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

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: A regulatory circuit operated by KDELR1 and KDELR3 fine-tunes the composition of the early secretory pathway

doi: 10.1007/s00018-025-06049-1

Figure Lengend Snippet: Different roles of human KDELRs. A Seventy-two hours after silencing with the indicated KDELR-specific duplexes, the supernatants of cells cultured for six hours were harvested and analysed by WB. Aliquots of the lysates and supernatants (the latter tenfold concentrated) of HeLa Milano cells were resolved under reducing conditions on a 4–12% pre-casted polyacrylamide gel run in MOPS buffer. After transfer to nitrocellulose, the membrane was first stained with anti-KDEL antibodies (clone 10C3, Enzo Life Sciences) and subsequently with anti-ERp44 and anti-AGR2, as indicated. Tubulin was used as a loading control. The simultaneous KD of all KDELRs (lanes 3 and 9) induced the extracellular release of all the ER-resident proteins visualized intracellularly. The identity of the two upper bands recognized by anti-KDEL (denoted X and Y) remains to be established. The intracellular signal corresponding to ERp44 and ERp46 (IN) disappeared almost completely upon downregulation of all KDELRs (lane 3), and partially under KDELR2 KD (lane 5). Noteworthy, instead, AGR2 increased dramatically upon downregulation of ERp44 or KDELR3 (lanes 2 and 6, respectively). See panel C for the relative quantifications. B Visual pulse-chase assays [ , ] (see also Materials and Methods for details) were used to compare the secretion rate of Halo-ERp44ΔRDEL (Halo-ERp44Δ), Halo-ERp46ΔKDEL (Halo-ERp46Δ), Halo-PDIΔKDEL (Halo-PDIΔ) and spHalo . The left panel shows the WB image of one representative experiment out of three, highlighting the signal of the fluorescent Halo-TMR used for the pulse (and visualized with a 532 nm laser). Densitometric quantifications are reported in the right panel (average of three independent experiments). The ratio of secreted/intracellular TMR-labelled signals was calculated for each time point (OUT/IN) and normalized to the ratio obtained for spHalo CTRL at 120 min time point, assumed to represent unassisted secretion . Statistical significance was calculated by Two-way ANOVA. The colours of the asterisks refer to the samples against which the comparison was performed. C Densitometric quantification of four independent WBs like the one shown in panel A. The bands specifically decorated by anti-AGR2 antibodies were quantified and normalized against tubulin. The ratios obtained were normalized to untreated cells. Statistical significance was calculated by an unpaired Student's t-test. D HeLa Milano cells were treated with duplexes specific for silencing ERp44 (44) ERp46 (46), or with control reagents (-) as indicated. Aliquots of their lysates were separated on 4–12% polyacrylamide gels under reducing conditions, blotted onto nitrocellulose and decorated with the antibodies indicated on the right-hand margin. Tubulin was used as loading control. Note the increase of AGR2 upon ERp44 KD but not ERp46 KD (compare lanes 2 and 3). The red asterisk points at a background band recognized by anti-ERp46 antibodies in this experiment. E HeLa Milano (left panel) and mucin-producing Caco-2 cells (right panel) were treated with ERp44- or AGR2- specific duplexes, or with control reagents (-) as indicated. Aliquots of their lysates (IN) and of their culture supernatants (OUT) ten-fold concentrated were separated on 4–12% polyacrylamide gels under reducing conditions, blotted onto nitrocellulose and decorated with the indicated antibodies. Ponceau signals provided loading controls

Article Snippet: AGR2 , Rabbit (Proteintech) , 1:500 in PBS 0.1% Tween.

