Journal: Cell Proliferation

Article Title: Liver cancer: WISP3 suppresses hepatocellular carcinoma progression by negative regulation of β‐catenin/TCF/LEF signalling

doi: 10.1111/cpr.12583

Figure Lengend Snippet: The expression of WISP3 was decreased in HCC cell lines and clinical samples. A, The protein level of WISP3 in two normal liver cell lines (QSG‐7701 and LO2) and five liver cancer cell lines (SMMC‐7721, SK‐HEP‐1, PVTT‐1, Huh7 and Hep3B) was examined by Western blot. Left panel: Result of Western blot. Right panel: Grey analysis results of the Western blot bands. B, The mRNA level of WISP3 in 71 HCC samples and paired normal liver tissues was detected by quantitative real‐time PCR. The expression of WISP3 was normalized to β‐actin. T, tumour samples; N, matched normal tissues. C, Protein level of WISP3 in 15 randomly chosen HCC samples and paired normal tissues; α‐tubulin was used as loading control. Left panel: Result of Western blot. Right panel: Grey analysis results of the Western blot bands. D, The mRNA levels of WISP3 in liver cancer samples in comparison with normal liver samples derived from GEPIA. * P < 0.05， *** P < 0.001 vs normal liver tissue

Article Snippet: For experiments with recombinant human WISP3 (rhWISP3), Huh7 and PVTT‐1 cells (2 × 10 5 ) with 400 ng/mL of rhWISP3 (Peprotech, Rocky Hill, NJ, USA) or with diluent in 1% foetal bovine serum were plated in the upper wells.

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Control, Comparison, Derivative Assay

Journal: Cell Proliferation

Article Title: Liver cancer: WISP3 suppresses hepatocellular carcinoma progression by negative regulation of β‐catenin/TCF/LEF signalling

doi: 10.1111/cpr.12583

Figure Lengend Snippet: Forced expression of WISP3 inhibited cell growth, migration and tumorigenesis. A, Overexpression of WISP3 in PVTT‐1 and Huh7 cells. Cells were infected with lentivirus expressing WISP3 or vector control, followed by selection with puromycin. Lysate was examined by Western blot. α‐tubulin was used as a loading control. B, The effects of WISP3 on the proliferation of PVTT‐1 and Huh7 cells were measured by MTT assay. C, The effects of WISP3 on the migration of PVTT‐1 and Huh7 cells were measured by Boyden Chamber assay. Left photograph : Boyden Chamber assay of PVTT‐1 and Huh7 cell lines after forced expression of WISP3. Right photograph : calculation of cells that migrated through the filter following eosin staining. Scale bar = 200 μm. D, The effects of WISP3 on growth of Huh7 in vivo. Left photograph : representative photograph of the xenografts. Right photograph : tumour weight. E, Overexpression of WISP3 inhibited HCC cell growth in distant organs. WISP3 overexpressing cells and control cells labelled with luciferase were injected into the left ventricle of nude mice. The metastatic lesions were measured by bioluminescent signals on days 0 and 28 (five mice for each group). Results were presented as mean ± SE of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 vs Lenti‐vector cells

Article Snippet: For experiments with recombinant human WISP3 (rhWISP3), Huh7 and PVTT‐1 cells (2 × 10 5 ) with 400 ng/mL of rhWISP3 (Peprotech, Rocky Hill, NJ, USA) or with diluent in 1% foetal bovine serum were plated in the upper wells.

Techniques: Expressing, Migration, Over Expression, Infection, Plasmid Preparation, Control, Selection, Western Blot, MTT Assay, Boyden Chamber Assay, Staining, In Vivo, Luciferase, Injection

Journal: Cell Proliferation

Article Title: Liver cancer: WISP3 suppresses hepatocellular carcinoma progression by negative regulation of β‐catenin/TCF/LEF signalling

doi: 10.1111/cpr.12583

Figure Lengend Snippet: Knockdown of WISP3 promoted cell growth and migration, as well as metastasis in vivo. A, Silencing the expression of WISP3 in QSG‐7701 and LO2 cells. Left panel: Result of Western blot. Right panel: Grey analysis results of the Western blot bands. B, Knockdown the expression of WISP3 promoted the growth of QSG‐7701 and LO2 cells by MTT assay. C, Knockdown the expression of WISP3 promoted the migration of QSG‐7701 and LO2 cells by Boyden Chamber assay. Left photograph : Boyden Chamber assay of QSG‐7701 and LO2 cell lines after knockdown the expression of WISP3. Right photograph : calculation of cells that migrated through the filter following eosin staining. Scale bar = 200 μm. D, Knockdown of WISP3 promoted cell growth in distant organs. The metastatic lesions were measured by bioluminescent signals on days 0 and 63 (five mice for each group). Results were presented as mean ± SE of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 vs Lenti‐vector cells

Article Snippet: For experiments with recombinant human WISP3 (rhWISP3), Huh7 and PVTT‐1 cells (2 × 10 5 ) with 400 ng/mL of rhWISP3 (Peprotech, Rocky Hill, NJ, USA) or with diluent in 1% foetal bovine serum were plated in the upper wells.

Techniques: Knockdown, Migration, In Vivo, Expressing, Western Blot, MTT Assay, Boyden Chamber Assay, Staining, Plasmid Preparation

Journal: Cell Proliferation

Article Title: Liver cancer: WISP3 suppresses hepatocellular carcinoma progression by negative regulation of β‐catenin/TCF/LEF signalling

doi: 10.1111/cpr.12583

Figure Lengend Snippet: WISP3 repressed β‐catenin/TCF transcriptional activity by inhibition of β‐catenin nuclear accumulation. A, WISP3 inhibited the activity of TOPflash reporter in a dose‐dependent manner in HEK293T cells. Left panel: WISP3 inhibited the activity of TOPflash reporter in a dose‐dependent manner in HEK293T cells. Right panel: WISP3 inhibited the activity of TOPflash reporter in a dose‐dependent manner in Huh7 cells. B, Overexpression of WISP3 inhibited the nuclear localization of β‐catenin. Left photograph : representative photomicrographs of PVTT‐1/Lenti‐WISP3 stable cells, and Huh7/Lenti‐WISP3 stable cells immunostained with anti‐β‐catenin antibody (red). Cell nuclei were counterstained with Hoechst (blue). Right photograph : protein level of both cytoplasmic and nuclear β‐catenin was further confirmed by Western blot. The targeted genes of β‐catenin/TCF signalling, cyclin D1 and MMP7 were detected. Scale bar = 20 μm. C, Knockdown of WISP3 promoted β‐catenin nuclear localization. Left photograph : representative photomicrographs of QSG‐7701/Lenti‐shWISP3 stable cells, and LO2/Lenti‐shWISP3 stable cells. Right photograph : translocation of β‐catenin and its target genes were further confirmed by Western blot. Scale bar = 20 μm. D, WISP3 regulated the phosphorylation of Akt/GSK3β. The effects of WISP3 on the phosphorylation of Akt and GSK3β were examined in PVTT‐1, Huh7, QSG‐7701, LO2 cells.

Article Snippet: For experiments with recombinant human WISP3 (rhWISP3), Huh7 and PVTT‐1 cells (2 × 10 5 ) with 400 ng/mL of rhWISP3 (Peprotech, Rocky Hill, NJ, USA) or with diluent in 1% foetal bovine serum were plated in the upper wells.

Techniques: Activity Assay, Inhibition, Over Expression, Western Blot, Knockdown, Translocation Assay, Phospho-proteomics