rat small intestinal crypt epithelial cell line  (ATCC)

Journal: Experimental & molecular medicine

Article Title: Neurotensin inhibits AMPK activity and concurrently enhances FABP1 expression in small intestinal epithelial cells associated with obesity and aging.

doi: 10.1038/s12276-025-01461-w

Figure Lengend Snippet: Fig. 5 AMPK activity is correlated with FABP1 expression. a Western blot analysis of protein extracted from monolayers of male WT mice fed a LFD or HFD for 6 weeks at weaning. Densitometric analysis of p-AMPKα (n = 5 mice per group) and FABP1 (n = 4 mice per group) was performed by ImageJ and normalized by AMPKα and β-actin, respectively. **P < 0.01. b Western blot analysis of protein extracted from monolayers of male WT mice (4 months old); cells were treated with PA at different concentrations for 24 h. c, d Western blot analysis of protein extracted from jejunal monolayers of male Nts+/+ and Nts−/−mice fed a LFD or HFD for 6 weeks at weaning. Densitometric analysis of FABP1 was performed by ImageJ and normalized by β-actin. (n = 6 mice per group). *P < 0.05. e, f Western blot (e) and qPCR (f) analysis of protein and RNA, respectively, extracted from monolayers of male young and aged mice on NC; cells were treated with Met at different concentrations for 24 h. n = 3 mice per group. *P < 0.001 versus control in young and aged, respectively; †P < 0.001 versus 0.1 mM in young and aged, respectively; ‡P < 0.001 versus 0.5 mM in young and aged, respectively; $P < 0.01 versus young 0.5 mM; &P < 0.001 versus young 1.0 mM. g IF staining of IEC-6 cells; images were taken by confocal microscopy.

Article Snippet: Cell culture and stable cell lines The normal rat small intestinal epithelial cell line IEC-6 and the human embryonic kidney cell line HEK-293, purchased from ATCC, were maintained in DMEM, supplemented with 10% fetal bovine serum. pEGFP-N1 was a gift from Antony K. Chen (Addgene plasmid #172281)27. mRFP1-N1 was a gift from Robert Campbell, Michael Davidson and Roger Tsien (Addgene plasmid #54635)28.

Techniques: Activity Assay, Expressing, Western Blot, Control, Staining, Confocal Microscopy

Journal: Acta Physiologica (Oxford, England)

Article Title: Enteric glial NLRP3 inflammasome contributes to gut mucosal barrier alterations in a mouse model of diet‐induced obesity

doi: 10.1111/apha.14232

Figure Lengend Snippet: Incubation of PA and LPS increases trans‐epithelial permeability and induces enteric gliosis. (A) Schematic representation of culture experiment on IEC‐6 cells. Intestinal epithelial cells were treated with PA and LPS for 5 days. At day 6, cells were lysed and culture media were collected. (B, C) A scatter plot representing the densitometric analysis and related representative blot of ZO‐1 and occludin assessed by Western blot assay in IEC‐6 cells treated with PA and LPS. (D) Schematic representation of FITC‐dextran experiment on IEC‐6 cells. For experimental protocol details see “Section ” and Figure . (E) FITC‐dextran fluorescence intensity measured in IEC‐6 cells treated with PA and LPS. Dots show values per individual experiments ( n = 5–6 independent experiments performed in duplicate) whereas black bars indicate means ± SEM. * p < 0.05. One‐way ANOVA. EGC, enteric glial cells; GFAP, glial fibrillary acidic protein; IEC‐6, intestinal epithelial cells; LPS, lipopolysaccharide; PA, palmitate; ZO‐1, zonulin‐1.

Article Snippet: Rat intestinal epithelial cell line (IEC‐6) was acquired from ATCC (IEC‐6; ATCC CRL‐1592, Manassas, VA, USA).

Techniques: Incubation, Permeability, Western Blot, Fluorescence

Journal: Acta Physiologica (Oxford, England)

Article Title: Enteric glial NLRP3 inflammasome contributes to gut mucosal barrier alterations in a mouse model of diet‐induced obesity

doi: 10.1111/apha.14232

Figure Lengend Snippet: Incubation of PA and LPS triggers NLRP3 inflammasome activation in activated EGCs. (A) Schematic representation of coculture experiments. For experimental protocol details see “Section ” and Figure . (B) A scatter plot representing the densitometric analysis and related representative blot of GFAP in cocultured EGCs treated with PA and LPS. (C–G) A scatter plot representing the densitometric analysis and related representative blot of (C) NLRP3, (D) ASC, (E–G) pro‐caspase and (F, G) caspase‐1 in cocultured EGCs treated with PA and LPS. (H–J) A scatter plot representing the IL‐1β levels in the supernatants of (H) mixing medium, (I) conditioned medium derived from cocultured EGCs and (J) conditioned medium derived from cocultured IEC‐6 cells. Dots show values per individual experiments ( n = 5–6 independent experiments performed in duplicate) whereas black bars indicate means ± SEM. * p < 0.05 and ‡ p < 0.001. One‐way ANOVA. EGC, enteric glial cells; IEC‐6, intestinal epithelial cells; IL‐1β, interleukin‐1β; LPS, lipopolysaccharide; NLRP3, nucleotide‐binding oligomerization domain leucine rich repeat and pyrin domain containing protein 3; PA, palmitate.

