rat small intestinal crypt epithelial cell line  (ATCC)

Journal: Burns & Trauma

Article Title: Glutamine promotes the proliferation of intestinal stem cells via inhibition of TP53-induced glycolysis and apoptosis regulator promoter methylation in burned mice

doi: 10.1093/burnst/tkae045

Figure Lengend Snippet: Gln supplementation promoted crypt and IEC6 cell proliferation. Immunofluorescence staining of pathological sections of the small intestinal samples from the sham, burn, and burn + Gln groups at 3 days post-burn injury using antibodies against ( a ) Lgr5 and ( b ) Ki67. Expression of ( c ) stemness-related and ( d ) proliferation-related genes after burn injury was assessed using RT–qPCR (Scale bar: 50 μm). ( e ) Concentration of ROS in IEC-6 cells was determined by flow cytometry using an H2DCFA probe. The cells were exposed to LPS (10 μg/ml) for 24 h and cultured in the absence or presence of 2 mM Gln. ( f ) CCK-8 and ( g ) EdU assays were used to determine the viability and proliferation of IEC-6 cells (Scale bar: 100 μm). Cells were exposed to LPS (10 μg/ml) for 24 h and cultured in the absence or presence of 2 mM Gln. ( h ) Expression of Ki67 and PCNA was quantified using RT–qPCR in IEC–6 cells cultured in the absence or presence of LPS (10 μg/ml, 24 h) or Gln (2 mM, 24 h). ( i ) Effects of LPS (10 μg/ml, 48 h) and Gln (2 mM, 48 h) on the growth of small intestinal organoids. Immunofluorescence staining was performed to analyze the proliferation of small intestinal organoids using antibodies against ( j ) Lgr5 and ( k ) Ki67 after culture in the absence or presence of 10 μg/ml LPS and 2 mM Gln for 48 h (Scale bar: 100 μm). Statistical analyses were conducted using one-way ANOVA. Data are presented as the mean ± SD; * p < 0.05, * * p < 0.01, * * * p < 0.001. Cont control, SD standard deviation, DAPI 4',6-diamidino-2-phenylindole, EdU 5-Ethynyl-2’-deoxyuridine, LPS lipopolysaccharide, Gln glutamine, CCK-8 cell counting kit-8, RT–qPCR real-time quantitative polymerase chain reaction, IEC-6 intestine epithelial cell 6, PCNA proliferating cell nuclear antigen

Article Snippet: The rat small intestinal epithelial cell line IEC-6 was obtained from American Type Culture Collection (ATCC), while 293 T cells were maintained in our laboratory.

Techniques: Immunofluorescence, Staining, Expressing, Quantitative RT-PCR, Concentration Assay, Flow Cytometry, Cell Culture, CCK-8 Assay, Control, Standard Deviation, Cell Counting, Real-time Polymerase Chain Reaction

Journal: Burns & Trauma

Article Title: Glutamine promotes the proliferation of intestinal stem cells via inhibition of TP53-induced glycolysis and apoptosis regulator promoter methylation in burned mice

doi: 10.1093/burnst/tkae045

Figure Lengend Snippet: Gln promotes the expression of TIGAR after burn injury. ( a, b ) Expression of TIGAR protein and mRNA in the crypts of the intestine at 1, 3, 5, and 7 days post-injury in the sham, burn, and burn + Gln groups. Two-way repeated measures ANOVA revealed that Gln supply ( p < 0.001) and time ( p < 0.001) both had a statistically significant effect on TIGAR expression. There was statistically significant interaction between Gln supply and time ( p < 0.05). ( c ) Immunohistochemical staining of intestinal sections using antibodies against TIGAR at 3 days post-injury (Scale bar: 50 μm). Expression of TIGAR mRNA and protein in IEC-6 cells treated with LPS (10 μg/ml) and Gln (2 mM) for 24 h was assessed by ( d ) RT–qPCR and ( e ) western blotting. Statistical analysis was conducted using one-way ANOVA. ( f ) Immunofluorescence staining was performed to analyze the changes in TIGAR in intestinal organoids (Scale bar: 50 μm). Data are presented as mean ± SD; * p < 0.05, * * p < 0.01, * * * p < 0.001. NS not significant, SD standard deviation, DAPI 4',6-diamidino-2-phenylindole, LPS lipopolysaccharide, Gln glutamine, RT–qPCR real-time quantitative polymerase chain reaction, Cont control, IEC-6 intestine epithelial cell 6, TIGAR TP53-induced glycolysis and apoptosis-regulator

