Journal: Frontiers in Pharmacology

Article Title: Vitamin D/vitamin D receptor protects intestinal barrier against colitis by positively regulating Notch pathway

doi: 10.3389/fphar.2024.1421577

Figure Lengend Snippet: Inhibition of the Notch pathway aggravates DSS-induced colitis. CT: control, LY411,575 (Notch inhibitor): 10 mg/kg LY411,575 gavage, DSS: 2.5% DSS water, DSS-LY411,575: LY411,575 was intragastric administrated at the last 4 days of DSS feeding (once per day). (A) DAI scores of each group. DAI scores were compared by the repeated measures ANOVA with Bonferroni post hoc multiple comparisons. (B) Serum FD-4 concentration in each group. Statistical analysis was performed using ANOVA with Tukey’s post-test. (C) Histological scores of distal colons in each group. Statistical analysis was performed using ANOVA with Tukey’s post-test. (D) Representative colonic H&E staining image of each group (scale bar = 50 μm). (E) Representative colonic IF staining image of claudin-1 ( green ) and claudin-3 ( green ) (scale bar = 25 μm). (F) Representative colonic IF staining image and quantification of N1ICD ( red ) and Hes1 ( red ) (scale bar = 25 μm). Quantification of N1ICD and Hes1 were determined by the ratio of positive staining cells per crypt. Data are presented as the mean ± SEM from 6-8 mice in each study group. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Article Snippet: The sections were blocked in 5% goat serum (SL038; Solarbio) at 37°C for 1 h and were then incubated overnight with anti-claudin-1 (1:100; YT0942; Immunoway, TX, United States), anti-claudin-3 (1:100; 34–1700; Invitrogen, Shanghai, China), anti-cleaved Notch-1 (1:100; YC0067; N1ICD, Immunoway), and anti-Hes1 (1:200; GTX108356; GeneTex, Alton, IL, United States) antibodies in a humid environment at 4°C.

Techniques: Inhibition, Control, Concentration Assay, Staining

Journal: Frontiers in Pharmacology

Article Title: Vitamin D/vitamin D receptor protects intestinal barrier against colitis by positively regulating Notch pathway

doi: 10.3389/fphar.2024.1421577

Figure Lengend Snippet: Notch pathway is inhibited in the colon under VDd or VDR KO conditions. (A, B) Representative colonic IF staining image and quantification of N1ICD ( red ) and Hes1 ( red ) in VDR KO (A) and VDd (B) mice (scale bar = 25 μm). (C, D) Real-time PCR analysis of the gene expression of Notch ligands, receptors, and effectors in the colon in VDR KO (C) and VDd (D) mice. Expression levels (VDR KO vs. WT or VDd vs. CT) were compared using paired t -test. Data are presented as the mean ± SEM from 6-8 mice in each study group. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Article Snippet: The sections were blocked in 5% goat serum (SL038; Solarbio) at 37°C for 1 h and were then incubated overnight with anti-claudin-1 (1:100; YT0942; Immunoway, TX, United States), anti-claudin-3 (1:100; 34–1700; Invitrogen, Shanghai, China), anti-cleaved Notch-1 (1:100; YC0067; N1ICD, Immunoway), and anti-Hes1 (1:200; GTX108356; GeneTex, Alton, IL, United States) antibodies in a humid environment at 4°C.

Techniques: Staining, Real-time Polymerase Chain Reaction, Expressing

Journal: Frontiers in Pharmacology

Article Title: Vitamin D/vitamin D receptor protects intestinal barrier against colitis by positively regulating Notch pathway

doi: 10.3389/fphar.2024.1421577

Figure Lengend Snippet: VD/VDR modulates the Notch pathway in colitis. (A, B) Representative colonic IF staining image and quantification of N1ICD ( red ) and Hes1 ( red ) in VDd-DSS and respective control colitis mice (scale bar = 25 μm). (C, D) Representative colonic IF staining image and quantification of N1ICD ( red ) and Hes1 ( red ) in VDR KO-DSS and respective control colitis mice. To activate VDR, mice were administered paricalcitol (PAR) 7 days before drinking DSS water and were sustained until sacrifice. (E) DAI scores of each group. DAI scores were compared by the repeated measures ANOVA with Bonferroni post hoc multiple comparisons. (F) Histological scores of distal colons in each group. Statistical analysis was performed using ANOVA with Tukey’s post-test. (G) Representative colonic H&E staining image of each group (scale bar = 50 μm). (H) Representative colonic IF staining image of claudin-1 ( green ), claudin-3 ( green ), and Hes1 ( red ) (scale bar = 25 μm). Data are presented as the mean ± SEM from 6-8 mice in each study group. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Article Snippet: The sections were blocked in 5% goat serum (SL038; Solarbio) at 37°C for 1 h and were then incubated overnight with anti-claudin-1 (1:100; YT0942; Immunoway, TX, United States), anti-claudin-3 (1:100; 34–1700; Invitrogen, Shanghai, China), anti-cleaved Notch-1 (1:100; YC0067; N1ICD, Immunoway), and anti-Hes1 (1:200; GTX108356; GeneTex, Alton, IL, United States) antibodies in a humid environment at 4°C.

Techniques: Staining, Control

Journal: Frontiers in Pharmacology

Article Title: Vitamin D/vitamin D receptor protects intestinal barrier against colitis by positively regulating Notch pathway

doi: 10.3389/fphar.2024.1421577

Figure Lengend Snippet: VD/VDR signaling regulates the Notch pathway and TJs in Caco-2 cells. TNF-α (100 ng/mL) was added to mimic inflammatory stimulation, and 100 nM PAR was used to activate VDR; the solvate was added as control (CT). Specific siRNA of VDR (SiVDR) was used to downregulate VDR, negative control (NC) sequence was used as control (scale bar = 10 μm). (A) Representative IF staining image of claudin-1 ( green ) and claudin-3 ( green ) in Caco-2 cells treated with or without PAR before TNF-α intervention. (B) Representative IF staining image of N1ICD ( red ) and Hes1 ( red ) in Caco-2 cells with or without VDR downregulation before TNF-α intervention. (C) Representative IF staining image of claudin-1 ( green ), claudin-3 ( green ), N1ICD ( red ), and Hes1 ( red ) in Caco-2 cells after VDR downregulation.

