Journal: Chinese Medical Journal

Article Title: “One stone three birds”: Andrographis paniculata -derived exosome-like nanoparticles mitigate dextran sulfate sodium-induced colitis

doi: 10.1097/CM9.0000000000003651

Figure Lengend Snippet: APELNs protect the intestinal barrier in DSS-induced colitis. (A) AB/PAS (Alcian Blue/Periodic Acid-Schiff stain) staining of colon sections. (B) Quantification of goblet cells in colonic crypts. (C) APELNs reduced intestinal permeability. (D–G) qRT-PCR analysis of Claudin-1, ZO-1, MUC2, and OCLN expression in colon samples. (H, I) IF showing the localization of Claudin-1, ZO-1, MUC2, and OCLN. (J, K) WB analysis and relative protein expression of Claudin-1, ZO-1, MUC2, and OCLN in colon tissues. Data were shown as mean ± standard deviation. * P <0.05, † P <0.01, ‡ P <0.001, § P <0.0001. AB/PAS: Alcian blue/periodic acid-schiff stain; APELN: Andrographis paniculata -derived exosome-like nanoparticle; DAPI: 4′,6-diamidino-2-phenylindole; DSS: Dextran sodium sulfate; FITC-Dex: Fluorescein isothiocyanate-dextran permeability assay; IF: Immunofluorescence; MUC2: Mucin2; ns: Not significant; OCLN: Occludin; qRT-PCR: Quantitative reverse transcription polymerase chain reaction; WB: Western blotting; ZO-1: Zonula occludens-1.

Article Snippet: Samples were incubated overnight at 4°C with primary antibodies, including anti-Mucin2 (MUC2) (GB11344, Servicebio, China), anti-occludin (OCLN) ( GB111401 , ab216327, Servicebio), anti-ZO-1 ( GB111402 , Servicebio), and anti-Claudin-1 (GB15032, Servicebio).

Techniques: Staining, Permeability, Quantitative RT-PCR, Expressing, Standard Deviation, Derivative Assay, FITC-Dextran Permeability Assay, Immunofluorescence, Reverse Transcription, Polymerase Chain Reaction, Western Blot

Journal: Chinese Medical Journal

Article Title: “One stone three birds”: Andrographis paniculata -derived exosome-like nanoparticles mitigate dextran sulfate sodium-induced colitis

doi: 10.1097/CM9.0000000000003651

Figure Lengend Snippet: APELNs enhance TJ protein expression in IECs. (A, B) qRT-PCR analysis of Claudin-1, ZO-1, MUC2, and OCLN in NCM460 and HCT116 cells. (C, D, E) WB analysis and relative protein expression of Claudin-1, ZO-1, MUC2, and OCLN in NCM460 and HCT116 cells. (F, G) IF images showing the expression of Claudin-1, ZO-1, MUC2, and OCLN. Bar: 20 μm. * P <0.05, † P <0.01, ‡ P <0.001, § P <0.0001. APELN: Andrographis paniculata -derived exosome-like nanoparticle; DAPI: 4′,6-diamidino-2-phenylindole; IEC: Intestinal epithelial cell; LPS: Lipopolysaccharide; MUC2: Mucin2; OCLN: Occludin; qRT-PCR: Quantitative reverse transcription polymerase chain reaction; TJ: Tight junction; WB: Western blotting; ZO-1: Zonula occludens-1.

Article Snippet: Samples were incubated overnight at 4°C with primary antibodies, including anti-Mucin2 (MUC2) (GB11344, Servicebio, China), anti-occludin (OCLN) ( GB111401 , ab216327, Servicebio), anti-ZO-1 ( GB111402 , Servicebio), and anti-Claudin-1 (GB15032, Servicebio).

Techniques: Expressing, Quantitative RT-PCR, Derivative Assay, Reverse Transcription, Polymerase Chain Reaction, Western Blot

Journal: eLife

Article Title: Lacticaseibacillus rhamnosus P118 enhances host tolerance to Salmonella infection by promoting microbe-derived indole metabolites

doi: 10.7554/eLife.101198

Figure Lengend Snippet: ( A ) Experimental design. ( B ) Survival curve of mice infected with S . Typhimurium. ( C ) Body weight. ( D ) Spleen index. ( E ) Representative images of spleen and intestine. ( F ) Liver index. ( G ) S . Typhimurium burden in tissues and shedding in feces. ( H ) Representative images of H&E staining, TEM, and immunostaining (DAPI, Ki67, lysozyme, Muc2, F4/80, Nos2) in the ileum. Different lowercase letters indicate a significant difference (p<0.05). Significant differences *p<0.05, **p<0.01. C: PBS group; P: P118 administered group; S: S . Typhimurium-infected group; P+S: P118 protective group.

