Journal: iScience

Article Title: AF6 regulates intestinal IgA via crosstalk between intestinal epithelial cells and immune cells in inflammatory bowel disease

doi: 10.1016/j.isci.2025.112658

Figure Lengend Snippet: AF6 is decreased in the colitis of mice and human (A) qPCR was used to detect Af6 mRNA level in colon tissues of mice. (B) The protein expression of AF6 in IECs was detected by Western blot. (C) The mRNA level of AF6 in human colitis samples from GEO database. On the left is the comparison of AF6 expression in healthy controls ( n = 21), patients with UC ( n = 18), and patients with CD ( n = 37) in the GSE126124 dataset. On the right is a comparison of AF6 expression in healthy controls ( n = 21), moderately active ulcerative colitis ( n = 66), and severely active ulcerative colitis ( n = 40) in the GSE87473 dataset. (D) qPCR was used to detect the relative expression of AF6 mRNA in patients with UC and CD and healthy controls. The patient samples were collected from the Tenth People’s Hospital of Tongji University in Shanghai. n = 6–8 mice per group. Data are expressed as mean ± SEM. Pairwise comparisons between groups were conducted using two-tailed non-paired Student’s t tests. p -values were shown in the panel, and p < 0.05 indicates a significant difference.

Article Snippet: Mouse: B6 Af6 flox/flox , Shanghai Model organisms , custom.

Techniques: Expressing, Western Blot, Comparison, Two Tailed Test

Journal: iScience

Article Title: AF6 regulates intestinal IgA via crosstalk between intestinal epithelial cells and immune cells in inflammatory bowel disease

doi: 10.1016/j.isci.2025.112658

Figure Lengend Snippet: AF6 deficiency in IECs exacerbates DSS-induced colitis and C . rodentium infection-induced enteritis (A–F) (A) Schematic diagram of the ulcerative colitis model, which was conducted using both Af6 f/f and Af6 ΔIEC mice. Age matched Af6 f/f ( n = 6) and Af6 ΔIEC ( n = 6) mice (8–10 weeks old) were maintained for 7 days on drinking water containing 2.5% DSS, during which time body weight ((B); expressed as percent baseline) and conditions of the feces of the mice were recorded daily. After 7 days, the mice were euthanized, and the disease activity index was scored according to the changes in body weight, feces, and blood in the stool (C). The length of the colon tissue was measured at necropsy (D), and the tissue was then fixed for hematoxylin and eosin (HE) staining and histological scoring (E), Scale bar = 100um. (F) Schematic diagram of C . rodentium infection model in Af6 f/f ( n = 6) and Af6 ΔIEC ( n = 6) mice. (G) Changes in body weight over time in C . rodentium -infected mice. (H) Weights of ceca and colon (combined) and spleen of C . rodentium -infected mice at Day 11. (I) Quantification of C . rodentium in feces of C . rodentium -infected mice at Day 9. (J) Quantification of C . rodentium in the spleens of C . rodentium -infected mice at Day 11. Data are expressed as mean ± SEM. Pairwise comparisons between groups were conducted using two-tailed non-paired Student’s t tests. p -values were shown in the panel, and p < 0.05 indicates a significant difference.

Article Snippet: Mouse: B6 Af6 flox/flox , Shanghai Model organisms , custom.

Techniques: Infection, Activity Assay, Staining, Two Tailed Test

Journal: iScience

Article Title: AF6 regulates intestinal IgA via crosstalk between intestinal epithelial cells and immune cells in inflammatory bowel disease

doi: 10.1016/j.isci.2025.112658

Figure Lengend Snippet: AF6 deficiency in IECs impairs B cell signaling and IgA production in mice with colitis (A) GO pathway enrichment analysis of colonic tissue transcriptomes in Af6 f/f and Af6 ΔIEC mice maintained on 2.5% DSS for 7 days. (B) GSEA analysis, based on GO, of colonic tissue transcriptomes in Af6 f/f and Af6 ΔIEC mice maintained on 2.5% DSS for 7 days. NES, standardized enrichment scores. (C) Flow cytometric analysis was used to measure the proportion of IgA + B220 - cells in the colon lamina propria of Af6 f/f and Af6 ΔIEC mice maintained on 2.5% DSS for 7 days. (D and E) (D) IgA content was measured by ELISA in feces (after 3 days on DSS-containing drinking water) and serum (after 7 days on DSS-containing drinking water) of Af6 f/f and Af6 ΔIEC mice. Flow cytometric analysis was used to measure the proportion of (E) MHCII + CD11c + cells and (F) CD44 + CD62L − in the lamina propria of Af6 f/f and Af6 ΔIEC mice maintained on 2.5% DSS for 7 days, n = 5–8 mice per group. Data are expressed as mean ± SEM. Pairwise comparisons between groups were conducted using two-tailed non-paired Student’s t tests. p -values were shown in the panel, and p < 0.05 indicates a significant difference.

