Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Prolonged Absence of Mechanoluminal Stimulation in Human Intestine Alters the Transcriptome and Intestinal Stem Cell Niche

doi: 10.1016/j.jcmgh.2016.12.008

Figure Lengend Snippet: Primary and Secondary Antibodies

Article Snippet: LGR5 , Rabbit , Miltenyi (San Diego, CA) , 130-104-945 , 1:200.

Techniques: Transduction, Plasmid Preparation

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Prolonged Absence of Mechanoluminal Stimulation in Human Intestine Alters the Transcriptome and Intestinal Stem Cell Niche

doi: 10.1016/j.jcmgh.2016.12.008

Figure Lengend Snippet: qPCR Primer List

Article Snippet: LGR5 , Rabbit , Miltenyi (San Diego, CA) , 130-104-945 , 1:200.

Techniques: Sequencing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Prolonged Absence of Mechanoluminal Stimulation in Human Intestine Alters the Transcriptome and Intestinal Stem Cell Niche

doi: 10.1016/j.jcmgh.2016.12.008

Figure Lengend Snippet: Absence of mechanoluminal stimulation increases LGR5 mRNA expression and downstream Wnt signaling gene expression. ( A and B ) Quantification and in situ hybridization of LGR5 ISCs within the crypts of fed and unfed intestine. Inset : High-magnification photographs label LGR5-positive in situ hybridization with purple arrows . qPCR comparison of ( C ) LGR5 , ( D ) CCND1 , and ( E ) MYC mRNA expression between 6 and 7 pairs of matched fed vs unfed intestine with outliers excluded. ( F ) Representative images of Western blot analysis of STAT3 phosphorylation from 3 pairs of matched tissue. Images were obtained on an upright Leica DM5500B immunofluorescence microscope using Leica Suite Advanced Fluorescence (LAS AF) 6000 software, processed with ImageJ software. Scale bars : 50 μm. * P < .05. Grey bars on plots indicate median with 95% confidence interval.

Article Snippet: LGR5 , Rabbit , Miltenyi (San Diego, CA) , 130-104-945 , 1:200.

Techniques: Expressing, Gene Expression, In Situ Hybridization, Comparison, Western Blot, Phospho-proteomics, Immunofluorescence, Microscopy, Fluorescence, Software

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Prolonged Absence of Mechanoluminal Stimulation in Human Intestine Alters the Transcriptome and Intestinal Stem Cell Niche

doi: 10.1016/j.jcmgh.2016.12.008

Figure Lengend Snippet: Global decrease of LGR5+, SOX9+, and OLFM4+ intestinal stem cell populations occurs in the absence of mechanoluminal flow. ( A–H ) Quantification and immunofluorescence staining of LGR5+ rapidly cycling intestinal stem cells per crypt with proliferating cell nuclear antigen counterstain in fed vs unfed intestine. ( C and F ) LYZ+ Paneth cells with E-cadherin (Ecad) counterstain of same crypt shown in panels A and D , respectively, for comparison. ( I–N ) Quantification and immunofluorescence staining of SOX9 and OLFM4 intestinal stem cells per crypt in fed vs unfed intestine. Images were obtained on an upright Leica DM5500B immunofluorescence microscope using Leica Suite Advanced Fluorescence (LAS AF) 6000 software, processed with ImageJ software. Scale bars : ( B , C , E , and F ) 50 μm, and ( A , D , I , J , L , and M ) 100 μm. * P < .05. Grey bars on plots indicate median with 95% confidence interval. PCNA, proliferating cell nuclear antigen.

Article Snippet: LGR5 , Rabbit , Miltenyi (San Diego, CA) , 130-104-945 , 1:200.

Techniques: Immunofluorescence, Staining, Comparison, Microscopy, Fluorescence, Software

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Lgr5+CD44+EpCAM+ Strictly Defines Cancer Stem Cells in Human Colorectal Cancer.

doi: 10.1159/000488743

Figure Lengend Snippet: Fig. 1. Flow cytometric sorting of cancer stem cells from DLD- 1 cells. (A) Sorting of S1-S5 subpopulations. (B) EpCAM+CD44+ cells generated more spheres than EpCAM-CD44- cells, as as sessed by sphere-forming assay; Bar, 100 μm. (C) Lgr5+ cells from both EpCAM+CD44+ and EpCAM-CD44- subpopulations gener ated more spheres than did Lgr5- cells. The Lgr5+EpCAM+CD44+ subpopulation generated more spheres and colonies than did the Lgr5+EpCAM-CD44- subpopulation. Bar, 100 μm. *P<0.05 com pared to control.