Article Snippet: For the immunofluorescent assay, the paraffin-embedded ileum samples were incubated with primary antibodies against Ki67 ( GB121141 , Servicebio, China), lysozyme (GB11345, Servicebio), Muc2 (GB11344, Servicebio), F4/80 ( GB113373 , Servicebio), Nos2 (GB11119, Servicebio), Il1b (GB11113, Servicebio), Il6 (GB11117, Servicebio), Tnfa (GB11188, Servicebio) overnight at 4°C and then further incubated with secondary antibodies (Alexa Fluor 488-conjugated goat anti-rabbit/mouse IgG) or HRP-conjugated goat anti-rabbit secondary antibody (GB23303, Servicebio) for 60 min at room temperature.

Techniques: Infection, Staining, Immunostaining

Journal: Nature Communications

Article Title: DHX9 maintains epithelial homeostasis by restraining R-loop-mediated genomic instability in intestinal stem cells

doi: 10.1038/s41467-024-47235-2

Figure Lengend Snippet: a DHX9 protein expression in normal intestinal tissues ( n = 4) and biopsies from patients with ulcerative colitis (UC) ( n = 5). Quantifications are presented on the right. b DHX9 protein expression in normal intestinal tissues ( n = 5) and biopsies from patients with Crohn’s disease (CD) ( n = 6). Quantifications are presented on the right. c-f Experimental design depicting the induction of colitis using DSS, Dhx9 fl//fl ( n = 6) and Dhx9 ΔIEC ( n = 4) mice were treated with 2.5% DSS for 6 days ( c ), with monitoring of body weight changes throughout the experimental period ( d ); measurement of colon length upon sacrifice on day 9 ( e ); and representative hematoxylin and eosin (H&E) staining of colonic tissue, Scale bar, 400 μm, histological scores are presented on the right ( f ). g Schematic representation of TNBS-induced colitis in Dhx9 fl//fl and Dhx9 ΔIEC mice ( n = 5 per group). h Body weight changes in Dhx9 fl//fl and Dhx9 ΔIEC mice following TNBS treatment ( n = 5 per group). i Measurement of colon length upon sacrifice on day 3, with quantifications provided on the right ( Dhx9 fl//fl , n = 5; Dhx9 ΔIEC , n = 3). j Survival rates of mice after TNBS treatment (two times results were summarized for statistics, n = 10 per group). k Representative H&E staining of colon sections. Scale bar, 200 μm. Corresponding histological scores are presented on the right ( Dhx9 fl//fl , n = 5; Dhx9 ΔIEC , n = 3). Results are representative of data generated in at least two independent experiments, error bars show means ± SEM. Statistical analyses of body weight changes were performed using two-way ANOVA analysis with Tukey’s multiple comparisons ( d and h ). Survival curves was determined by the log-rank test ( j ). Two-tailed unpaired Student’s t -test were used for other analyses. Source data are provided as a Source Data file.

Article Snippet: For IF staining, primary antibodies against Ki67 (GB111141, Servicebio, 1:500), Lysozyme (GB11345, Servicebio, 1:500), MUC2 (GB11344, Servicebio, 1:500), E-cadherin (GB12082, Servicebio, 1:500), DHX9 (ab26271, Abcam, 1:200), DHX9 (67153, Proteintech, 1:200), Cleaved Caspase-3 (9661, Cell Signaling Technology, 1:200) were used.

Techniques: Expressing, Staining, Generated, Two Tailed Test

Journal: Nature Communications

Article Title: DHX9 maintains epithelial homeostasis by restraining R-loop-mediated genomic instability in intestinal stem cells

