Journal: eLife

Article Title: β-Catenin-NF-κB-CFTR interactions in cholangiocytes regulate inflammation and fibrosis during ductular reaction

doi: 10.7554/eLife.71310

Figure Lengend Snippet: ( A ) Experimental design showing WT2 and KO2 on 2 weeks of CDE diet and recovery on normal diet for up to 6 months with analysis at intermediate time points as indicated. ( B ) Serum alanine aminotransferase (ALT), bilirubin, and alkaline phosphatase (ALP) in the two groups over time (one-way ANOVA, **p<0.01, ****p<0.0001, n = 3–6 per group). ( C ) β-Catenin immunohistochemistry in WT2 and KO2 mice at 3 months and 6 months of recovery showing β-catenin-positive BECs and hepatocytes in KO2 and WT2 (100×). ( D ) Ctnnb1 gene expression in WT2 and KO2 mice during recovery from CDE diet (one-way ANOVA, *p<0.05, **p<0.01, ****p<0.0001. n = 3–6 per group; individual animal values represented by dots).

Article Snippet: In brief, 23–25-day-old Ctnnb1 flox/flox ;Rosa-stop flox/flox -EYFP mice were injected intraperitoneally with 1 × 10 12 genome copies (GCs) of adeno-associated virus serotype 8 encoding Cre recombinase under the hepatocyte-specific thyroid binding globulin promoter (AAV8-TBG-Cre; Addgene) followed by a 12-day washout period.

Techniques: Immunohistochemistry, Gene Expression

Journal: eLife

Article Title: β-Catenin-NF-κB-CFTR interactions in cholangiocytes regulate inflammation and fibrosis during ductular reaction

doi: 10.7554/eLife.71310

Figure Lengend Snippet: ( A ) Experimental design showing WT1 and KO1 on 2 weeks of CDE diet and recovery on normal diet for up to 6 months with analysis at intermediate time points as indicated. ( B ) Serum alanine aminotransferase (ALT), bilirubin, and alkaline phosphatase (ALP) in the two groups over time (one-way ANOVA, **p<0.01, ****p<0.0001, n = 3–5 per group). ( C ) β-Catenin immunohistochemistry in WT1 and KO1 mice at 3 months and 6 months of recovery showing absence of β-catenin in biliary epithelial cells (BECs) and hepatocytes in KO1 (100×). ( D ) Ctnnb1 gene expression in WT1 and KO1 mice during recovery from CDE diet shows continued β-catenin absence over time in KO1 (one-way ANOVA, *p<0.05, **p<0.01, ****p<0.0001, n = 3–5 per group, individual animal values represented by dots).

Article Snippet: In brief, 23–25-day-old Ctnnb1 flox/flox ;Rosa-stop flox/flox -EYFP mice were injected intraperitoneally with 1 × 10 12 genome copies (GCs) of adeno-associated virus serotype 8 encoding Cre recombinase under the hepatocyte-specific thyroid binding globulin promoter (AAV8-TBG-Cre; Addgene) followed by a 12-day washout period.

Techniques: Immunohistochemistry, Gene Expression

Journal: eLife

Article Title: β-Catenin-NF-κB-CFTR interactions in cholangiocytes regulate inflammation and fibrosis during ductular reaction

