Journal: Cell reports

Article Title: Mutual Zonated Interactions of Wnt and Hh Signaling Are Orchestrating the Metabolism of the Adult Liver in Mice and Human.

doi: 10.1016/j.celrep.2019.11.104

Figure Lengend Snippet: Figure 2. Lobular Distribution of Wnt/Hh Pathway Protein Expression in Humans (A) IHC of Wnt proteins WNT5A, FZD4, LGR5, AXIN2, and CTNNB1 on human liver biopsy slides. (B) IHC of and Hh pathway proteins IHH, GLI1, GLI2, and GLI3 on human liver biopsy slides. See also Table S1.

Article Snippet: The following primary antibodies were used: anti AXIN2 (1:1000; Abcam, ab32197, UK), anti CTNNB1 (1:500; Transduction Laboratories, 610054, USA), anti FZD2 (1:700; Alomone, AFR-022, Israel), anti FZD4 (1:500; LSBio, LS-B5723, USA) anti GLI1 (1:1500; GeneTex, GTX124274, USA), anti GLI2 (1:140; Biozol, ORB378810, Germany) anti GLI3 (1:1000; GeneTex, GTX26050, USA), Sigma, Germany), anti GS (1:1000; Abcam, ab49873, UK), anti IHH (1:600; Abcam, ab39634, UK), anti PTCH1 (1:1000; LSBio, LS-C122292, USA), anti PTCH2 (1:1000; LSBio, LS-B301, USA), anti WNT5a (1:500; St. John’s Laboratory, StJ26117, UK).

Techniques: Expressing

Journal: Journal of Clinical Investigation

Article Title: Norrin mediates tumor-promoting and -suppressive effects in glioblastoma via Notch and Wnt

doi: 10.1172/jci128994

Figure Lengend Snippet: Figure 1. NDP is expressed in a wide range of cancers, and is enriched in CNS tumors. (A and B) Analysis of NDP (A) and FZD4 (B) expression levels in primary human tumors from TCGA using the cBioportal web server. NDP expression was significantly enriched in GBM and lower-grade glioma relative to the average of all cancer types (upper graph), while FZD4 expression was comparable to other tumor types. ACC, adrenocortical carcinoma; AML, acute myeloid leukemia; DLBC, lymphoid neoplasm diffuse large B cell lymphoma; PCPG, pheochromocytoma and paraganglioma; CS, carci- nosarcoma; ccRCC, clear cell renal cell carcinoma; chRCC, chromophobe renal cell carcinoma; pRCC, papillary renal cell carcinoma; VUS, variant of uncertain significance.

Article Snippet: For cell treatments we used anti-FZD4 blocking antibody (Lexicon Pharmaceuticals); anti-KLH blocking antibody (Lexicon Pharmaceuticals); IWP-2 (Sigma-Aldrich, I0536); XAV939 (Sigma-Aldrich, X3004); rhNorrin (R&D Systems, 3014-NR); rhWNT3a (R&D Systems, 5036-WN); DKK1 (R&D Systems, 5439-DK); DAPT (GSI; Sigma-Aldrich, D5942); CHIR (CHIR 99021: 6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1Himidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3 pyridinecarbonitrile; Axon Medchem, CT99021); and BIO ((2′Z,3′E)-6-bromoindirubin-3′-oxime; Sigma-Aldrich, B1686).

Techniques: Expressing, Variant Assay

Journal: Journal of Clinical Investigation

Article Title: Norrin mediates tumor-promoting and -suppressive effects in glioblastoma via Notch and Wnt

doi: 10.1172/jci128994

Figure Lengend Snippet: Figure 2. NDP is expressed in GSCs and correlates with survival in neurological tumors. (A) Gene set enrichment analysis reveals correla- tion between NDP expression and “Glioblastoma Classical” and “Aging Brain” gene sets. (B) Kaplan-Meier analysis correlating NDP expression with patient survival in neurological cancers. (C) Expression of compo- nents of the NDP/FZD4 signaling axis in a panel of 9 patient-derived GSCs (left) and 3 primary fetal hNSC lines (right). Blue boxes, ASCL1lo GSC lines; red, ASCL1hi GSC lines indicate the GSC lines selected for functional analysis.

Article Snippet: For cell treatments we used anti-FZD4 blocking antibody (Lexicon Pharmaceuticals); anti-KLH blocking antibody (Lexicon Pharmaceuticals); IWP-2 (Sigma-Aldrich, I0536); XAV939 (Sigma-Aldrich, X3004); rhNorrin (R&D Systems, 3014-NR); rhWNT3a (R&D Systems, 5036-WN); DKK1 (R&D Systems, 5439-DK); DAPT (GSI; Sigma-Aldrich, D5942); CHIR (CHIR 99021: 6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1Himidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3 pyridinecarbonitrile; Axon Medchem, CT99021); and BIO ((2′Z,3′E)-6-bromoindirubin-3′-oxime; Sigma-Aldrich, B1686).