doi: 10.1038/s41467-024-47235-2

Figure Lengend Snippet: a Gene ontology analysis revealing the down-regulated expressed genes in IECs of Dhx9 ΔIEC mice compared to Dhx9 fl//fl mice ( P < 0.05; right-tailed Fischer’s exact t -test). b Heat map analysis of the RNA-seq dataset displaying marker genes for intestinal epithelial cell lines in Dhx9 ΔIEC mice versus Dhx9 fl//fl mice. c Volcano plot analysis of the RNA-seq dataset illustrating the top upregulated and downregulated genes in IECs of Dhx9 ΔIEC mice compared to Dhx9 fl//fl mice ( P < 0.05; right-tailed Fischer’s exact t -test). d Gene set enrichment analysis (GSEA) comparing the gene expression profiles of intestinal cells based on RNA-seq data in Dhx9 ΔIEC compared to Dhx9 fl//fl IECs. Two-tailed t -test with the Benjamini–Hochberg correction for an adjusted P -value. e Single-cell RNA sequencing of IECs from Dhx9 ΔIEC and their Dhx9 fl//fl littermates. Visualized as a Uniform Manifold Approximation and Projection (UMAP) plot, categorizing 30,252 cells from the small intestine into 15 distinct clusters based on their gene expression profiles. f Graphical representation of the proportion of each identified cell cluster relative to the total population in the analyzed samples. g – j Littermate 8 week-old male Dhx9 fl//fl and Dhx9 ΔIEC mice were subjected to immunofluorescence staining. Representative staining of Ki67 in colon (upper panel) and ileum (lower panel) sections (at least 30 crypt-villus axes were counted, colon of Dhx9 fl//fl , n = 32; colon of Dhx9 ΔIEC , n = 31; ileum of Dhx9 fl//fl , n = 33; ileum of Dhx9 ΔIEC , n = 31) ( g ); Lysozyme and E-cadherin in ileum sections (at least 30 crypt-villus axes were counted, Dhx9 fl//fl , n = 30; Dhx9 ΔIEC , n = 34) ( h ); Muc2 and E-cadherin in colon sections (at least 30 crypt axes were counted, Dhx9 fl//fl , n = 35; Dhx9 ΔIEC , n = 32) ( i ); Muc2 and E-cadherin in ileum sections (at least 30 crypt-villus axes were counted, Dhx9 fl//fl , n = 31; Dhx9 ΔIEC , n = 32) ( j ). Scale bars represent 100 μm. Each experiment was performed on n = 3 mice individually, with similar results. Error bars show means ± SEM. Statistical analyses were performed by two-tailed unpaired Student’s t -test. Source data are provided as a Source Data file.

Article Snippet: For IF staining, primary antibodies against Ki67 (GB111141, Servicebio, 1:500), Lysozyme (GB11345, Servicebio, 1:500), MUC2 (GB11344, Servicebio, 1:500), E-cadherin (GB12082, Servicebio, 1:500), DHX9 (ab26271, Abcam, 1:200), DHX9 (67153, Proteintech, 1:200), Cleaved Caspase-3 (9661, Cell Signaling Technology, 1:200) were used.

Techniques: RNA Sequencing, Marker, Gene Expression, Two Tailed Test, Immunofluorescence, Staining

Journal: Nature Communications

Article Title: DHX9 maintains epithelial homeostasis by restraining R-loop-mediated genomic instability in intestinal stem cells

doi: 10.1038/s41467-024-47235-2

Figure Lengend Snippet: a Representative transmission electron microscopy (TEM) images of ileal crypt bases from Dhx9 fl//fl and Dhx9 ΔIEC mice. ISCs are highlighted in green, and Paneth cells are indicated in brown. Scale bar, 10 μm. Experiment was performed on n = 3 mice individually, with similar results. b Representative brightfield images of Dhx9 fl//fl and Dhx9 ΔIEC ileum organoids after 2 days of culture ( n = 10 per group). Scale bar, 100 μm. Quantifications provided on the right. Three individual experiments were performed, with similar results. c Propidium iodide (PI) staining of organoids derived from Dhx9 fl//fl and Dhx9 ΔIEC mice. Scale bar, 200 μm. The panel to the right of the images provides a statistical analysis, quantifying the relative area of PI staining per organoid ( Dhx9 fl//fl , n = 16; Dhx9 ΔIEC , n = 17). Three individual experiments were performed, with similar results. d Comparative analysis of the growth status of intestinal organoids from Dhx9 fl//fl and Dhx9 ΔIEC mice at different time points (2 days, 4 days, and 6 days). Scale bar, 100 μm. The growth curves of intestinal organoids provided on the right ( n = 11 per group). Three individual experiments were performed, with similar results. e – g Dhx9 fl//fl and Dhx9 ΔIEC organoids were cultured for 6 days and underwent two serial passages, with images captured for each generation to illustrate morphological changes, scale bar, 500 μm ( e ) analysis of the budding rate in each generation of the organoids, n = 8 per group ( f ) and quantitative analysis of Dhx9 mRNA levels in the original crypts and across each generation of organoids, n = 4 per group ( g ). Data are representative of three independent experiments. Error bars show means ± SEM. Statistical analyses of organoid size changes were performed using two-way ANOVA analysis with Tukey’s multiple comparisons ( d ). Two-tailed unpaired Student’s t -test were used for other analyses. Source data are provided as a Source Data file.