Article Snippet: Rat intestinal epithelial cell line (IEC‐6) was acquired from ATCC (IEC‐6; ATCC CRL‐1592, Manassas, VA, USA).

Techniques: Incubation, Activation Assay, Derivative Assay, Binding Assay

Journal: Acta Physiologica (Oxford, England)

Article Title: Enteric glial NLRP3 inflammasome contributes to gut mucosal barrier alterations in a mouse model of diet‐induced obesity

doi: 10.1111/apha.14232

Figure Lengend Snippet: Glial NLRP3‐mediated IL‐1β contributes to further exacerbate the mucosal barrier dysfunctions associated with obesity. (A) Representative blot of NLRP3 expression assessed by Western blot assay in EGCs silenced for NLRP3 with siRNA, and subsequent incubation with PA and LPS. (B–E) A scatter plot representing the densitometric analysis and related representative blot of (B–D) ZO‐1 and (B–E) occludin assessed by Western blot assay in IEC‐6 cells incubated with conditioned medium derived from silenced‐NLRP3 EGCs or with PA plus LPS, in the absence or in the presence of anakinra, in single culture and in coculture. (F) Schematic representation of coculture experiments and FITC‐dextran experiment protocols. For experimental protocol details see “Section ” and Figure . (G) FITC‐dextran fluorescence intensity measured in IEC‐6 treated with conditioned medium derived from silenced‐NLRP3 EGCs or with PA and LPS, in the absence or in the presence of anakinra, in single culture and in coculture. Dots show values per individual experiments ( n = 5–6 independent experiments performed in duplicate) whereas black bars indicate means ± SEM. * p < 0.05, † p < 0.01 and ‡ p < 0.001. One‐way ANOVA. (H) Schematic representation of pathophysiological mechanisms underlying the impairment of IEB integrity and function associated with obesity: Interplay between intestinal epithelium and enteric glial cells in obesity. (1) HFD exposure, besides to induce alterations in IEB structure, determinates enteric gliotic processes (2) characterized by a hyperactivation of NLRP3 inflammasome (3) with consequent release of IL‐1β (4). The release of a massive levels of IL‐1β contributes to further exacerbate the disruption of mucosal barrier integrity with consequent increase of epithelial permeability (5). EGC, enteric glial cells; HFD, high fat diet; IEC‐6, intestinal epithelial cells; LPS, lipopolysaccharide; PA, palmitate; TJs, tight junctions; ZO‐1, zonulin‐1.

Article Snippet: Rat intestinal epithelial cell line (IEC‐6) was acquired from ATCC (IEC‐6; ATCC CRL‐1592, Manassas, VA, USA).

Techniques: Expressing, Western Blot, Incubation, Derivative Assay, Fluorescence, Disruption, Permeability

Journal: Acta Physiologica (Oxford, England)

Article Title: Enteric glial NLRP3 inflammasome contributes to gut mucosal barrier alterations in a mouse model of diet‐induced obesity

doi: 10.1111/apha.14232

Figure Lengend Snippet: Schematic representation of coculture experiments and FITC‐dextran experiment protocols. (A) IEC‐6 cells were seeded in the apical chamber of the transwell plate. In parallel, EGCs were plated in a separate six well plates. After attachment, transwell inserts with IEC‐6 cells were transferred into wells containing EGCs. For the first 2 days, treatment with PA plus LPS was incubated in contact with IEC‐6 cells to mimic the in vivo contact of intestinal epithelium with HFD diet. After 48 h, culture media of apical and lower side of transwell plate were mixed until day 5 to allow the released soluble to interact with both cell lines. At day 5, mixing media were collected and cell lines were separated for 24 h. The next day, cells were lysed, and the soluble molecules released in the medium (designed as “conditioned medium”) have been identified and associated with the respective cell line. (B) In FITC‐dextran experiments, IEC‐6 cells were seeded in the upper side of transwell chambers on 24‐well plates. Cells were treated with: Medium containing PA plus LPS or conditioned medium derived from cocultured EGCs, previously treated with PA and LPS, in the absence or in the presence of anakinra (IL‐1β receptor antagonist). After 48 h, FITC‐dextran was added in the apical side of trans‐well insert and following 2 h of incubation, the medium in the basolateral side was collected and the absorbance of fluorescence was quantified. EGC, enteric glial cells; HFD, high fat diet; IEC‐6, intestinal epithelial cells; LPS, lipopolysaccharide; PA, palmitate.

Article Snippet: Rat intestinal epithelial cell line (IEC‐6) was acquired from ATCC (IEC‐6; ATCC CRL‐1592, Manassas, VA, USA).

Techniques: Incubation, In Vivo, Derivative Assay, Fluorescence