Article Snippet: Mouse: B6 Af6 flox/flox , Shanghai Model organisms , custom.

Techniques: Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: iScience

Article Title: AF6 regulates intestinal IgA via crosstalk between intestinal epithelial cells and immune cells in inflammatory bowel disease

doi: 10.1016/j.isci.2025.112658

Figure Lengend Snippet: AF6 deficiency in IECs is associated with decreased MHC II expression in mice with colitis (A) GSEA analysis, based on GO, of colonic tissue transcriptomes in Af6 f/f and Af6 ΔIEC mice maintained on 2.5% DSS for 7 days. NES, standardized enrichment scores. (B) Transcriptome analysis of colon tissues from Af6 f/f and Af6 ΔIEC mice maintained on 2.5% DSS for 7 days. Heat maps show a significant decrease in expression (in Af6 ΔIEC mice compared to Af6 f/f animals) of genes involved in antigen processing and presentation pathways. (C–E) (C) qPCR was used to measure the expression of genes encoding MHC II-related functions in intestinal epithelial cells (IECs) of Af6 f/f and Af6 ΔIEC mice maintained on 2.5% DSS for 7 days. MHCII production in (IECs) of Af6 f/f and Af6 ΔIEC mice maintained on 2.5% DSS for 7 days was assessed by (D) flow cytometric analysis and (E) immunofluorescence staining, Scale bar = 100 μm. (F) Schematic diagram of the co-culture of organoids with T cells. (G) Flow cytometric analysis of T cell effector factors in co-cultures. n = 5–8 mice per group, and for co-culture, there were 2 organoids in each group. Data are expressed as mean ± SEM. Pairwise comparisons between groups were conducted using two-tailed non-paired Student’s t tests. p -values were shown in the panel, and p < 0.05 indicates a significant difference.

Article Snippet: Mouse: B6 Af6 flox/flox , Shanghai Model organisms , custom.

Techniques: Expressing, Immunofluorescence, Staining, Co-Culture Assay, Two Tailed Test

Journal: iScience

Article Title: AF6 regulates intestinal IgA via crosstalk between intestinal epithelial cells and immune cells in inflammatory bowel disease

doi: 10.1016/j.isci.2025.112658

Figure Lengend Snippet: AF6 regulates the expression of MHC II by modulating the expression of STAT1 in intestinal epithelial cells (IECs) (A) The protein levels of components of the IFN-γ-related signaling pathway were detected by immunoblotting of colon tissues from Af6 f/f and Af6 ΔIEC mice maintained on 2.5% DSS for 7 days. (B) qPCR was used to assess STAT1 transcript levels in colon tissues from Af6 f/f and Af6 ΔIEC mice maintained on 2.5% DSS for 7 days. (C and D) (C) The mRNA levels of STAT1 and its downstream target genes (as assessed by qPCR) and (D) the protein levels of STAT1 and proteins encoded by its downstream target genes (as assessed by immunoblotting) were determined in organoids generated from colon tissues of Af6 f/f and Af6 ΔIEC mice, as measured before and after IFN-γ treatment. (E) Co-immunoprecipitation (co-IP) was used to detect endogenous interactions between AF6 and IRF1 in IECs. (F) 293T cells were co-transfected with constructs encoding hemagglutinin-tagged AF6 (HA-AF6) and Flag peptide-tagged IRF1 (Flag-IRF1); their interaction domains were detected by co-IP. (G) Immunoblotting was used to assess the expression of IRF1 and proteins encoded by its downstream genes in HT29 cells with or without IFN-γ exposure. (H) HT29 cells were transfected with constructs encoding AF6 with no or 3 nuclear localization signaling (NLS) domains (ΔNLS-AF6 and 3×NLS-AF6, respectively), and the expression and localization of IRF1 and proteins encoded by its downstream genes were assessed by immunoblotting following the separation of the nuclear and cytoplasmic fractions. Data are expressed as mean ± SEM. Pairwise comparisons between groups were conducted using two-tailed non-paired Student’s t tests. p -values were shown in the panel, and p < 0.05 indicates a significant difference.

Article Snippet: Mouse: B6 Af6 flox/flox , Shanghai Model organisms , custom.

