Journal: Frontiers in cellular and infection microbiology

Article Title: Reprogrammed fecal and mucosa-associated intestinal microbiota and weakened mucus layer in intestinal goblet cell- specific Piezo1-deficient mice.

doi: 10.3389/fcimb.2022.1035386

Figure Lengend Snippet: FIGURE 1 Expression of Piezo1 in colon. (A) Expression of Piezo1 in colon. (B) Relative mRNA expression of Piezo1. (C) The co-localization of AGR2 and GC Piezo1 in colon (400×). Data are expressed as the mean ± SEM (n =4-5). *p < 0.05.

Article Snippet: Anti-Piezo1 antibody (1:200, Proteintech, 15939-1-AP), anti-AGR2 antibody (1:200, R&D Systems, AF6068) anti-muc2 antibody (1:200, Genetex, GTX100664) and were diluted at a ratio of 1 to 400 for staining.

Techniques: Expressing

Journal: Frontiers in cellular and infection microbiology

Article Title: Reprogrammed fecal and mucosa-associated intestinal microbiota and weakened mucus layer in intestinal goblet cell- specific Piezo1-deficient mice.

doi: 10.3389/fcimb.2022.1035386

Figure Lengend Snippet: FIGURE 3 Decreased GC numbers and weakened mucus layer in GC Piezo1-/- mice. (A) The mucus layer in colon tissue by AB-PAS staining. 200×. (B) the number of goblet cells and the thickness of the inner mucus layer from two groups. (C) The typical immunofluorescent images of colon tissues were stained with DAPI (blue) and Mucin2 (red) or AGR2 (red) and were observed using a confocal laser-scanning microscope. 400×. (D) The relative mean fluorescence intensity of AGR2, Mucin2. (E) The relative mRNA expression of Mucin2, AGR2, and TFF3 in colonic tissue. (F) The relative mRNA expression of DOCK4, Hes1, Spdef, and Klf4 in colonic tissue. Data are expressed as the mean ± SEM. (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001;. ns, no significance.

Article Snippet: Anti-Piezo1 antibody (1:200, Proteintech, 15939-1-AP), anti-AGR2 antibody (1:200, R&D Systems, AF6068) anti-muc2 antibody (1:200, Genetex, GTX100664) and were diluted at a ratio of 1 to 400 for staining.

Techniques: Staining, Laser-Scanning Microscopy, Expressing

Journal: International journal of molecular sciences

Article Title: Slowed Intestinal Transit Induced by Less Mucus in Intestinal Goblet Cell Piezo1-Deficient Mice through Impaired Epithelial Homeostasis.

doi: 10.3390/ijms241814377

Figure Lengend Snippet: Figure 1. Identification of Piezo1∆GC mice. (A) Immunofluorescence co-localization of Piezo1 and Agr2 (label goblet cells) in WT and Piezo1∆GC mouse colons. The yellow box shows a magnified localized image. Scale bar: 100 µm. (B) mRNA level of Piezo1 in WT and Piezo1∆GC mouse colon crypts (normalized to GAPDH). At least three independent experiments were conducted. Data are expressed as the mean ± SEM. (n = 5 mice). ** p < 0.01.

Article Snippet: The antibodies used in our experiments included the following: Piezo1 (1:200, 15939-1-AP, Proteintech, Chicago, IL, USA), Agr2 (1:200, AF6068, R & D Systems, Minnesota, USA), Ki67 (1:200, GB111141, Servicebio, Wuhan, China), cKit (1:150, ab256345, Abcam, Cambridge, England), Alpi (1:200, A0514, Abclonal, Woburn, MA, USA), and Chga (1:200, A9576, Abclonal).

Techniques:

Journal: International journal of molecular sciences

Article Title: Slowed Intestinal Transit Induced by Less Mucus in Intestinal Goblet Cell Piezo1-Deficient Mice through Impaired Epithelial Homeostasis.

doi: 10.3390/ijms241814377

Figure Lengend Snippet: Figure 2. Decreased GC numbers and thinner mucus layer in Piezo1∆GC mouse colons. (A) Im- munofluorescence staining of Agr2 in WT and Piezo1∆GC mouse colons. Scale bar: 100 µm. The crypt was divided equally into three parts: upper, middle, and base. The white arrow indicates a goblet cell. (B) Statistical analysis of goblet cells/epithelial cells in (A). (C) Statistical analysis of goblet cells in different parts/epithelial cells in (A). (D) RNA level of Mucin2 in colon tissues from WT and Piezo1∆GC mice (normalized to GAPDH). (E) AB-PAS staining of mucus in WT and Piezo1∆GC mouse colons. The red arrows indicate the mucus layer. Scale bar: 50 µm. (F) Statistical analysis of mucus layer thickness in (D). At least three independent experiments were conducted. Data are expressed as the mean ± SEM. (n = 5 mice). ns, not significant; ** p < 0.01; *** p < 0.001.