Techniques: Cell Culture, Membrane, Staining, Control, Pulse Chase, Comparison

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: A regulatory circuit operated by KDELR1 and KDELR3 fine-tunes the composition of the early secretory pathway

doi: 10.1007/s00018-025-06049-1

Figure Lengend Snippet: Opposite effects of KDELR1 and KDELR3 on AGR2. HeLa Milano cells were silenced as indicated and analysed as described in Figs. and . Proteins were quantified in cell lysates (IN) and in 6h culture supernatants (OUT) as described in legends to Fig. (panels B-C). To quantify mRNAs, RT-qPCR analyses were performed with AGR2-specific oligos (panels A, and D-F). GAPDH was used to normalize the results obtained. A AGR2 mRNAs increases upon ERp44 KD and/or KDELR3 KD . RT-qPCR data are expressed as Log2FC with respect to control (-) conditions (that, by definition, correspond to 0). The data of the single experiments are shown in the plot, together with the average ± SEM. Statistical significance was calculated by an unpaired Student's t-test. B and C . Aliquots from cell lysates (IN) and 10X supernatants (OUT) were analysed as described above. Tubulin was used as a loading control. Note that AGR2 protein accumulates also when both KDELR3 and ERp44 are silenced (panel B), upon combined silencing of all the KDELRs, as well as upon combined silencing of KDELR1 and KDELR2, a condition in which most ERp44 is secreted. D AGR2 mRNAs increase upon double KDELR silencing. The panel shows the AGR2 transcript levels in cells treated as in C. mRNAs were quantified as in panel A. Average of five or more independent experiments ± SEM. Statistical significance was calculated by an unpaired Student's t-test. E ERp44 rescues AGR2 inhibition in HeLa ERp44KO cells via KDELR3. HeLa ERp44KO cells, stably expressing doxycycline-inducible Halo-ERp44, were silenced for 72 h for KDELR3, and then treated with 100 µM doxycycline overnight. Transcripts were quantified as described in panels A and D. Average of three or more independent experiments ± SEM. An unpaired Student's t-test was performed. F Neither Halo-RDEL nor ERp46 activate KDELR3-dependent AGR2 inhibition. HeLa ERp44KO cells were transfected either with an empty vector or with vectors encoding for Halo-ERp44, Halo-ERp46, and Halo-RDEL. After 48 h, RNAs were extracted and quantified by RTq-PCR (see panel A). Average of three independent experiments ± SEM. Statistical significance was calculated by an unpaired Student's t-test

Article Snippet: AGR2 , Rabbit (Proteintech) , 1:500 in PBS 0.1% Tween.

Techniques: Quantitative RT-PCR, Control, Inhibition, Stable Transfection, Expressing, Transfection, Plasmid Preparation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: A regulatory circuit operated by KDELR1 and KDELR3 fine-tunes the composition of the early secretory pathway

doi: 10.1007/s00018-025-06049-1

Figure Lengend Snippet: Transcriptional patterns of HeLa cells upon KDELRs downregulation. A Principal Component Analysis Score plot of PC1 vs PC2. The graph shows clear separation of the five experimental conditions. B Overlap analysis of differentially expressed genes after silencing of KDEL receptors or ERp44. Left panel: genes up-regulated; central panel: genes down-regulated; right panel: genes up-regulated upon silencing of KDELR3 and ERp44 and down-regulated upon KDELR1 (thus sharing the same behaviour as AGR2). C Functional Enrichment using Enrichr of Gene Ontology Cellular Components and the Databases of Jensen Compartments of the 41 genes behaving like AGR2 showing enrichment in “Extracellular Space”, “Extracellular matrix”, and “Endoplasmic Reticulum Lumen”

Article Snippet: AGR2 , Rabbit (Proteintech) , 1:500 in PBS 0.1% Tween.

Techniques: Functional Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: A regulatory circuit operated by KDELR1 and KDELR3 fine-tunes the composition of the early secretory pathway

doi: 10.1007/s00018-025-06049-1

Figure Lengend Snippet: Functional specialization of human KDELRs. As summarized in the right part of the cartoon, ERp44 and KDELR3 act epistatically to inhibit AGR2 transcription, while KDELR1 (on the left) can be stimulated by different chaperones to promote AGR2 transcription. In HeLa cells, the more abundant KDELR2 and KDELR1 sustain the retrieval activity of diverse ER chaperones and enzymes. Considering the essential roles of AGR2 in assisting mucin folding and inhibiting the gut specific ER-stress sensor Ire1ß, this pathway may be important in mucin biogenesis

Article Snippet: AGR2 , Rabbit (Proteintech) , 1:500 in PBS 0.1% Tween.

Techniques: Functional Assay, Activity Assay