Article Snippet: The fixed cells were then permeabilized with 0.1% Triton X-100 at room temperature for 15 min, followed by incubation at 4°C for 10 h with primary antibodies against Vimentin (1:200, Cell Signaling Technology, MA, USA) and Lgr5 (1:200, Santa Cruz Biotechnology, CA, USA).

Techniques: Generated, Control

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Lgr5+CD44+EpCAM+ Strictly Defines Cancer Stem Cells in Human Colorectal Cancer.

doi: 10.1159/000488743

Figure Lengend Snippet: Fig. 3. Characterization of self-re newal and stem-like properties. (A) When cultured in serum-free me dium, Lgr5-negative cells (S4 and S5) formed smaller spheres at a sig nificantly lower frequency than did Lgr5-positive cells (S1, S2 and S3). (B) The CSC median frequencies of the S1 (10/121), S2 (10/133), and S3 (10/137) subpopulations were higher than those of the S4 (10/238) and S5 (10/250) subpopulations, as assessed by limiting dilution assay. Bar, 100 μm. #P<0.05 compared to any other group.

Article Snippet: The fixed cells were then permeabilized with 0.1% Triton X-100 at room temperature for 15 min, followed by incubation at 4°C for 10 h with primary antibodies against Vimentin (1:200, Cell Signaling Technology, MA, USA) and Lgr5 (1:200, Santa Cruz Biotechnology, CA, USA).

Techniques: Cell Culture, Limiting Dilution Assay

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Lgr5+CD44+EpCAM+ Strictly Defines Cancer Stem Cells in Human Colorectal Cancer.

doi: 10.1159/000488743

Figure Lengend Snippet: Fig. 4. Characterization of colony formation and prolif eration properties. (A) Soft agar assays showed that Lgr5- positive cells (S1, S2 and S3) generated more colonies than did Lgr5-negative cells (S4 and S5). (B) Colony formation as says showed that Lgr5-positive cells (S1, S2 and S3) generated more colonies than did Lgr5- negative cells (S4 and S5). (C) The CCK-8 assay indicated that Lgr5-positive cells exhibited a significantly higher prolif erative capacity than did Lgr5- negative cells. #P<0.05 com pared to any other group.

Article Snippet: The fixed cells were then permeabilized with 0.1% Triton X-100 at room temperature for 15 min, followed by incubation at 4°C for 10 h with primary antibodies against Vimentin (1:200, Cell Signaling Technology, MA, USA) and Lgr5 (1:200, Santa Cruz Biotechnology, CA, USA).

Techniques: Generated, CCK-8 Assay

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Lgr5+CD44+EpCAM+ Strictly Defines Cancer Stem Cells in Human Colorectal Cancer.

doi: 10.1159/000488743

Figure Lengend Snippet: Fig. 5. Characterization of epi thelial-mesenchymal transition profiles. (A) Mesenchymal genes, such as Vimentin, Snail, Slug, and Twist, were more highly expressed in Lgr5-positive cells (S1, S2 and S3) than in Lgr5-negative cells (S4 and S5), while the epithelial genes E-cadherin and ZO-1 were more highly expressed in Lgr5-negative cells, as assessed by qRT-PCR. (B) Immunofluorescence staining and laser confocal microscopy demon strated the co-expression of Lgr5 and Vimentin in S1 cells. (C) Lgr5- positive cells exhibited a higher ca pacity for migration and invasion than did Lgr5-negative cells, as as sessed by real-time migration and invasion assays. Bar, 30 μm. #P<0.05 compared to any other group. The S5 subpopulation was used as a control group.

Article Snippet: The fixed cells were then permeabilized with 0.1% Triton X-100 at room temperature for 15 min, followed by incubation at 4°C for 10 h with primary antibodies against Vimentin (1:200, Cell Signaling Technology, MA, USA) and Lgr5 (1:200, Santa Cruz Biotechnology, CA, USA).