Techniques: Expressing, Western Blot, Generated, Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection, Construct, Two Tailed Test

Journal: iScience

Article Title: AF6 regulates intestinal IgA via crosstalk between intestinal epithelial cells and immune cells in inflammatory bowel disease

doi: 10.1016/j.isci.2025.112658

Figure Lengend Snippet: AF6 deficiency in intestinal epithelial cells (IECs) is associated with the decreased expression of Pigr (A and B) The levels of (A) Pigr transcript (as assessed by qPCR) and (B) pIgR protein (as assessed by immunoblotting) were measured in IECS from Af6 f/f and Af6 ΔIEC mice maintained on DSS. (C) The mRNA levels of Pigr (as assessed by qPCR) and (D) the protein levels of p-NF-κB and pIgR (as assessed by immunoblotting) were determined in organoids generated from colon tissues of Af6 f/f and Af6 ΔIEC mice, as measured before and after IFN-γ treatment. (E) The protein levels of pIgR were determined (by immunoblotting) in an Af6 -silenced HT29 cell line (generated by stable transfection with PLKO and shAF6 constructs) grown in the presence of various concentrations of IFN-γ. (F and G) (F) The mRNA levels of Pigr (as assessed by qPCR) and (G) the protein levels of pIgR (as assessed by immunoblotting) were determined in organoids generated from colon tissues of Af6 f/f and Af6 ΔIEC mice, as measured before and after IFN-γ treatment. Data are expressed as mean ± SEM. Pairwise comparisons between groups were conducted using two-tailed non-paired Student’s t tests. p -values were shown in the panel, and p < 0.05 indicates a significant difference.

Article Snippet: Mouse: B6 Af6 flox/flox , Shanghai Model organisms , custom.

Techniques: Expressing, Western Blot, Generated, Stable Transfection, Construct, Two Tailed Test

Journal: iScience

Article Title: AF6 regulates intestinal IgA via crosstalk between intestinal epithelial cells and immune cells in inflammatory bowel disease

doi: 10.1016/j.isci.2025.112658

Figure Lengend Snippet: AF6 affects the composition of the intestinal microbiota (A) Bar chart of the phylum-level composition of the fecal microbiota in Af6 f/f and Af6 ΔIEC mice. (B) Boxplots of α diversity of fecal microbiota in Af6 f/f and Af6 ΔIEC mice, show the Shannon index, Simpson index, inverse Simpson index, richness index, and evenness index. (C) Plots show the β diversity analyses of fecal microbiota in Af6 f/f and Af6 ΔIEC mice. Upper panel: PCA analysis; lower panel, PCoA analysis based on the Bray-Curtis distance algorithm. (D) The LDA scores were calculated based on the differences in fecal microbiota abundances between Af6 f/f and Af6 ΔIEC mice. The feature selection criterion was log (LDA score) > 4. n = 4 mice per group. Data are expressed as mean ± SEM. Pairwise comparisons between groups were conducted using two-tailed non-paired Student’s t tests. p -values were shown in the panel, and p < 0.05 indicates a significant difference.

Article Snippet: Mouse: B6 Af6 flox/flox , Shanghai Model organisms , custom.

Techniques: Selection, Two Tailed Test

Journal: iScience

Article Title: AF6 regulates intestinal IgA via crosstalk between intestinal epithelial cells and immune cells in inflammatory bowel disease

doi: 10.1016/j.isci.2025.112658

Figure Lengend Snippet: IgA administration in mice with DSS-induced colitis restores both the normal sensitivity to the disease and the abundances of the gut microbiota (A) Schematic diagram of the experimental W27 IgA supplementation model. Age matched Af6 f/f ( n = 6) and Af6 ΔIEC ( n = 6) mice (8–10 weeks old) were giving W27 supplemented water for 3 days then maintained for 7 days on drinking water containing 1.5% DSS, during which time body weight ((B); expressed as percent baseline) and conditions of the feces of the mice were recorded daily. After 7 days, the mice were euthanized, and the disease activity index was scored according to the changes in body weight, feces, and blood in the stool (C). The length of the colon tissue was measured at necropsy (D), and the tissue was then fixed for hematoxylin and eosin (HE) staining (E), Scale bar = 300um. (F) The effects of W27 supplementation on the intestinal microbiota of Af6 ΔIEC mice were detected by qPCR. Data are expressed as mean ± SEM. Pairwise comparisons between groups were conducted using two-tailed non-paired Student’s t tests. p -values were shown in the panel, and p < 0.05 indicates a significant difference.

Article Snippet: Mouse: B6 Af6 flox/flox , Shanghai Model organisms , custom.

Techniques: Activity Assay, Staining, Two Tailed Test

Journal: mBio

Article Title: Standard mouse diets lead to differences in severity in infectious and non-infectious colitis

doi: 10.1128/mbio.03302-24

Figure Lengend Snippet: Antibodies used for flow cytometry experiments

Article Snippet: MHC-II (I-Ab) , AF6-120.1 , eF450 , 48–5320-82 , 200 , eBioscience.

Techniques: Flow Cytometry

Journal: Cell reports

Article Title: Defective removal of invariant chain peptides from MHC class II suppresses tumor antigen presentation and promotes tumor growth

doi: 10.1016/j.celrep.2024.115150

Figure Lengend Snippet:

Article Snippet: Rat anti-mouse MHC-I (H-2Kb) FITC (clone AF6-88.5.5.3) , ThermoFisher , Cat # 11-5958-82; RRID: AB_11149502.

Techniques: Purification, Recombinant, Protease Inhibitor, Western Blot, Isolation, Cell Isolation, Software