Article Snippet: The rat small intestinal epithelial cell line IEC-6 was obtained from American Type Culture Collection (ATCC), while 293 T cells were maintained in our laboratory.

Techniques: Expressing, Immunohistochemical staining, Staining, Quantitative RT-PCR, Western Blot, Immunofluorescence, Standard Deviation, Real-time Polymerase Chain Reaction, Control

Journal: Burns & Trauma

Article Title: Glutamine promotes the proliferation of intestinal stem cells via inhibition of TP53-induced glycolysis and apoptosis regulator promoter methylation in burned mice

doi: 10.1093/burnst/tkae045

Figure Lengend Snippet: TIGAR facilitates the proliferation of IEC-6 cells. ( a ) Western blot analysis was performed to assess the expression of TIGAR in IEC-6 cells transfected with the indicated plasmid. ( b ) EdU, ( c ) colony formation and ( d ) CCK-8 assays were conducted in IEC-6 cells after overexpression (oe) of TIGAR (Scale bar: 200 μm). ( e ) Relative expression levels of Ki67 and PCNA in IEC-6 cells were assessed by RT–qPCR following TIGAR overexpression. ( f ) Knockdown efficiency was assessed by western blot analysis following transfection of four TIGAR siRNAs in IEC-6 cells. ( g ) Colony formation, ( h ) EdU, and ( i ) CCK-8 assays were used to investigate the proliferation of IEC-6 cells after TIGAR depletion (Scale bar: 100 μm). ( j ) Relative expression levels of Ki67 and PCNA in IEC-6 cells were assessed by RT–qPCR after TIGAR knockdown. Statistical analyses in (a, c, d and e) were performed using Student’s t test; one-way ANOVA was used in others. Data are presented as mean ± SD; * p < 0.05, * * p < 0.01, * * * p < 0.001. Cont control, DAPI 4',6-diamidino-2-phenylindole, EdU 5-Ethynyl-2’-deoxyuridine, LPS lipopolysaccharide, Gln glutamine, CCK-8 cell counting kit-8, RT–qPCR real-time quantitative polymerase chain reaction, IEC-6 intestine epithelial cell 6, oe over expression, shNC short hairpin negative control, TIGAR TP53-induced glycolysis and apoptosis-regulator, PCNA proliferating cell nuclear antigen, GAPDH glyceraldehyde-3-phosphate dehydrogenase, SD standard deviation

Article Snippet: The rat small intestinal epithelial cell line IEC-6 was obtained from American Type Culture Collection (ATCC), while 293 T cells were maintained in our laboratory.

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, CCK-8 Assay, Over Expression, Quantitative RT-PCR, Knockdown, Control, Cell Counting, Real-time Polymerase Chain Reaction, Negative Control, Standard Deviation

Journal: Burns & Trauma

Article Title: Glutamine promotes the proliferation of intestinal stem cells via inhibition of TP53-induced glycolysis and apoptosis regulator promoter methylation in burned mice