Techniques: Quantitative RT-PCR, Immunofluorescence, Staining, Confocal Microscopy, Expressing, Migration, Control

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Human Intestinal Enteroids: New Models to Study Gastrointestinal Virus Infections

doi: 10.1007/7651_2017_1

Figure Lengend Snippet: RT-qPCR probes used to analyze differentiation status of HIEs

Article Snippet: N = 3 jejunal HIEs table ft1 table-wrap mode="anchored" t5 caption a7 Marker Changes during differentiation Cat. No. (Life Technologies) CD44 Down Hs01075861_m1 LGR5 Down Hs00969422_m1 MUC2 Up Hs03005103_g1 PCNA Down Hs00427214_g1 Sucrase isomaltase Up Hs00356112_m1 Open in a separate window RT-qPCR probes used to analyze differentiation status of HIEs table ft1 table-wrap mode="anchored" t5 caption a7 Marker Cell type marked Company (concentration) Sucrase isomaltase Enterocytes Santa Cruz (1:100) Muc2 Goblet cells Santa Cruz (1:500) Chromagranin A Enteroendocrine cells Novus Biologicals (1:100) UEA-1 lectin Histo-blood group antigens Sigma-Aldrich (1:500) E-cadherin Adherens junctions BD Biosciences (1:100) Lysozyme Paneth cells Dako (1:100) Open in a separate window Antibodies used to analyze differentiation status of HIEs 3.2 Rotavirus Infection and Analysis of Infected 3D HIEs 3.2.1 Rotavirus Infection of Differentiated 3D HIEs list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Preparation : Make rotavirus incubation media by dissolving 0.25 mg/ml pancreatin in Differentiation Media.

Techniques:

Journal: Theranostics

Article Title: Me6TREN targets β-catenin signaling to stimulate intestinal stem cell regeneration after radiation

doi: 10.7150/thno.46415

Figure Lengend Snippet: Me6 promoted intestinal stem cell proliferation and crypt regeneration post irradiation. ( A ) Representative HE-stained sections and the quantification of the villus and crypt length of small intestine at 24 and 96 h after 14 Gy WBI and different treatments (* p < 0.05; Scale bar = 100 µm). ( B-C ) Proliferation of the crypts of small intestine at 24 and 96 h after 14 Gy WBI and different treatments, as assessed by Ki67 (B) and CyclinD1 (C) staining, respectively (* p < 0.05, ** p < 0.01; Scale bar = 100 µm). Three mice were used in each group. ( D-E ) Heat map showing the proliferation (D) and ISC-related genes (E) that are upregulated in the intestinal crypts from Me6-treated mice at 96 h after 14 Gy WBI. Microarray was performed using mRNA isolated from the intestinal crypts. ( F ) qPCR detection for ISC-related gene expression in the intestinal crypts at 96 h after 14 Gy WBI and different treatments (** p < 0.01). ( G ) Representative Lgr5-stained intestinal sections and the Lgr5 + cell numbers in the crypts at 24 and 96 h after 14 Gy WBI and different treatments (** p < 0.01; Scale bar = 100 µm). Three mice were used in each group. ( H ) Representative images of in situ hybridization for Lgr5 mRNA in the intestinal crypts at 96 h after 14 Gy WBI and different treatments. ( I ) Representative Sox9-stained sections and the quantification of Sox9 + cells in the crypts of the small intestine after 14 Gy WBI and different treatments (** p < 0.01; Scale bar = 100 µm). Three mice were used in each group. ( J ) The quantification of regenerated crypts at 96 h after 14 Gy WBI and different treatments, as indicated by BrdU incorporation for 2 h (** p < 0.01; Scale bar = 100 µm). Three mice were used in each group. ( K ) The number of crypt fission per filed on murine jejunum sections. Six mice were used in each group (** p < 0.01).

Article Snippet: The antibodies used were as follows: anti-Lgr5 (R&D, MAB8240), anti-β-catenin (CST, 8480S), anti-PH3 (CST, 53348), anti-CyclinD1 (CST, 2978), anti-BrdU (CST, 5292), anti-Ki67 (CST, 9129), anti-p-AKT (CST, 4060), anti-p-ERK1/2 (CST, 4370), anti-Muc2 (Gene Tex, GTX100664), anti-Chga (Gene Tex, GTX113165), anti-Lysozyme (Abcam, ab108508).