Article Snippet: The antibodies used in our experiments included the following: Piezo1 (1:200, 15939-1-AP, Proteintech, Chicago, IL, USA), Agr2 (1:200, AF6068, R & D Systems, Minnesota, USA), Ki67 (1:200, GB111141, Servicebio, Wuhan, China), cKit (1:150, ab256345, Abcam, Cambridge, England), Alpi (1:200, A0514, Abclonal, Woburn, MA, USA), and Chga (1:200, A9576, Abclonal).

Techniques: Staining

Journal: International journal of molecular sciences

Article Title: Slowed Intestinal Transit Induced by Less Mucus in Intestinal Goblet Cell Piezo1-Deficient Mice through Impaired Epithelial Homeostasis.

doi: 10.3390/ijms241814377

Figure Lengend Snippet: Figure 6. Abnormal intestinal epithelial cell composition and impaired colon stem cell niche in Piezo1∆GC mice. (A) RNA levels of Ki67 and stem cell markers (Lgr5, Sox9, and EphB2) in WT and Piezo1∆GC mouse colon crypts (normalized to GAPDH). (B) Immunohistochemistry of Ki67 in WT and Piezo1∆GC mouse colons. Scale bar: 100 µm. (C) Statistical analysis of Ki67 positive cell ratio in (B). (D) RNA levels of stem cell niche marker (cKit), differentiated colonocyte marker (Alpi), goblet cell marker (Agr2), and enteroendocrine cell marker (Chga) in WT and Piezo1∆GC mouse colon crypts (normalized to GAPDH). (E) Immunohistochemistry of cKit in WT and Piezo1∆GC mouse colons. The white dashed lines mark the crypt borders, and the red arrow indicates a cKit-positive cell. Scale bar: 50 µm. (F) Statistical analysis of cKit-positive cell ratio in (E). (G,H) Immunofluorescence staining of Alpi (G) and Chga (H) in WT and Piezo1∆GC mouse colons. The white dashed lines mark crypt borders, and the white arrow indicates a differentiated colonocyte in (G) and an enteroendocrine cell in (H). Scale bar: 25 µm. (I,J) Statistical analysis of differentiated colonocyte ratio in (G) and enteroendocrine cell ratio in (H). At least three independent experiments were conducted. Data are presented as the mean ± SEM. (n = 5 mice). ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The antibodies used in our experiments included the following: Piezo1 (1:200, 15939-1-AP, Proteintech, Chicago, IL, USA), Agr2 (1:200, AF6068, R & D Systems, Minnesota, USA), Ki67 (1:200, GB111141, Servicebio, Wuhan, China), cKit (1:150, ab256345, Abcam, Cambridge, England), Alpi (1:200, A0514, Abclonal, Woburn, MA, USA), and Chga (1:200, A9576, Abclonal).

Techniques: Immunohistochemistry, Marker, Staining

Journal: International journal of molecular sciences

Article Title: Slowed Intestinal Transit Induced by Less Mucus in Intestinal Goblet Cell Piezo1-Deficient Mice through Impaired Epithelial Homeostasis.

doi: 10.3390/ijms241814377

Figure Lengend Snippet: Figure 7. Decreased self-renewal capacity of colon stem cells from Piezo1∆GC mice. (A) Representative images of colonoids from WT and Piezo1∆GC mice at day 5. Images of one colonoid on day 1, 3, and 5 are below, reflecting the growth process of a colonoid. Scale bar: 100 µm. (B–D) Indicators related to colonoids growth: number of buds per colonoid (B), surface area per colonoid (C), and percentage of colonoids with buds per well (D). (E) Immunofluorescence staining of EDU and Ki67 in colonoids from WT and Piezo1∆GC mice. Scale bar: 100 µm. (F) Statistical analysis of EDU and Ki67 positive cell ratio in (E). (G) Immunofluorescence staining of differentiated colonocyte (Alpi), goblet cell (Agr2), and enteroendocrine cell (Chga) in colonoids from WT and Piezo1∆GC mice. Scale bar: 100 µm. (H) Statistical analysis of Alpi, Agr2, and Chga positive cell ratio in (G). (I) RNA levels of Ki67 from WT and Piezo1∆GC mouse colonoids (normalized to GAPDH). (J) AB-PAS staining of colonoids from WT and Piezo1∆GC mice. The red arrows indicate mucus secreted by goblet cells in colonoids. Scale bar: 100 µm. At least three independent experiments were conducted. Data are presented as the mean ± SEM. (n = 5 mice). ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The antibodies used in our experiments included the following: Piezo1 (1:200, 15939-1-AP, Proteintech, Chicago, IL, USA), Agr2 (1:200, AF6068, R & D Systems, Minnesota, USA), Ki67 (1:200, GB111141, Servicebio, Wuhan, China), cKit (1:150, ab256345, Abcam, Cambridge, England), Alpi (1:200, A0514, Abclonal, Woburn, MA, USA), and Chga (1:200, A9576, Abclonal).

