Claudin 3 Antibody - #AF0129

Figure 3. |USP5 promotes EMT in hepatocellular carcinoma. (A) Motif analysis of SLUG ChIP-Seq cited from ChIPBASE. (B) PLC-PRF-5 and Hep3B cells were treated with different amounts of USP5 siRNA. Cellular extracts were prepared for ChIP assays with anti-SLUG. (C) PLC-PRF-5 and Hep3B cells were transfected with E-cadherin - dependent reporter gene plasmids. Luciferase activity was measured when cells overexpressed or knocked down USP5. (D) WB analysis of USP5, SLUG and EMT related markers in PLC-PRF-5 and Hep3B cells under USP5 knocked down or overexpressed treatment.

Figure 4. COMP promotes EMT in colorectal cancer. (A) Cell phenotypic changes in cells treated with COMP siRNA and COMP overexpression. (B) WB analysis of COMP and EMT-related markers in HCT116 cells under COMP knockdown or overexpression. (C) Cell wound scratch assay of HCT116, HCT-8, and SW620 cells treated with COMP siRNA or COMP overexpression vectors. (D) Transwell assay of HCT116, HCT-8, and SW620 cells treated with COMP siRNA or COMP overexpression vectors. (E) Immunofluorescence assay of HCT116, HCT-8, and SW620 cells treated with COMP siRNA or COMP overexpression vectors. Image J software was used to analyze the relative intensity of E-cadherin and Vimentin. (F) HCT116 and HCT-8 cells with knocked down or overexpressed COMP were transplanted on nude mice. Tumor volumes were measured every 4 days. (G) Tumor weight in the control, COMP knockdown, and COMP overexpression groups. (H) The in situ spleen model of the colorectal cancer cell line SW620 showed that overexpression of COMP promoted liver metastasis of colorectal cancer, while downregulation of COMP inhibited liver metastasis of colorectal cancer. (I) IHC staining to identify EMT biomarkers and COMP-related proteins in the control, COMP knockdown, and COMP overexpression groups. COMP knockdown displayed strong E-cadherin staining and reduced Vimentin, Snail1, Twist1, and MMP-2 staining. The expression levels of other proteins were identical to those observed in WB experiments. (J) Staining indices of COMP, E-cadherin, Vimentin, Snail1, Twist1, and MMP2. The error bars in all graphs represented SD, and each experiment was repeated three times. * and ** stand for P<0.05 and P<0.01, respectively.

Figure 5Commensal Roseburia ameliorates gut-dysbiosis-induced mastitis in mice

Fig. 5 MenSCs promoted TJ- and bile transport function-related protein expression and reduced fibrosis-related protein expression. A Western blot analysis protein levels of Claudin-1, Claudin-3, Claudin-5, Claudin-7 and Occludin in liver tissue of different groups (n = 3 for each group). B BSEP, OATP2 and NTCP1 expression in liver tissues of different groups (n = 3 for each group). C, D Liver COL1A1, α-SMA, TGF-β1 and β-catenin expression among different groups (n = 3 for each group). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Figure 1 Unilateral Vag induces mastitis and disrupts the BMB in mice (A) Experimental design. The mice were randomly divided into three groups: the control group, the Vag group, and the vagus nerve stimulation group (n = 7). After 3 days, the mice were sacrificed for the collection of mammary tissue. (B) Representative H&E-stained images of mammary tissues from three different groups (scale bar, 50 μm). (C) Histopathological score of the mammary gland (n = 7). ns, not significant. (D–F) Inflammatory cytokines, including TNF-α (D) and IL-1β (E) concentrations and MPO activity (F) in the three groups of mammary tissues, were detected by the kit (n = 7). (G–J) The expression levels of the TJ proteins ZO-1, Occludin, and Claudin-3 in representative mammary tissues as detected by western blotting (G) and the relative intensity of the three proteins using β-actin as an internal reference (H–J) (n = 3). (K) The localization and expression of TJ proteins in the mammary gland was determined by immunohistochemistry (scale bar, 20 μm). (L and M) Serum levels of TNF-α (L) and IL-1β (M) (n = 7). Data are presented as means ± SDs (C–F, H–J, L, and M), and one-way ANOVA was performed for statistical analysis (B–F and H–J). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; and ∗∗∗∗p < 0.0001 indicate significant differences. See also Figures S1 and S2.