Techniques: Irradiation, Staining, Microarray, Isolation, Expressing, In Situ Hybridization, BrdU Incorporation Assay

Journal: Theranostics

Article Title: Me6TREN targets β-catenin signaling to stimulate intestinal stem cell regeneration after radiation

doi: 10.7150/thno.46415

Figure Lengend Snippet: Me6 promoted intestinal organoid expansion and ISC proliferation in vitro . ( A ) Representative colony staining image and the colony numbers formed by IEC-6 cells after 10 Gy irradiation and the addition of PBS or Me6 (100 µM) to the culture medium (** p < 0.01). ( B ) The percentage analysis of BrdU incorporation for 48 h in IEC-6 cells after 10 Gy irradiation with or without the addition of Me6 (100 µM) to the medium (** p < 0.01). ( C ) Representative phase contrast microscopic images of intestinal organoids cultured with Me6 at different concentrations and the quantification of the organoid numbers per well, the bud numbers and the surface areas per organoid (* p < 0.05, ** p < 0.01, Scale bar = 100 µm). ( D ) Immunostaining analysis for Ki67 in organoids treated with or without Me6 (** p < 0.01, Scale bar = 50 µm). ( E ) Representative dot plots and percentages of GFP + cells in the intestinal organoids with or without Me6 (100 µM) treatment for 4 days. The intestinal crypts were isolated from Lgr5-EGFP-IRES-creERT2 mice (* p < 0.05). ( F ) qPCR for proliferation- and ISC-related gene expression in the cultured organoids with or without Me6 (100 µM) supplementation of the medium for 7 days (* p < 0.05, ** p < 0.01). ( G ) Western blotting detection of ISC-related protein expression in the cultured organoids with or without Me6 (100 µM) supplementation of the medium for 7 days. ( H ) The morphology and quantification analysis of the small intestine organoids, including the numbers of organoids per well, the bud numbers and the surface areas per organoid at day 7 after irradiation (** p < 0.01). The intestinal organoids received 0 or 6 Gy irradiation and then were cultured in the standard organoid medium with or without Me6 (100 µM) supplementation.

Article Snippet: The antibodies used were as follows: anti-Lgr5 (R&D, MAB8240), anti-β-catenin (CST, 8480S), anti-PH3 (CST, 53348), anti-CyclinD1 (CST, 2978), anti-BrdU (CST, 5292), anti-Ki67 (CST, 9129), anti-p-AKT (CST, 4060), anti-p-ERK1/2 (CST, 4370), anti-Muc2 (Gene Tex, GTX100664), anti-Chga (Gene Tex, GTX113165), anti-Lysozyme (Abcam, ab108508).

Techniques: In Vitro, Staining, Irradiation, BrdU Incorporation Assay, Cell Culture, Immunostaining, Isolation, Expressing, Western Blot

Journal: Theranostics

Article Title: Me6TREN targets β-catenin signaling to stimulate intestinal stem cell regeneration after radiation

doi: 10.7150/thno.46415

Figure Lengend Snippet: In vivo knockdown of β-catenin abolished the role of Me6 on crypt regeneration from irradiated mice. ( A ) Scheme of the time schedule for scramble control siRNA (scramble)/siβ-catenin and Me6/PBS injection in mice after 14 Gy abdominal irradiation. ( B ) Analysis of the expression of β-catenin in the crypts at day 7 from irradiated mice with injection of scramble or β-catenin siRNA. Each group was treated with or without Me6 (** p < 0.01, Scale bar = 100 µm). Three mice were used in each group. ( C ) Representative HE-stained sections and the quantification of the crypt length at day 7 after irradiation (** p < 0.01, Scale bar = 100 µm). Three mice were used in each group. ( D ) Representative Tunel-stained sections and the quantification of the Tunel+ cells in the crypt of small intestine at day 7 after 14 Gy irradiation and different treatments (** p < 0.01, Scale bar = 100 µm). Three mice were used in each group. ( E ) Representative CyclinD1/PH3/Lgr5-stained sections and their quantification of them in the crypt of small intestine at day 7 after 14 Gy irradiation and different treatments (** p < 0.01, Scale bar = 100 µm). Three mice were used in each group. ( F ) The number of regenerated crypts after 14 Gy AIR, which was indicated by BrdU incorporation for 2 h (** p < 0.01, Scale bar = 100 µm). Three mice were used in each group.

Article Snippet: The antibodies used were as follows: anti-Lgr5 (R&D, MAB8240), anti-β-catenin (CST, 8480S), anti-PH3 (CST, 53348), anti-CyclinD1 (CST, 2978), anti-BrdU (CST, 5292), anti-Ki67 (CST, 9129), anti-p-AKT (CST, 4060), anti-p-ERK1/2 (CST, 4370), anti-Muc2 (Gene Tex, GTX100664), anti-Chga (Gene Tex, GTX113165), anti-Lysozyme (Abcam, ab108508).

Techniques: In Vivo, Irradiation, Injection, Expressing, Staining, TUNEL Assay, BrdU Incorporation Assay