Techniques: Staining

Journal: British Journal of Cancer

Article Title: Improved early detection of ovarian cancer using longitudinal multimarker models

doi: 10.1038/s41416-019-0718-9

Figure Lengend Snippet: a AGR2 serum levels; b LRG1 serum levels; c CHI3L1 serum levels; d FSTL1 serum levels; e SLPI serum levels; f DNAH17 serum levels; g PEBP4 serum levels; h CA125 serum levels; i HE4 serum levels; j glycodelin serum levels. * P < 0.05, ** P < 0.001 from either t test or Mann–Whitney test. Data for borderline cases are omitted. Axis labels: C = controls; I = Type I; II = Type II; L = late; E = early.

Article Snippet: The kits used, catalogue numbers, dilutions, and intra-assay coefficient of variations were: Human AGR2 ELISA Kit (ElabScience; E-EL-H0298; 1:20; 18%), CA125 ECLIA assay (Roche; Elecsys CA 125 II; 1:1; 4%), CHI3L1 Quantikine ELISA Kit (R&D Systems; DC3L10; 1:50; 14%), DNAH17 (human) ELISA Kit (EIAab; E5886h, 1:5; 17%), FSTL1 ELISA Kit (USCN; SEJ085Hu; 1:100; 11%), Glycodelin/PP14 Elisa Kit (Bioserv Diagnostics; BS-30-20; 1:2; 22%), HE4 ECLIA assay (Roche; Elecsys HE 4; 1:1; 8%), LRG1 ELISA Kit (IBL; 27769; 1:2000; 16%), Human PEBP4 ELISA Kit (ElabScience; E-EL-H5440; 1:200; 20%), and SLPI Quantikine ELISA Kit (R&D Systems; DP100, 1:50; 12%).

Techniques: MANN-WHITNEY

Journal: British Journal of Cancer

Article Title: Improved early detection of ovarian cancer using longitudinal multimarker models

doi: 10.1038/s41416-019-0718-9

Figure Lengend Snippet: Performance of top models based on leave-one-out-cross-validation.

Article Snippet: The kits used, catalogue numbers, dilutions, and intra-assay coefficient of variations were: Human AGR2 ELISA Kit (ElabScience; E-EL-H0298; 1:20; 18%), CA125 ECLIA assay (Roche; Elecsys CA 125 II; 1:1; 4%), CHI3L1 Quantikine ELISA Kit (R&D Systems; DC3L10; 1:50; 14%), DNAH17 (human) ELISA Kit (EIAab; E5886h, 1:5; 17%), FSTL1 ELISA Kit (USCN; SEJ085Hu; 1:100; 11%), Glycodelin/PP14 Elisa Kit (Bioserv Diagnostics; BS-30-20; 1:2; 22%), HE4 ECLIA assay (Roche; Elecsys HE 4; 1:1; 8%), LRG1 ELISA Kit (IBL; 27769; 1:2000; 16%), Human PEBP4 ELISA Kit (ElabScience; E-EL-H5440; 1:200; 20%), and SLPI Quantikine ELISA Kit (R&D Systems; DP100, 1:50; 12%).

Techniques: Biomarker Discovery

Journal: Nature

Article Title: A gene-environment induced epigenetic program initiates tumorigenesis

