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. 2020 Mar 18;146(5):1153–1167. doi: 10.1007/s00432-020-03182-1

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© The Author(s) 2020

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — Restored SFRP1 expression affects WNT signaling activity and HB tumor cell characteristics. a mRNA expression of SFRP1 in transient pcDNA3.1- and pSFRP1-transfected HuH-6 cells after 48 and 72 h was determined by qRT-PCR and calculated as normalized mRNA expression (fold change) to pcDNA3.1 control (n = 2). b Cell growth of transient pcDNA3.1- and pSFRP1-transfected HuH-6 cells was assessed by MTT assay at indicated time points. Values were normalized to zero hour time point and shown as mean ± SEM (n = 2). Slope difference was analyzed by linear regression, ****p < 0.0001. c mRNA expression and representative immunoblot image of SFRP1 in stable pcDNA3.1- and pSFRP1-transfected HuH-6 cell clone 2. GAPDH served as loading control in immunoblot analysis (n = 2). Gene expression was determined by qRT-PCR and calculated as normalized mRNA expression (fold change) to pcDNA3.1 control (n = 3). d Cell growth of stable pcDNA3.1- and pSFRP1-transfected HuH-6 cells was assessed by MTT assay at indicated time points. Values were normalized to zero hour time point and shown as mean ± SEM (n = 4). Slope difference was analyzed by linear regression, *p < 0.05. e Representative pictures and quantification of number of colonies per well of stable pcDNA3.1- and pSFRP1-transfected HuH-6 cells (n = 3). f Representative pictures and quantification of cell migration at indicated time points were normalized to zero hour time points. Slope difference was analyzed by linear regression, ****p < 0.0001. g TOP/FOP reporter plasmid activity was assessed by a relative luciferase activity in stable pcDNA3.1- and pSFRP1-transfected HuH-6 cells 48 h after co-transfection (n = 5). h mRNA expression of MYC and CCND1 in stable pcDNA3.1- and pSFRP1-transfected HuH-6 cell clone 2 was determined by qRT-PCR and calculated as normalized mRNA expression (fold change) to pcDNA3.1 control (n = 4). i Immunofluorescence staining of β-catenin (CTNNB1, green) and DAPI nuclear staining (blue) in stable pcDNA3.1- and pSFRP1-transfected HuH-6 cells. Scale bars: 100 µm. All data are represented as mean ± SEM; unless otherwise stated p values were calculated by two-tailed, unpaired Student’s t test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001