Article Snippet: For IF staining, primary antibodies against Ki67 (GB111141, Servicebio, 1:500), Lysozyme (GB11345, Servicebio, 1:500), MUC2 (GB11344, Servicebio, 1:500), E-cadherin (GB12082, Servicebio, 1:500), DHX9 (ab26271, Abcam, 1:200), DHX9 (67153, Proteintech, 1:200), Cleaved Caspase-3 (9661, Cell Signaling Technology, 1:200) were used.

Techniques: Transmission Assay, Electron Microscopy, Staining, Derivative Assay, Cell Culture, Two Tailed Test

Journal: Nature Communications

Article Title: DHX9 maintains epithelial homeostasis by restraining R-loop-mediated genomic instability in intestinal stem cells

doi: 10.1038/s41467-024-47235-2

Figure Lengend Snippet: a Schematic diagram illustrating the strategy of tamoxifen administration. Dhx9 iΔISC ( Dhx9 fl//fl Lgr5-EGFP-IRES-cre ERT2 ) mice received tamoxifen injections for 5 consecutive days. b Representative images of ileums from corn oil-injected (control) and tamoxifen-treated Dhx9 iΔISC mice. ( n = 5 mice per group). Scale bar, 200 μm. Quantification of Lgr5-EGFP + crypts in the small intestine of mice is provided on the right. c Representative flow cytometry plots showing the percentages of EGFP + crypt cells from corn oil-injected (control) and tamoxifen-treated Dhx9 iΔISC mice ( n = 6 mice per group). Quantification of EGFP + crypt cells is provided on the right. d Representative images of Dhx9 iΔISC ileum organoids after 2 days of culture treated with EtOH (control) or 4-OH. Scale bar, 200 μm. Quantification of EGFP + organoids were provided on the right ( n = 8 per group). e Representative flow cytometry plots showing the percentages of EGFP + organoid cells, obtained from organoids cultured for 2 days that treated with EtOH ( n = 5) or 4-OH ( n = 4). Quantification of EGFP + and DAPI + organoid cells is provided on the right. f – i Experimental design depicting the induction of colitis using DSS. Dhx9 iΔISC mice ( n = 5) and their Dhx9 fl//fl littermates ( n = 3) were injected with tamoxifen from 7 days before DSS treatment (5 consecutive injections), followed by exposure to 2.5% DSS for 6 days ( f ) monitoring of body weight changes throughout the experimental period ( g ); measurement of colon length upon sacrifice on day 9 ( h ); and representative H&E staining of colonic tissue, Scale bar, 400 μm ( i ). Data are representative of three independent experiments; error bars show means ± SEM. Statistical analyses of body weight changes were performed using two-way ANOVA analysis with Tukey’s multiple comparisons ( g ). Two-tailed unpaired Student’s t -test were used for other analyses. Source data are provided as a Source Data file.

Article Snippet: For IF staining, primary antibodies against Ki67 (GB111141, Servicebio, 1:500), Lysozyme (GB11345, Servicebio, 1:500), MUC2 (GB11344, Servicebio, 1:500), E-cadherin (GB12082, Servicebio, 1:500), DHX9 (ab26271, Abcam, 1:200), DHX9 (67153, Proteintech, 1:200), Cleaved Caspase-3 (9661, Cell Signaling Technology, 1:200) were used.