doi: 10.7554/eLife.71310

Figure Lengend Snippet: ( A ) Luciferase reporter assay shows successful knockdown of Ctnnb1 in small cholangiocyte cell (SMCC) line by TOPFlash assay (left), which stimulates p65 transcriptional activity with or without 100 ng/ml lipopolysaccharide (LPS) (right) (unpaired t-test, ns: no significance, *p<0.05, **p<0.01, ****p<0.0001, n = 3 biological replication). ( B ) Luciferase reporter assay shows expression of constitutively active S45Y-β-catenin enhances TOPFlash (left) and suppresses p65 transcriptional activity with or without 100 ng/ml LPS (right) (unpaired t-test, ns: no significance, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n = 3 biological replication). ( C ) Representative WB from two independent experiment shows knockdown of Ctnnb1 increases p65 nuclear translocation with or without 500 ng/ml LPS. ( D ) Quantification of nuclear β-catenin (left) and nuclear p65 (right) to HH3 (blots in ( C ) were technically quantified three times and p-value was calculated using unpaired t-test, ns: no significance, **p<0.01, ***p<0.001, ****p<0.0001). ( E ) Identification of RELA and NFKB1 among the top 15 transcription factors identified by applying the 335 differentially expressed genes (DEGs) to JASPAR. ( F ) qPCR shows knockdown of Ctnnb1 in SMCCs induces Ccl2 (left) and Cxcl5 (right) expression (unpaired t-test, ns: no significance, *p<0.05, **p<0.01, n = 3 biological replication). ( G ) qPCR shows Ccl2 (left) and Cxcl5 (right) are induced in KO1 after 6-month recovery of choline-deficient, ethionine-supplemented (CDE) diet (unpaired t-test, *p< 0.05, n = 3–4 biological replication). ( H ) Representative immunoprecipitation (IP) image from two independent experiment shows p65 is strongly associated with β-catenin in SMCC. ( I ) IP shows that p65 is associated with β-catenin in whole liver lysate (L: liver; S: SMCC; P: equal amount of liver and SMCC lysate). ( J ) Quantification of colocalization of p65 and β-catenin is significantly diminished in KO1 compared to WT1 (unpaired t-test, **p<0.01, n = 3–4 biological replication). Figure 7—source data 1. WB shows knockdown of Ctnnb1 increases p65 nuclear translocation with or without 500 ng/ml lipopolysaccharide (LPS). Figure 7—source data 2. Immunoprecipitation (IP) image shows p65 is strongly associated with β-catenin in small cholangiocyte cell (SMCC). Figure 7—source data 3. Immunoprecipitation (IP) shows that p65 is associated with β-catenin in whole liver lysate.

Article Snippet: In brief, 23–25-day-old Ctnnb1 flox/flox ;Rosa-stop flox/flox -EYFP mice were injected intraperitoneally with 1 × 10 12 genome copies (GCs) of adeno-associated virus serotype 8 encoding Cre recombinase under the hepatocyte-specific thyroid binding globulin promoter (AAV8-TBG-Cre; Addgene) followed by a 12-day washout period.

Techniques: Luciferase, Reporter Assay, Knockdown, TOPFlash assay, Activity Assay, Expressing, Translocation Assay, Immunoprecipitation

Journal: eLife

Article Title: β-Catenin-NF-κB-CFTR interactions in cholangiocytes regulate inflammation and fibrosis during ductular reaction

doi: 10.7554/eLife.71310

Figure Lengend Snippet: ( A ) Reporter assay shows knockdown of Ctnnb1 stimulates p65 transcriptional activity (left) while transfection of stable S45Y-β-catenin represses p65 reporter activity (right) in MzChA human cholangiocarcinoma cells (unpaired t-test, ***p<0.001). ( B ) Reporter assay shows knockdown of Ctnnb1 stimulates p65 transcriptional activity (left) while expression of constitutively active S45Y-β-catenin suppresses p65 transcriptional activity (right) in HuCCT1 human cholangiocarcinoma cells (unpaired t-test, *p<0.05, **p<0.01, ****p<0.0001). ( C ) Heatmap of the differentially expressed genes in Ctnnb1 loss of function in small cholangiocyte cell (SMCC). ( D ) Heatmap of the differentially expressed genes in CTNNB1 gain of function in SMCC. ( E ) Very few common differentially expressed genes between Ctnnb1 loss- and gain-of-function models were identified, although altogether 335 genes were altered. These genes were assessed for transcription factor (TF) binding profiles by JASPAR (presented in ). ( F ) Representative confocal images showing the expression of p65 (red), β-catenin (green), and DAPI (white) in WT1 and KO1 livers (left). To visualize colocalization for p65 (red) and β-catenin (green), 3D images were reconstructed using Zen blue 2012 software (right). The reconstruction was performed with nine confocal z-stacks with 0.8 µm increments, and yellow region indicates colocalization of p65 (red) and β-catenin (green) in the projection on the X, Y, and Z planes.