doi: 10.1038/s41586-020-03147-x

Figure Lengend Snippet: a, Schematic representation of the experimental design to interrogate the impact of recombinant IL-33 (rIL-33) on the transcriptional, chromatin and phenotypic state of the pancreatic epithelium from Kras-mutant (KC-GEMM) or wild-type (C-GEMM) mice. Molecular analyses were performed in lineage-traced (mKate2+) pancreatic epithelial cells purified by FACS-sorting from of rIL-33 or vehicle treated mice at day 0 (ATAC-seq), or day 0 and day 21 days (RNA-seq) after treatment. b, GSEA comparing the expression of the early chromatin activated gene program identified in analyses (left), or of genes overexpressed in human PDAC specimens compared to normal pancreas (Moffitt et al. dataset) (right), in Kras-mutant cells isolated from rIL-33 treated vs PBS-treated mice (day 21 time point). The chromatin activated genes queried are the chromatin-dynamic DEGs identified to be upregulated during injury-accelerated neoplasia ( Kras*+Injury ) and in advanced disease ( PDAC ) but not during normal regeneration ( Injury alone) and blunted by Brd4 suppression in metaplastic Kras-mutant cells (KC sh : Kras+Injury ). c-d, GSEA comparing the expression of genes induced by the combination of mutant Kras + rIL-33 in either shBrd4 vs shRen Kras-mutant pancreatic epithelial cells (mKate2+) isolated from KC sh -GEMM ( Kras*+Injury ) (c) or in Kras-mutant populations isolated from caeruelin-treated ( Kras*+Injury ) vs resting ( Kras* ) KC mice (d). The queried gene sets were identified as significantly upregulated in Kras-mutant pancreatic epithelial cell populations (mKate2+) isolated from rIL-33 (vs PBS) treated mice ( KC+rIL-33 vs KC+Veh ) at either day 0 (d0) or day 21 (d21) time points. e, qRT-PCR analysis of rIL-33 effects in the mRNA levels of acinar differentiation ( Cpa1 ), metaplasia ( Sox9 ) and Kras-dependent neoplasia ( Agr2 , Muc6 ) markers in pancreatic epithelial cell (mKate2+) populations isolated from Kras-WT (C) or Kras-mutant (KC) mice (n=2 each) treated with rIL-33 or Vehicle (PBS) and analyzed 21 days thereafter. f, GSEA comparing the expression of genes induced by the combination of mutant Kras + rIL-33 in human PDAC specimens vs human normal pancreas (Moffitt et al. dataset) . g, Volcano plots comparing the chromatin accessibility landscape of Kras-mutant pancreatic epithelium of rIL-33-treated vs vehicle-treated mice, as assessed by ATAC-seq performed at the day 0 time-point. h, Top-scoring motifs identified by HOMER de novo analysis in accessibility- GAIN peaks identified in Kras-mutant pancreatic epithelial cells (mKate2+) isolated from rIL-33-treated mice vs from PBS-treated counterparts, assessed by ATAC-seq analyses performed at the day 0 time point. The significance of the enrichment is shown in brackets. i , Metagene representation of the mean ATAC-seq signal (n=3 mice per condition) at accessibility- GAIN regions driven by injury in the Kras-mutant pancreatic epithelium ( Kras*+Injury vs Kras* ) (top) or at accessibility- GAIN regions linked to the neoplasia-specific gene activation program (identified in analyses, right) in Kras-mutant pancreatic epithelial cells (mKate2+) from isolated from rIL-33 treated vs PBS-treated mice (n=3 each, day 0 time point). rIL-33 treatment promotes accessibility at injury-sensitive sites. p-values were determined by Kolmogorov–Smirnov test. j, Quantification of the relative number of ADM and PanIN lesions in pancreata from Kras wild-type (C-GEMM) or Kras mutant (KC-GEMM) mice treated with rIL-33 or vehicle (PBS) and analyzed at the indicated time points in days (d) after treatment. Data are presented as means ± s.e.m and significance was assessed by unpaired two-tailed Student’s t-test (ns, not significant). n=3, 4, 4, 5, 3 or 4 (from left to right) independent animals per experimental condition. k, Representative immunofluorescence stains of IL-33 protein (green) co-stained with the lineage-tracer marker mKate2 (red) marking pancreatic epithelial cells from mice (n=3 per group) harbouring wild-type (Normal) or mutant Kras in the indicated tissue states. Scale bar, 100 μm. l, Relative mRNA levels (RNA-seq tpm counts) of Il33 (left) or the indicated mutant Kras effector ( Agr2 ), middle) or acinar TF ( Cpa1 , right) in FACS-sorted mKate2+ pancreatic epithelial cell populations isolated from rIL-33-treated or PBS-treated mice harbouring WT or mutant Kras . n=3, 4, 4, 4, 5 or 4 (from left to right) biological replicates (independent mice) per group; median and upper/lower quantile values per group are indicated.

Article Snippet: The following primary antibodies were used: mKate2 (Evrogen, AB233, 1:1000), GFP (ab13970, Abcam, 1:500; and 2956S, Cell Signaling Technology, 1:200), Brd4 (HPA015055, Sigma-Aldrich, 1:100), Myc (ab32072, Abcam, 1:100), CPA1 (AF2765, R&D, 1:400), Clusterin (sc-6419, SCBT, 1:200), SOX9 (AB5535, Millipore, 1:1000), Amylase (sc-31869, SCBT, 1:1000), KRT19 (Troma III, Developmental Studies Hybridoma Bank, 1:500), FOSL1 (sc-376148, SCBT, 1:100), JUNB (sc-8051, SCBT, 1:100), AGR2 (NBP2–27393, Novus Biologicals, 1:200), DCLK1 (ab109029, Abcam, 1:200), Ki67 (BD Biosciences 550609, 1:200) and IL-33 (AF3626, R&D, 1:150).

Techniques: Recombinant, Mutagenesis, Purification, RNA Sequencing Assay, Expressing, Isolation, Quantitative RT-PCR, Activation Assay, Two Tailed Test, Immunofluorescence, Staining, Marker