Figure 1 Unilateral Vag induces mastitis and disrupts the BMB in mice (A) Experimental design. The mice were randomly divided into three groups: the control group, the Vag group, and the vagus nerve stimulation group (n = 7). After 3 days, the mice were sacrificed for the collection of mammary tissue. (B) Representative H&E-stained images of mammary tissues from three different groups (scale bar, 50 μm). (C) Histopathological score of the mammary gland (n = 7). ns, not significant. (D–F) Inflammatory cytokines, including TNF-α (D) and IL-1β (E) concentrations and MPO activity (F) in the three groups of mammary tissues, were detected by the kit (n = 7). (G–J) The expression levels of the TJ proteins ZO-1, Occludin, and Claudin-3 in representative mammary tissues as detected by western blotting (G) and the relative intensity of the three proteins using β-actin as an internal reference (H–J) (n = 3). (K) The localization and expression of TJ proteins in the mammary gland was determined by immunohistochemistry (scale bar, 20 μm). (L and M) Serum levels of TNF-α (L) and IL-1β (M) (n = 7). Data are presented as means ± SDs (C–F, H–J, L, and M), and one-way ANOVA was performed for statistical analysis (B–F and H–J). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; and ∗∗∗∗p < 0.0001 indicate significant differences. See also Figures S1 and S2.

Fig. 2 Treatment with sf-GFP-S. maltophilia destroys the integrity of the blood-milk barrier. After the mice were treated with 1010 CFU S. maltophilia for a week, mammary gland tissues were collected and the integrity of blood-milk barrier was detected. A Expression of tight junction proteins ZO-1, Occludin, Claudin-3 in mouse mammary gland. B FITC-albumin fluorescence staining of mammary gland tissue to determine the degree of albumin infiltration. The data are presented as mean ± SD. One-way analysis of variance was performed for statistical analysis. *P < 0.05, **P < 0.01 and ***P < 0.001 indicate significance from each group. ns, no significance

Fig 2 HEX improves S. aureus-caused disruption of the blood-milk barrier. A-D. Western blotting was performed to detect the protein levels of ZO-1, Occludin, and Claudin-3 in the mammary gland (n = 5). E. Representative immunohistochemical image of the mammary glands by ZO-1, Occludin, and Claudin-3 antibodies staining (scale bars, 20 μm). Data are expressed as mean ± SD (B-D). ns, no significance, *p < 0.05, **p < 0.01, and ***p < 0.001 indicate significant statistical difference by one-way ANOVA (B-D).

Fig 2 HEX improves S. aureus-caused disruption of the blood-milk barrier. A-D. Western blotting was performed to detect the protein levels of ZO-1, Occludin, and Claudin-3 in the mammary gland (n = 5). E. Representative immunohistochemical image of the mammary glands by ZO-1, Occludin, and Claudin-3 antibodies staining (scale bars, 20 μm). Data are expressed as mean ± SD (B-D). ns, no significance, *p < 0.05, **p < 0.01, and ***p < 0.001 indicate significant statistical difference by one-way ANOVA (B-D).

Fig 3 HEX enhances barrier integrity and limits the NF-κB signaling pathway in MMECs. A-D. The protein levels of ZO-1, Occludin, and Claudin-3 were assessed after HEX pretreatment (0, 2.5, and 5 μM) for 2 h and S. aureus treatment (105 CFU) for 24 h (n = 5). E. Immunofluorescence staining was performed to detect TJs protein ZO-1, Occludin, and Claudin-3 expressions in MMECs (scale bars, 10 μm). F-H. Protein levels in the NF-κB signaling pathway, including IκB, p65, and phosphorylation levels of IκB and p65, were detected with HEX pretreatment (0, 2.5, and 5 μM) for 2 h and S. aureus treatment (105 CFU) for 24 h (n = 5). One-way ANOVA (B-D and G-H) was used, and values are presented as the mean ± SD (B-D and G-H). *p < 0.05, **p < 0.01, and ***p < 0.001 indicate significantly different from each group. ns, no significance.