per plate). After 3 weeks, colonies were stained with crystal violet and counted. (B) PLC/PRF/5 cells were transfected with control (siGL) or CTGF specific (siCTGF) siRNAs. After 48h cells were harvested and subcutaneously injected into nude mice (10 × 106 cells per mouse). Tumor volumes (mm3) were monitored over time. Data are means ± SEM (n-10 mice per group). Asterisks indicate p<0.05 vs. siCTGF.
HEP_24587_sm_SuppFig5.tif185KSupporting Information Figure 5. Validation of selected genes identified by microarray analysis as differentially expressed in Hep3B cells upon CTGF knockdown. Quantitative real-time PCR analysis of gene expression in total RNA samples from Hep3B cells 48h after transfection with siCTGF or control siGL siRNAs. Values are expressed in relative transcript levels as compared with control siGL siRNA. (A) Genes downregulated upon CTGF knockdown included G protein-coupled receptor 160 (GPR160), connexin 43/GJA1 (GJA), lysyl oxidase (LOX), orosomucoid 1 and 2 (ORM1, ORM2) and fibroblast growth factor receptor 2 (FGFR2). (B) Genes upregulated upon CTGF knockdown included bile acid CoA: amino acid N-acyltransferase (BAAT), the PR-Set7 histone methyltransferase (SETD8), latent TGFβ binding protein-1 (LTBP1), p21-activated protein kinase-2 (PAK2), lysine acetyltransferase 2B (KAT2B/PCAF), UDP-glucuronosyltransferase-2B15 (UGT2B15), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor 2 (TNFRSF10B or TRAIL-R2) and tryptophan dioxygenase (TDO). Differences were statistically significant (p values at least <0.05) with respect control transfections for all genes shown.
HEP_24587_sm_SuppInfo.doc47KSupporting Information
HEP_24587_sm_SuppTabS1.doc57KSupporting Information Table 1. Selection of genes with differential expression in Hep3B cells upon CTGF knockdown identified by microarray analysis.
HEP_24587_sm_SuppTabS2.doc71KSupporting Information Table 2. Primers used in this study to measure gene expression by quantitative RT-PCR.

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