Article Snippet: In brief, 23–25-day-old Ctnnb1 flox/flox ;Rosa-stop flox/flox -EYFP mice were injected intraperitoneally with 1 × 10 12 genome copies (GCs) of adeno-associated virus serotype 8 encoding Cre recombinase under the hepatocyte-specific thyroid binding globulin promoter (AAV8-TBG-Cre; Addgene) followed by a 12-day washout period.

Techniques: Reporter Assay, Knockdown, Activity Assay, Transfection, Expressing, Binding Assay, Software

Journal: Developmental cell

Article Title: Tumor Suppressor Inactivation of GDF11 Occurs by Precursor Sequestration in Triple-Negative Breast Cancer

doi: 10.1016/j.devcel.2017.10.027

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Cross) N/A Experimental Models: Organisms/Strains Mouse: SCID Beige (CB17.Cg-Prkdc scid Lyst bg-J /Crl) Charles River Strain #250, RRID: IMSR_CRL:250 Mouse: BALB/c (BALB/cAnNCrl) Charles River Strain #028, RRID: IMSR_CRL:28 Oligonucleotides Primers for quantitative PCR, see Table S6 This paper N/A Recombinant DNA TET-pLKO.1 puro shLuc This paper Addgene #83092 TET-pLKO.1 puro shGFP This paper Addgene #83085 TET-pLKO.1 puro shLacZ This paper Addgene #98632 TET-pLKO.1 puro shGDF11 #1 This paper Addgene #83083 TET-pLKO.1 puro shGDF11 #2 This paper Addgene #83084 TET-pLKO.1 puro shSMAD4 #1 This paper Addgene #83089 TET-pLKO.1 puro shSMAD4 #2 This paper Addgene #83091 TET-pLKO.1 puro shID2 #1 This paper Addgene #83086 TET-pLKO.1 puro shID2 #2 This paper Addgene #83087 TET-pLKO.1 puro shId2 #1 This paper Addgene #83088 TET-pLKO.1 puro shId2 #2 This paper Addgene #83090 pLX304 LacZ-2′V5 blast This paper Addgene #83099 pLX304 GDF11-V5 blast This paper Addgene #83098 pLX304 PCSK5-V5 blast This paper Addgene #83100 pLX304 PCSK5 (T288P)-V5 blast This paper Addgene #83101 pLX304 PCSK5 (A270T)-V5 blast This paper Addgene #83102 pLX304 PCSK5 (H516P)-V5 blast This paper Addgene #83103 pLX304 PCSK5 (R511C)-V5 blast This paper Addgene #83104 pLX304 Luciferase-V5 blast This paper Addgene #98580 pLX302 ID2-V5 puro This paper Addgene #83096 pLX302 GDF11-V5 puro This paper Addgene #83097 pLX302 PCSK5-V5 puro This paper Addgene #98389 pLX302 PCSK5 (T288P)-V5 puro This paper Addgene #98709 pLX302 LacZ-2′V5 blast This paper Addgene #98579 pEN_TT_miRc2 3xFLAG This paper Addgene #83274 pEN_TT_miRc2 3xFLAG LacZ This paper Addgene #83093 pEN_TT_miRc2 3xFLAG Luciferase This paper Addgene #98391 pEN_TT_miRc2 3XFLAG SMAD4 This paper Addgene #83094 pEN_TT_miRc2 3XFLAG ID2 This paper Addgene #98390 pSLIK_TT 3xFLAG LacZ neo This paper Addgene #83105 pSLIK_TT 3xFLAG LacZ hygro This paper Addgene #98395 pSLIK_TT 3xFLAG Luciferase neo This paper Addgene #98392 pSLIK_TT 3xFLAG SMAD4 neo This paper Addgene #83273 pSLIK_TT 3xFLAG ID2 neo This paper Addgene #98393 pSLIK_TT 3xFLAG ID2 hygro This paper Addgene #98394 pBabe TGFBR3-HA neo This paper Addgene #83095 pBabe Venus neo This paper Addgene #98396 pBabe mRFP1 neo This paper Addgene #98397 pLKO.1 shTGFBR3 puro Wang et al., 2014 .

Techniques: In Situ, Recombinant, Enzyme-linked Immunosorbent Assay, Mutagenesis, Amplification, Expressing, Real-time Polymerase Chain Reaction, Luciferase, Software