doi: 10.1093/burnst/tkae045

Figure Lengend Snippet: TIGAR promotes IEC-6 cell proliferation by facilitating the nuclear translocation of YAP. ( a ) Expression levels of YAP and p-YAP in intestinal crypts at 1, 3, 5, and 7 days were assessed by western blot analysis in the sham, burn, and burn + Gln groups. Changes in the expression of YAP and p-YAP in IEC-6 cells were assessed by western blot analysis following TIGAR overexpression (oe) ( b ) and knockdown ( c ), as well as LPS (10 μg/ml, 24 h) and Gln (2 mM, 24 h) treatment. Nuclear and cytoplasmic fractions of IEC-6 cells were isolated following TIGAR overexpression or knockdown, and the subcellular localization of YAP was assessed by ( d, e ) western blot and ( f, g ) immunofluorescence analyses (Scale bar: 20 μm). ( h ) IEC-6 cell viability was determined by CCK-8 assays after 24 h of treatment with different concentrations of the YAP inhibitor CA3. Proliferation of IEC-6 cells after TIGAR overexpression and treatment with CA3 (0.5 μM, 24 h) was evaluated using ( i ) EdU and ( j ) CCK-8 assays (Scale bar: 100 μm). Statistical analyses were conducted using one-way ANOVA, except in (d and e) where analyses were performed using Student’s t test. Data are presented as mean ± SD; * p < 0.05, * * p < 0.01, * * * p < 0.001. NS not significant, SD standard deviation, DAPI 4',6-diamidino-2-phenylindole, LPS lipopolysaccharide, Gln glutamine, CCK-8 cell counting kit-8, IEC-6 intestine epithelial cell 6, oe over expression, shNC short hairpin negative control, TIGAR TP53-induced glycolysis and apoptosis-regulator, GAPDH glyceraldehyde-3-phosphate dehydrogenase, YAP yes-associated protein, H3 histone 3

Article Snippet: The rat small intestinal epithelial cell line IEC-6 was obtained from American Type Culture Collection (ATCC), while 293 T cells were maintained in our laboratory.

Techniques: Translocation Assay, Expressing, Western Blot, Over Expression, Knockdown, Isolation, Immunofluorescence, CCK-8 Assay, Standard Deviation, Cell Counting, Negative Control

Journal: Burns & Trauma

Article Title: Glutamine promotes the proliferation of intestinal stem cells via inhibition of TP53-induced glycolysis and apoptosis regulator promoter methylation in burned mice

doi: 10.1093/burnst/tkae045

Figure Lengend Snippet: TIGAR inhibits ferroptosis in IEC-6 cells. ( a ) Concentration of Fe 2+ , ( b ) NADPH/NADP + ratio and ( c ) GSH/GSSG ratio were determined in intestinal crypts from the sham, burn, and burn + Gln groups. ( d ) Western blot analysis was performed to investigate the expression of GPX4 in intestinal crypts from the sham, burn, and burn + Gln groups. ( e ) Concentration of Fe 2+ , ( f ) NADPH/NADP + ratio, and ( g ) GSH/GSSG ratio were determined in IEC-6 cells following exposure to LPS (10 μg/ml, 24 h) and culture with Gln (2 mM, 24 h). ( h ) Expression of GPX4 in IEC-6 cells treated with LPS (10 μg/ml, 24 h) and Gln (2 mM, 24 h) was assessed by western blotting. Levels of Fe 2+ in IEC-6 cells after TIGAR overexpression (oe) and depletion were measured using ( i ) a Ferro Orange probe, and ( j ) a cell ferrous iron colorimetric assay kit (Scale bar: 200 μm). ( k ) Levels of ROS, ( l ) the NADPH/NADP + ratio, and ( m ) the GSH/GSSG ratio were assessed in IEC-6 cells after overexpression or knockdown of TIGAR. ( n ) Western blotting was used to determine the expression of GPX4 in TIGAR-overexpressing and TIGAR-knockdown IEC-6 cells. Statistical analyses were conducted using one-way ANOVA, except in overexpressing TIGAR, where analyses were performed using Student’s t test. Data are presented as the means ± SD; * p < 0.05, * * p < 0.01, * * * p < 0.001. LPS lipopolysaccharide, Gln glutamine, CCK-8 cell counting kit-8, IEC-6 intestine epithelial cell 6, oe over expression, shNC short hairpin negative control, GSH glutathione, GSSG oxidized glutathione, NADPH nicotinamide adenine dinucleotide phosphate, TIGAR TP53-induced glycolysis and apoptosis-regulator, GAPDH glyceraldehyde-3-phosphate dehydrogenase, GPX4 glutathione peroxidase 4, SD standard deviation

Article Snippet: The rat small intestinal epithelial cell line IEC-6 was obtained from American Type Culture Collection (ATCC), while 293 T cells were maintained in our laboratory.