Article Snippet: The sections were blocked in 5% goat serum (SL038; Solarbio) at 37°C for 1 h and were then incubated overnight with anti-claudin-1 (1:100; YT0942; Immunoway, TX, United States), anti-claudin-3 (1:100; 34–1700; Invitrogen, Shanghai, China), anti-cleaved Notch-1 (1:100; YC0067; N1ICD, Immunoway), and anti-Hes1 (1:200; GTX108356; GeneTex, Alton, IL, United States) antibodies in a humid environment at 4°C.

Techniques: Control, Negative Control, Sequencing, Staining

Journal: Frontiers in Pharmacology

Article Title: Vitamin D/vitamin D receptor protects intestinal barrier against colitis by positively regulating Notch pathway

doi: 10.3389/fphar.2024.1421577

Figure Lengend Snippet: VD/VDR-mediated TJ protection is partly dependent on the Notch pathway. LY411,575 (1 μM) was used to inhibit the Notch pathway in Caco-2 cells, and 100 nM PAR was used to activate VDR. The solvate was added as control (CT). Representative IF staining image of N1ICD ( red ), Hes1 ( red ), claudin-1 ( green ), and claudin-3 ( green ) in Caco-2 cells treated with or without LY411,575 before PAR intervention (scale bar = 10 μm).

Article Snippet: The sections were blocked in 5% goat serum (SL038; Solarbio) at 37°C for 1 h and were then incubated overnight with anti-claudin-1 (1:100; YT0942; Immunoway, TX, United States), anti-claudin-3 (1:100; 34–1700; Invitrogen, Shanghai, China), anti-cleaved Notch-1 (1:100; YC0067; N1ICD, Immunoway), and anti-Hes1 (1:200; GTX108356; GeneTex, Alton, IL, United States) antibodies in a humid environment at 4°C.

Techniques: Control, Staining

Journal: Frontiers in Pharmacology

Article Title: Vitamin D/vitamin D receptor protects intestinal barrier against colitis by positively regulating Notch pathway

doi: 10.3389/fphar.2024.1421577

Figure Lengend Snippet: VD/VDR signaling positively regulates the Notch-1/Hes1 pathway. (A) Representative IF staining image of claudin-1 ( green ), claudin-3 ( green ), N1ICD ( red ), and Hes1 ( red ) in cultured intestinal organoids (scale bar = 10 μm). (B) Western blot analysis of N1ICD and Hes1 proteins in cultured intestinal organoids from WT and VDR KO mice. (C) Real-time PCR analysis of the expression of Notch ligands, receptors, and Hes1 in cultured intestinal organoids from WT and VDR KO mice. (D) Real-time PCR analysis of the expression of Notch ligands, receptors, and Hes1 in Caco-2 cells after TNF-α intervention. (E, F) Real-time PCR analysis of the expression of Notch ligands, receptors, and Hes1 in Caco-2 cells after downregulation of VDR with siVDR (E) or activation of VDR with PAR (F) . Data are presented as the mean ± SEM from three separate experiments, the statistical analysis performed with paired t -test. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Article Snippet: The sections were blocked in 5% goat serum (SL038; Solarbio) at 37°C for 1 h and were then incubated overnight with anti-claudin-1 (1:100; YT0942; Immunoway, TX, United States), anti-claudin-3 (1:100; 34–1700; Invitrogen, Shanghai, China), anti-cleaved Notch-1 (1:100; YC0067; N1ICD, Immunoway), and anti-Hes1 (1:200; GTX108356; GeneTex, Alton, IL, United States) antibodies in a humid environment at 4°C.

Techniques: Staining, Cell Culture, Western Blot, Real-time Polymerase Chain Reaction, Expressing, Activation Assay

Journal: Frontiers in Pharmacology

Article Title: Vitamin D/vitamin D receptor protects intestinal barrier against colitis by positively regulating Notch pathway

doi: 10.3389/fphar.2024.1421577

Figure Lengend Snippet: VD/VDR positively regulates Notch-1 transcription. (A) Western blot analysis of VDR, Notch-1, N1ICD, and Hes1 in Caco-2 cells after siVDR and PAR intervention. (B) Graphs of the predicted VDR binding region in the Notch-1 promoter. (C) Dual-luciferase reporter assay of VDR to Notch-1 promoter. Notch-1 promoter (Notch-1) and negative control (CON) were constructed in a GV238 vector. The VDR cDNA and vector (pcDNA3.1) were linked to upregulate VDR (pVDR). Data are presented as the mean ± SEM from three separate experiments. Statistical analysis was performed using ANOVA with Tukey’s post-test. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Article Snippet: The sections were blocked in 5% goat serum (SL038; Solarbio) at 37°C for 1 h and were then incubated overnight with anti-claudin-1 (1:100; YT0942; Immunoway, TX, United States), anti-claudin-3 (1:100; 34–1700; Invitrogen, Shanghai, China), anti-cleaved Notch-1 (1:100; YC0067; N1ICD, Immunoway), and anti-Hes1 (1:200; GTX108356; GeneTex, Alton, IL, United States) antibodies in a humid environment at 4°C.

Techniques: Western Blot, Binding Assay, Luciferase, Reporter Assay, Negative Control, Construct, Plasmid Preparation