Techniques: Injection, Control, Flow Cytometry, Cell Culture, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: DHX9 maintains epithelial homeostasis by restraining R-loop-mediated genomic instability in intestinal stem cells

doi: 10.1038/s41467-024-47235-2

Figure Lengend Snippet: a Western Blotting analysis of γH2AX and GAPDH (loading control) in lysates from Dhx9 fl//fl and Dhx9 ΔIEC IECs. Three individual experiments were performed, with similar results. b Expression pattern of γH2AX in the ileum of Dhx9 fl//fl and Dhx9 ΔIEC mice. γH2AX is shown in red, and nuclei are stained with DAPI (blue). n = 20 villus axes were counted. Scale bar, 100 μm. Experiment was performed on n = 3 mice individually, with similar results. c Neutral comet assay of Dhx9 fl//fl and Dhx9 ΔIEC IECs. Representative images of comet tails in IECs stained with SYBR green. Scale bar, 10 μm. Quantifications are provided on the right ( Dhx9 fl//fl , n = 13; Dhx9 ΔIEC , n = 16). Three individual experiments were performed, with similar results. d , e Dot blot analysis to quantify R-loops in IECs from Dhx9 ΔIEC and Dhx9 fl//fl mice ( n = 3 per group). RNase H1 treatment was included as a negative control ( d ). Quantitative analysis of the dot blot results ( e ). f IF staining of HeLa cells stably transfected with a doxycycline (Dox)-inducible RNase H1 expression plasmid, which concurrently expresses zsGreen. These cells underwent treatment with specified siRNAs for 48 h and were either induced with Dox for 24 h or left uninduced. Staining includes the S9.6 antibody (red) to detect R-loops and the nucleolin antibody (cyan) for nucleolar identification. Scale bar, 10 μm. g Upper panel: WB analysis of Flag-RNase H1 and Actin in lysates from Dox+ and Dox- HeLa cells from ( f ). Lower panel: Quantitative analysis of the dot blot results from panel ( f ) (SiNC, n = 23; SiDHX9, n = 27; Dox + SiNC, n = 25; Dox + SiDHX9, n = 27). Three individual experiments were performed, with similar results. h , i Monitoring of body weight changes over 16 weeks ( h ), and weight of spleen at 20 weeks ( i ) in Dhx9 fl/fl Apc min/+ and Dhx9 ΔIEC Apc min/+ mice ( n = 3 mice per group). j The number of tumors in the intestine was calculated for Dhx9 fl/fl Apc min/+ ( n = 6) and Dhx9 ΔIEC Apc min/+ mice ( n = 5). Arrows indicate the tumors. Error bars show means ± SEM. Statistical analyses for ( h ) were conducted using two-way ANOVA analysis with Tukey’s multiple comparisons. Two-tailed unpaired Student’s t -test were used for other analyses. Source data are provided as a Source Data file.

Article Snippet: For IF staining, primary antibodies against Ki67 (GB111141, Servicebio, 1:500), Lysozyme (GB11345, Servicebio, 1:500), MUC2 (GB11344, Servicebio, 1:500), E-cadherin (GB12082, Servicebio, 1:500), DHX9 (ab26271, Abcam, 1:200), DHX9 (67153, Proteintech, 1:200), Cleaved Caspase-3 (9661, Cell Signaling Technology, 1:200) were used.

Techniques: Western Blot, Control, Expressing, Staining, Neutral Comet Assay, SYBR Green Assay, Dot Blot, Negative Control, Stable Transfection, Transfection, Plasmid Preparation, Two Tailed Test

Journal: Nature Communications

Article Title: DHX9 maintains epithelial homeostasis by restraining R-loop-mediated genomic instability in intestinal stem cells

doi: 10.1038/s41467-024-47235-2

Figure Lengend Snippet: a Gene ontology analysis revealing the up-regulated expressed genes in IECs of Dhx9 ΔIEC mice compared to Dhx9 fl//fl mice, based on RNA-seq data ( P < 0.05; right-tailed Fischer’s exact t -test). b Volcano plot analysis of the RNA-seq dataset illustrating the top upregulated and downregulated genes in IECs of Dhx9 ΔIEC mice compared to Dhx9 fl//fl mice (The black dots represent genes with no statistically significant differences; the green dots and the red dots, P < 0.05; right-tailed Fischer’s exact t -test). c Interaction network of up-regulated expressed genes in IECs of Dhx9 ΔIEC compared to Dhx9 fl//fl , based on RNA-seq data. d Representative flow cytometry plots showing the colonic lamina propria-infiltrated myeloid cells of Dhx9 fl//fl ( n = 4) and Dhx9 ΔIEC mice ( n = 6) on day 5 after DSS treatment, including CD11b + , CD11b + Ly6c + , and CD11b + Ly6g + myeloid cells. Quantification is provided on the right. e RT-qPCR analysis was performed to assess the expression of downstream genes of the cGAS-STING pathway, including Oasl2 , Isg15 , Ifit1 , Il-18 , Cxcl10 , and Tnfaip2 , in the ileum of Dhx9 fl//fl , Dhx9 ΔIEC , Dhx9 ΔIEC Sting −/− , and Sting −/− mice at 8 weeks of age ( n = 6 per group). f – i Experimental design illustrating the induction of colitis using DSS. Dhx9 fl//fl ( n = 6), Dhx9 ΔIEC ( n = 6), Dhx9 ΔIEC Sting −/− ( n = 5), and Sting −/− ( n = 6) mice were treated with 2.5% DSS for 6 days ( f ), with monitoring of body weight changes throughout the experimental period ( g ); measurement of colon length upon sacrifice on day 9 ( h ); and representative H&E staining of colonic tissue, Scale bar, 200 μm ( i ). Data are representative of three independent experiments, and error bars show means ± SEM. Statistical analyses of body weight changes were performed using two-way ANOVA analysis with Tukey’s multiple comparisons ( f ). Two-tailed unpaired Student’s t -test were used for other analyses ( d , e , h , and i ). Source data are provided as a Source Data file.