Techniques: Concentration Assay, Western Blot, Expressing, Over Expression, Colorimetric Assay, Knockdown, CCK-8 Assay, Cell Counting, Negative Control, Standard Deviation

Journal: Burns & Trauma

Article Title: Glutamine promotes the proliferation of intestinal stem cells via inhibition of TP53-induced glycolysis and apoptosis regulator promoter methylation in burned mice

doi: 10.1093/burnst/tkae045

Figure Lengend Snippet: Glutamine promotes the proliferation of IEC-6 cells by inhibiting TIGAR promoter methylation. ( a, b ) Levels of 5mC and 5hmC in the crypt cells of sham, burn, and burn + Gln groups of mice 3 days after burn injury were assessed using dot blot analysis. ( c ) Methylation/unmethylation ratio in the crypt cells of the sham, burn and burn + Gln groups of mice at 3 days post-burn injury was determined by methylation-specific PCR. Expression of ( d ) DNMTs and ( e ) TETs in intestinal crypts was investigated by RT–qPCR. ( f ) IEC-6 cell viability was assessed by CCK-8 assays following treatment with different concentrations of the DNMT inhibitor RG108. ( g, h ) mRNA and protein expression of TIGAR was determined using RT-qPCR and western blotting following a 24 h incubation with LPS (10 μg/ml) and RG108 (0.5 μM). ( i ) EdU assay was used to evaluate the proliferation of IEC-6 cells treated with LPS (10 μg/ml) and RG108 (0.5 μM) for 24 h. ( j ) Immunofluorescence was performed to assess the level of TIGAR in the intestinal organoids treated with LPS (10 μg/ml) and RG108 (0.5 μM) for 48 h. ( k ) CCK-8 assay was used to determine IEC-6 cell viability after treatment with different concentrations of the TET inhibitor Bobcat339 for 24 h. ( l ) Colony formation assays were conducted in IEC-6 cells treated with 50 μM Bobcat339. ( m, n ) RT-qPCR and western blotting were used to assess the levels of TIGAR mRNA and protein in IEC-6 cells cultured with different concentrations of Bobcat339. ( o ) Level of TIGAR in the organoids treated with 50 μM Bobcat339 for 24 h was analyzed by immunofluorescence. Statistical analyses were conducted using one-way ANOVA, except in (l) where analysis was performed using Student’s t test. Data are presented as mean ± SD, * p < 0.05, * * p < 0.01, * * * p < 0.001. NS not significant, DAPI 4',6-diamidino-2-phenylindole, EdU 5-Ethynyl-2’-deoxyuridine, LPS lipopolysaccharide, Gln glutamine, CCK-8 cell counting kit-8, IEC-6 intestine epithelial cell 6, oe over expression, shNC short hairpin negative control, Cont control, 5mC 5-methylcytosine, 5hmC 5-hydroxymethylcytosine, TIGAR TP53-induced glycolysis and apoptosis-regulator, GAPDH glyceraldehyde-3-phosphate dehydrogenase, TET ten-eleven translocation, DNMT DNA methyltransferase, SD standard deviation

Article Snippet: The rat small intestinal epithelial cell line IEC-6 was obtained from American Type Culture Collection (ATCC), while 293 T cells were maintained in our laboratory.

Techniques: Methylation, Dot Blot, Expressing, Quantitative RT-PCR, CCK-8 Assay, Western Blot, Incubation, EdU Assay, Immunofluorescence, Cell Culture, Cell Counting, Over Expression, Negative Control, Control, Translocation Assay, Standard Deviation