Journal: PLoS ONE

Article Title: Generation of Mice with Hepatocyte-Specific Conditional Deletion of Notum

doi: 10.1371/journal.pone.0150997

Figure Lengend Snippet: Huh7 cells were transfected with empty vector and pET-Notum6His as indicated, together with 50ng of TopFLASH Firefly Luciferase plasmid and 5ng of control Renillia Luciferase. Twenty-four hours after transfection, cells were stimulated or not with 50ng/ml recombinant human Wnt3a and analyzed for luciferase activity 24 hours later. Data represent means of three independent transfections with the standard deviations indicated. NOTUM silencing did not increase β-catenin activity. HuH7 cells were reverse-transfected as indicated with 100 pmol of NOTUM siRNA. Twenty four hours after, β-catenin luciferase reporters (TOPflash/FOPflash) were transfected and Wnt3a was added as indicated 16 hours post-transfection of the plasmids encoding luciferase. Luciferase activity was measured the following day.

Article Snippet: Recombinant human Wnt3a was obtained from R&D Systems (Minneapolis, MN).

Techniques: Transfection, Plasmid Preparation, Luciferase, Recombinant, Activity Assay

Journal: Cell Death & Disease

Article Title: Crosstalk between hepatic tumor cells and macrophages via Wnt/β-catenin signaling promotes M2-like macrophage polarization and reinforces tumor malignant behaviors

doi: 10.1038/s41419-018-0818-0

Figure Lengend Snippet: a , b BMDMs (M0) were treated with Wnt3a (100 ng/mL) or LiCl (10 mM) in advance. Cells were then stimulated with LPS + IFN-γ (M1) for 24 h. The mRNA levels of M1 markers such as TNF-α, IL-12, and iNOS ( a ), and M2 markers, including Arg1, MR and IL-10 ( b ), were determined by qRT-PCR, respectively. β-actin was used as an internal control ( n = 3). c , d BMDMs were polarized with IL-4 (M2) followed by Wnt3a (100 ng/mL) treatment or no treatment for 24 h. The mRNA level of M2 markers ( c ) and the downstream genes of Wnt signaling ( d ) were detected by qRT-PCR, and then were quantitatively compared ( n = 3). e , f Wnt signaling inhibitor ICG001 (10 μM) was added into BMDMs, and then BMDMs were polarized with IL-4 for 24 h. The mRNA level of M2 markers ( e ) and the downstream genes of Wnt signaling ( f ) were examined by qRT-PCR, and then compared quantitatively ( n = 3). g − j BMDMs were cultured on coverslips, and treated with Wnt3a or ICG001 followed by IL-4 stimulation for 24 h. Then cells were subjected to IF staining using anti-F4/80 and anti-MR ( g , h ) or anti-Arg1 ( i , j ) antibodies. Nuclei were counterstained with Hoechst ( n = 3). Mean florescence intensity was determined and compared. Bars, mean ± SD; * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: The supernatant was collected from cultured Hepa1-6 cells that had been transduced with sh-Wntless successfully, and then the concentration of Wnt3a in the supernatant was detected using a mouse Wnt3a ELISA kit (MyBioSource, California, USA) according to the instructions.

Techniques: Quantitative RT-PCR, Control, Cell Culture, Staining

Journal: Cell Death & Disease

Article Title: Crosstalk between hepatic tumor cells and macrophages via Wnt/β-catenin signaling promotes M2-like macrophage polarization and reinforces tumor malignant behaviors

doi: 10.1038/s41419-018-0818-0

Figure Lengend Snippet: a BMDMs were transfected with c-Myc siRNA (sic-Myc) or randomized siRNA control (siR) for 24 h, and then the cells were stimulated with IL-4 for 24 h. The expression of M2 markers was determined by qRT-PCR, and compared quantitatively among each group ( n = 3). b BMDMs were transfected with sic-Myc or siR for 24 h, and then the cells were polarized with IL-4 in the presence of Wnt3a for 24 h. The expression of M2 markers was determined by qRT-PCR, and compared quantitatively among each group ( n = 3). Bars, mean ± SD; * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: The supernatant was collected from cultured Hepa1-6 cells that had been transduced with sh-Wntless successfully, and then the concentration of Wnt3a in the supernatant was detected using a mouse Wnt3a ELISA kit (MyBioSource, California, USA) according to the instructions.

Techniques: Transfection, Control, Expressing, Quantitative RT-PCR