Article Snippet: For IF staining, primary antibodies against Ki67 (GB111141, Servicebio, 1:500), Lysozyme (GB11345, Servicebio, 1:500), MUC2 (GB11344, Servicebio, 1:500), E-cadherin (GB12082, Servicebio, 1:500), DHX9 (ab26271, Abcam, 1:200), DHX9 (67153, Proteintech, 1:200), Cleaved Caspase-3 (9661, Cell Signaling Technology, 1:200) were used.

Techniques: RNA Sequencing, Flow Cytometry, Quantitative RT-PCR, Expressing, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: DHX9 maintains epithelial homeostasis by restraining R-loop-mediated genomic instability in intestinal stem cells

doi: 10.1038/s41467-024-47235-2

Figure Lengend Snippet: DHX9 deficiency in ISCs leads to the abnormal accumulation of R-loops, subsequently inducing genomic instability. This directly results in a reduction in the number of ISCs and influences secretory cell lineage commitment, thereby affecting intestinal homeostasis. DNA damage amplifies cGAS-dependent inflammatory responses in both IECs and myeloid cell populations. R-loop-mediated genomic instability, in conjunction with the triggered activation of the cGAS-STING pathway, impairs ISC function and contributes to the pathogenesis of IBD.

Article Snippet: For IF staining, primary antibodies against Ki67 (GB111141, Servicebio, 1:500), Lysozyme (GB11345, Servicebio, 1:500), MUC2 (GB11344, Servicebio, 1:500), E-cadherin (GB12082, Servicebio, 1:500), DHX9 (ab26271, Abcam, 1:200), DHX9 (67153, Proteintech, 1:200), Cleaved Caspase-3 (9661, Cell Signaling Technology, 1:200) were used.

Techniques: Activation Assay

Journal: International immunopharmacology

Article Title: Melatonin alleviates necrotizing enterocolitis by reducing bile acid levels through the SIRT1/FXR signalling axis.

doi: 10.1016/j.intimp.2023.111360

Figure Lengend Snippet: Fig. 1. Disturbance of BA transporters emerges in NEC. (a)DEGs (2650 up-regulated and 2394 down-regulated) were shown in the Volcano Plot. (b)The metabolic pathways was significantly enriched in KEGG pathway enrichment analysis. (c-d)DEGs were enriched in the primary BA biosynthesis (17 DEGs) pathway and bile secretion(71 DEGs) pathway. (e-f)Heat map showed the expression of DEGs in the primary BA biosynthesis pathway and bile secretion pathway. (g-l)IHC and WB results showed that the expression of FXR, ASBT and IBABP was up-regulated, while SIRTI and OST-α were down-regulated in NEC compared with non-NEC (scale bar:1mm; n = 3 per group;). * P < 0.05, ** P < 0.01. Data are presented as the mean ± SEM.

Article Snippet: Sections were then incubated overnight at 4 ◦C with primary antibodies against MUC2 (1:200 dilution, #GB11344, Servicebio), ZO-1 (1:200 dilution, #21773–1-AP, Proteintech), ASBT (1:200 dilution, #GB112579, Servicebio), IBABP (1:200 dilution, #GB113145, Servicebio), FXR (1:200 dilution, #25055–1-AP, Proteintech) and SIRT1 (1:200 dilution, #Ab110304, Abcam).

