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n3:pmcid
PMC0
bibo:doi
10.1038%2Fnature16460
n8:contains
_:vb46931358
Subject Item
_:vb46931358
rdf:type
n8:Section
dc:title
methods
n8:contains
_:vb46931359 _:vb46931376 _:vb46931372 _:vb46931373 _:vb46931374 _:vb46931375 _:vb46931368 _:vb46931369 _:vb46931370 _:vb46931371 _:vb46931364 _:vb46931365 _:vb46931366 _:vb46931367 _:vb46931360 _:vb46931361 _:vb46931362 _:vb46931363
Subject Item
_:vb46931359
rdf:type
n3:Context
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C57BL/6 (B6, H-2b) and LP (H-2b) mice were obtained from Jackson Laboratory (Bar Harbor, USA). B6 Lgr5-LacZ and B6 lgr5-gfp-ires-CreERT2 (Lgr5-GFP) mice were kindly provided by H. Clevers>>1<<,10. Mouse maintenance and procedures were done in accordance with the institutional protocol guideline of the Memorial Sloan Kettering Cancer Center (MSKCC) Institutional Animal Care and Use Committee.
n3:mentions
n2:17934449
Subject Item
_:vb46931360
rdf:type
n3:Context
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1 mM N-acetyl cysteine (Sigma), B27 supplement (Invitrogen), N2 supplement (Invitrogen), 50 ng/ml mEGF (Peprotech), 100 ng/ml mNoggin (Peprotech) and 10% human R-spondin-1 conditioned medium from R-spondin-1-transfected HEK 293T cells>>31<< was added to small intestine crypt cultures10. For experiments evaluating organoid budding, the concentration of R-spondin was lowered to 1.25-5%.
n3:mentions
n2:19398967
Subject Item
_:vb46931361
rdf:type
n3:Context
rdf:value
Intestinal stem cells (ISCs) were isolated from Lgr5-GFP mice using a modified crypt isolation protocol with 20 minutes of 30 mM EDTA>>32<<,33 followed by several strainer steps and a 5 minunte incubation with TrypLE and 0.8 KU/ml DNase1 under minute-to-minute vortexing to make a single cells suspension.
n3:mentions
n2:23644405
Subject Item
_:vb46931362
rdf:type
n3:Context
rdf:value
Intestinal stem cells (ISCs) were isolated from Lgr5-GFP mice using a modified crypt isolation protocol with 20 minutes of 30 mM EDTA32,>>33<< followed by several strainer steps and a 5 minunte incubation with TrypLE and 0.8 KU/ml DNase1 under minute-to-minute vortexing to make a single cells suspension.
n3:mentions
n2:23928185
Subject Item
_:vb46931363
rdf:type
n3:Context
rdf:value
Topro3−CD45+CD11b−CD11c−CD90+ lamina propria lymphocytes (LPLs) from B6 WT and IL-22−/− mice and Topro3−CD45+CD3−RORγt+ group 3 innate lymphoid cells (ILCs)>>34<< from Rorc(γt)+ GFP mice (Jackson) were sorted for co-cultures with SI crypts.
n3:mentions
n2:23348417
Subject Item
_:vb46931364
rdf:type
n3:Context
rdf:value
A neutralizing monoclonal antibody against IL-22 (8E11, Genentech)>>35<< was used to abrogate IL-22-specific effects of ILCs.
n3:mentions
n2:18264109
Subject Item
_:vb46931365
rdf:type
n3:Context
rdf:value
Frozen passaged organoids from Lgr5CreER;Lgr5DTR (Lgr5GFP-DTR)>>25<< mice were used to culture organoids in which Lgr5+ stem cells could be depleted with daily administration of Diphtheria Toxin (DT, 1 ng/ul).
n3:mentions
n2:21927002
Subject Item
_:vb46931366
rdf:type
n3:Context
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For Paneth cell-deficient organoid cultures, frozen crypts from Atoh1fl/fl;Villin-CreERT2 mice>>36<< depleted of Paneth cells were used to culture organoids.
n3:mentions
n2:17570220
Subject Item
_:vb46931367
rdf:type
n3:Context
rdf:value
As previously described>>36<<, Atoh1fl/fl;Villin-CreERT2 mice (and littermate controls) were given an intraperitoneal (IP) injection of tamoxifen (1 mg/mouse, Sigma, dissolved in corn oil) for 5 consecutive days to achieve deletion of Atoh1 from intestinal epithelium.
n3:mentions
n2:17570220
Subject Item
_:vb46931368
rdf:type
n3:Context
rdf:value
R-spondin-1-transfected HEK293T cells>>31<< were kindly provided by Calvin Kuo, Department of Medicine, Stanford University.
n3:mentions
n2:19398967
Subject Item
_:vb46931369
rdf:type
n3:Context
rdf:value
BMT procedures were performed as previously described>>37<<. A minor histocompatibility antigen-mismatched BMT model (LP→B6, H-2b→H-2b) was utilized.
n3:mentions
n2:14523046
Subject Item
_:vb46931370
rdf:type
n3:Context
rdf:value
β-galactosidase (LacZ) staining was preformed as previously described by Barker et al>>1<<. Washed 2.5 cm sized small intestine fragments were incubated with an ice-cold fixative, consisting of 1% formaldehyde, 0.2% NP40 and 0.2% gluteraldehyde. After removing the fixative, organs were stained for the presence of LacZ according
n3:mentions
n2:17934449
Subject Item
_:vb46931371
rdf:type
n3:Context
rdf:value
Paneth cells were identified based on bright CD24 staining and side scatter granularity as described by Sato et al>>2<<.
n3:mentions
n2:21113151
Subject Item
_:vb46931372
rdf:type
n3:Context
rdf:value
To explore the association of ISC gene signatures (GSE33948 and GSE23672)>>16<< with Stat3-regulated genes, we performed GSEA in a Stat3ΔIEC murine DSS colitis dataset (GSE15955)12, comparing Stat3fl/fl;Villin-Cre− (WT) vs.
n3:mentions
n2:22692129
Subject Item
_:vb46931373
rdf:type
n3:Context
rdf:value
To explore the association of ISC gene signatures (GSE33948 and GSE23672)16 with Stat3-regulated genes, we performed GSEA in a Stat3ΔIEC murine DSS colitis dataset (GSE15955)>>12<<, comparing Stat3fl/fl;Villin-Cre− (WT) vs. Stat3fl/fl;Villin-Cre+ (Stat3ΔIEC) mice with DSS colitis (GSEA2-2.2.0; http:
n3:mentions
n2:19564350
Subject Item
_:vb46931374
rdf:type
n3:Context
rdf:value
org/gsea)>>39<<,40. A Paneth cell signature gene set was used as a negative control (DLL1+CD24hi, GSE39915)17.
n3:mentions
n2:16199517
Subject Item
_:vb46931375
rdf:type
n3:Context
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org/gsea)39,>>40<<. A Paneth cell signature gene set was used as a negative control (DLL1+CD24hi, GSE39915)17.
n3:mentions
n2:12808457
Subject Item
_:vb46931376
rdf:type
n3:Context
rdf:value
A Paneth cell signature gene set was used as a negative control (DLL1+CD24hi, GSE39915)>>17<<. Nominal P-values are shown.
n3:mentions
n2:23000963
Subject Item
_:vb594485917
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41
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n2:19329995
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35
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33
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32
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32
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n2:22921121
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