Techniques: Expressing, Derivative Assay, Functional Assay

Journal: Journal of Clinical Investigation

Article Title: Norrin mediates tumor-promoting and -suppressive effects in glioblastoma via Notch and Wnt

doi: 10.1172/jci128994

Figure Lengend Snippet: Figure 4. Divergent effects of NDP and FZD4 knockdown on the proliferation indices of GSC cultures. (A and B) Representative images of ICC staining for Ki67 (red) and SOX2 (green) after NDP or FZD4 knockdown in ASCL1lo (G564) (A) and ASCL1hi (G472) (B) GSCs. Scale bars: 50 μm. (C and D) Quantification of the frequency of Ki67+ and SOX2+ cells after NDP and FZD4 knockdown in G564 (C) and G472 (D) GSCs. n = 3 (with the exception of Ki67 in G564; n = 9 because the cells are very large and the number of cells per field or vessel was quite small). Data are presented as mean ± SEM. *P < 0.05 by 1-way ANOVA.

Article Snippet: For cell treatments we used anti-FZD4 blocking antibody (Lexicon Pharmaceuticals); anti-KLH blocking antibody (Lexicon Pharmaceuticals); IWP-2 (Sigma-Aldrich, I0536); XAV939 (Sigma-Aldrich, X3004); rhNorrin (R&D Systems, 3014-NR); rhWNT3a (R&D Systems, 5036-WN); DKK1 (R&D Systems, 5439-DK); DAPT (GSI; Sigma-Aldrich, D5942); CHIR (CHIR 99021: 6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1Himidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3 pyridinecarbonitrile; Axon Medchem, CT99021); and BIO ((2′Z,3′E)-6-bromoindirubin-3′-oxime; Sigma-Aldrich, B1686).

Techniques: Knockdown, Staining

Journal: Journal of Clinical Investigation

Article Title: Norrin mediates tumor-promoting and -suppressive effects in glioblastoma via Notch and Wnt

doi: 10.1172/jci128994

Figure Lengend Snippet: Figure 9. Effects of NDP on progression of GBM in vivo depend on ASCL1 expression status. (A and B) Representative H&E staining of orthotopically xenografted G411 (ASCL1lo) (A) and G523 (ASCL1hi) GSC (B) tumors (dotted line, tumor outline). Shown are tile scans taken at ×2.5 magnification (left) and boxed regions are shown at higher magnification (×20) on the right. G411 GSCs form localized and G523 GSCs form diffuse tumors. Scale bars: 10 μm and 50 μm, respectively. (C and D) Kaplan-Meier survival analysis of mice orthotopically transplanted with G411 GSCs after NDP or FZD4 knockdown (C) and overexpression (D). (E and F) Kaplan-Meier survival analysis of mice orthotopically transplanted with G523 GSCs after NDP knockdown (E) and overexpres- sion (F). P < 0.05 by log-rank test (C–F).

Article Snippet: For cell treatments we used anti-FZD4 blocking antibody (Lexicon Pharmaceuticals); anti-KLH blocking antibody (Lexicon Pharmaceuticals); IWP-2 (Sigma-Aldrich, I0536); XAV939 (Sigma-Aldrich, X3004); rhNorrin (R&D Systems, 3014-NR); rhWNT3a (R&D Systems, 5036-WN); DKK1 (R&D Systems, 5439-DK); DAPT (GSI; Sigma-Aldrich, D5942); CHIR (CHIR 99021: 6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1Himidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3 pyridinecarbonitrile; Axon Medchem, CT99021); and BIO ((2′Z,3′E)-6-bromoindirubin-3′-oxime; Sigma-Aldrich, B1686).

Techniques: In Vivo, Expressing, Staining, Knockdown, Over Expression

Journal: Journal of Clinical Investigation

Article Title: Norrin mediates tumor-promoting and -suppressive effects in glioblastoma via Notch and Wnt

doi: 10.1172/jci128994

Figure Lengend Snippet: Figure 10. NDP modulation results in selective advantage or disadvantage in xenografted tumors depending on ASCL1 expression status. (A and B) Representative IHC images of G411-derived tumors with NDP or FZD4 knockdown (A) or overexpression (B). Scale bars: 50 μm. (C and D) Representative IHC images of G523-derived tumors with NDP knockdown (C) or overexpression (D). Scale bars: 50 μm.

Article Snippet: For cell treatments we used anti-FZD4 blocking antibody (Lexicon Pharmaceuticals); anti-KLH blocking antibody (Lexicon Pharmaceuticals); IWP-2 (Sigma-Aldrich, I0536); XAV939 (Sigma-Aldrich, X3004); rhNorrin (R&D Systems, 3014-NR); rhWNT3a (R&D Systems, 5036-WN); DKK1 (R&D Systems, 5439-DK); DAPT (GSI; Sigma-Aldrich, D5942); CHIR (CHIR 99021: 6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1Himidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3 pyridinecarbonitrile; Axon Medchem, CT99021); and BIO ((2′Z,3′E)-6-bromoindirubin-3′-oxime; Sigma-Aldrich, B1686).

Techniques: Expressing, Derivative Assay, Knockdown, Over Expression