Techniques: Expressing

Journal: International immunopharmacology

Article Title: Melatonin alleviates necrotizing enterocolitis by reducing bile acid levels through the SIRT1/FXR signalling axis.

doi: 10.1016/j.intimp.2023.111360

Figure Lengend Snippet: Fig. 4. Melatonin regulates BA metabolism in NEC mice. (a-d) The expression of IBABP and ASBT in the ileum was detected by IF (n ≥6 per group,Scale bar:50 um). (e-h) WB detection of bile acid transporters (ASBT, OST-α, IBABP) and regulatory proteins (SIRT1, FXR) in ileum,and the degree of FXR acetylation (n ≥3 per group). (i)Transcript levels of the downstream genes of FXR(n = 3 per group). (j-k) Changes in serum and fecal bile acid subtypes in NEC mice after melatonin intervention (n ≥6 per group). * P < 0.05, ** P < 0.01, *** P < 0.001,**** P < 0.0001. At least three serial sections of each sample were analyzed. In each slice, statistics were evaluated in three random frames. Data are presented as the mean ± SEM.

Article Snippet: Sections were then incubated overnight at 4 ◦C with primary antibodies against MUC2 (1:200 dilution, #GB11344, Servicebio), ZO-1 (1:200 dilution, #21773–1-AP, Proteintech), ASBT (1:200 dilution, #GB112579, Servicebio), IBABP (1:200 dilution, #GB113145, Servicebio), FXR (1:200 dilution, #25055–1-AP, Proteintech) and SIRT1 (1:200 dilution, #Ab110304, Abcam).

Techniques: Expressing

Journal: International immunopharmacology

Article Title: Melatonin alleviates necrotizing enterocolitis by reducing bile acid levels through the SIRT1/FXR signalling axis.

doi: 10.1016/j.intimp.2023.111360

Figure Lengend Snippet: Fig. 5. Melatonin regulates BA metabolism through SIRT1 in NEC mice. (a-d) The expression of IBABP and ASBT in the ileum was detected by IF (n ≥6 per group,Scale bar:50 um). (e-f) WB detection of BA transporters (ASBT, OST-α, IBABP) and regulatory proteins (SIRT1, FXR) in ileum (n ≥3 per group). (g-h)the degree of ileal injury was detected by HE staining(n ≥6 per group,Scale bar:100 um). (i-j) Changes in serum and fecal bile acid subtypes in NEC mice after SIRT1 agonist/ inhibitor intervention alone or in combination with melatonin (n ≥6 per group). * P < 0.05, ** P < 0.01, *** P < 0.001,**** P < 0.0001. At least three serial sections of each sample were analyzed. In each slice, statistics were evaluated in three random frames. Data are presented as the mean ± SEM.

Article Snippet: Sections were then incubated overnight at 4 ◦C with primary antibodies against MUC2 (1:200 dilution, #GB11344, Servicebio), ZO-1 (1:200 dilution, #21773–1-AP, Proteintech), ASBT (1:200 dilution, #GB112579, Servicebio), IBABP (1:200 dilution, #GB113145, Servicebio), FXR (1:200 dilution, #25055–1-AP, Proteintech) and SIRT1 (1:200 dilution, #Ab110304, Abcam).

Techniques: Expressing, Staining

Journal: International immunopharmacology

Article Title: Melatonin alleviates necrotizing enterocolitis by reducing bile acid levels through the SIRT1/FXR signalling axis.

doi: 10.1016/j.intimp.2023.111360

Figure Lengend Snippet: Fig. 6. Melatonin regulates FXR acetylation through SIRT1. (a-b) The degree of FXR acetylation in the ileum was measured by WB (n ≥3 per group). (c) Transcript levels of the downstream genes of FXR(n = 3 per group). * P < 0.05, ** P < 0.01, *** P < 0.001,**** P < 0.0001. Data are presented as the mean ± SEM.

Article Snippet: Sections were then incubated overnight at 4 ◦C with primary antibodies against MUC2 (1:200 dilution, #GB11344, Servicebio), ZO-1 (1:200 dilution, #21773–1-AP, Proteintech), ASBT (1:200 dilution, #GB112579, Servicebio), IBABP (1:200 dilution, #GB113145, Servicebio), FXR (1:200 dilution, #25055–1-AP, Proteintech) and SIRT1 (1:200 dilution, #Ab110304, Abcam).

Techniques: