Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Circadian Clock Disruption Suppresses PDL1 + Intraepithelial B Cells in Experimental Colitis and Colitis-Associated Colorectal Cancer

doi: 10.1016/j.jcmgh.2021.02.008

Figure Lengend Snippet: Cell death of activated CD4 + T cells in DSS-treated Bmal1 -/- mice and the effects of PDL1 antagonist antibody. ( A ) Fluorescence-activated cell sorter (FACS) graphs and ( B ) proportions of CD8 + and ( C ) CD4 + T cells in the colon of DSS-treated Bmal1 -/- mice and WT mice. ( D ) Protein expression of Bmal1, activated caspase 3, Bcl-2 Associated X Protein, and Bcl2 in CD8 + T cells and ( E ) CD4 + T cells in the colons of DSS-treated Bmal1 -/- mice and WT mice. ( F ) FACS graphs of CD4 + Ki67 + cells, ( G ) proportions of CD4 + T cells, ( H ) CD4 + Ki67 + cells, and ( I ) CD8 + T cells in the IELs of Bmal1 -/- mice, Bmal1 -/- mice receiving B cells from WT mice, Bmal1 -/- mice receiving B cells from WT mice and PDL1 antagonist antibodies. ( J ) FACS graphs and proportion of ( K ) Annexin-v + Comp-7-AAD - B220 + or ( L ) Annexin-v + Comp-7-AAD + B220 + cells, ( M ) Annexin-v + Comp-7-AAD - CD4 + or ( N ) Annexin-v + Comp-7-AAD + CD4 + cells, and ( O ) Annexin-v + Comp-7-AAD - CD8 + or ( P ) Annexin-v + Comp-7-AAD + CD8 + cells in IELs from DSS-treated WT mice and Bmal1 -/- mice. ( Q ) FACS graphs and ( U ) proportions of CD4 + IL17 + cells in IELs. ( R ) FACS graphs and ( V ) proportions of CD4 + Foxp3 + cells in IELs. ( S ) Transcription expression of Bmal1, IL17, RORγt, IL21, and IL6 in IELs of DSS-treated WT and Bmal1 -/- mice. ( T ) Transcription expression of Forkhead Box P3, IL2, IL10, TGFβ, and IFNγ in IELs of DSS-treated WT and Bmal1 -/- mice. ∗ P < .05. APC, Allophycocyanin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: For others, 5 ng/mL TGFβ (7666-MB; RD) was added in DMEM medium for 72 hours, 50 ng/mL IL21 (210-21; Peprotech) for 72 hours, 30 ng/mL anti-IL-33 neutralizing antibody (AF3626; RD) for 48 hours, and 10 ng/mL IL33 (3626-ML/CF; RD) for an hour, respectively.

Techniques: Fluorescence, Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Circadian Clock Disruption Suppresses PDL1 + Intraepithelial B Cells in Experimental Colitis and Colitis-Associated Colorectal Cancer

doi: 10.1016/j.jcmgh.2021.02.008

Figure Lengend Snippet: The effects of IL33 in intestinal microenvironment on PDL1 + Breg cells in DSS-treated Bmal1 -/- mice. ( A ) Transcription levels of IL4, IL6, IL10, IL21, IL33, TGFβ, and MCP-1 in the IELs of DSS-treated and untreated Bmal1 -/- and WT mice among 24 hours. The P value was estimated by JTK cycle analysis. ( B ) Transcription levels of inflammatory cytokines IL4, IL6, IL10, IL21, IL33, TGFβ, and MCP-1 in the IELs of DSS-treated Bmal1 -/- and WT mice at the same time. ( C ) Protein expressions of Bmal1 and IL33, ( D ) proportion of B220 + IL33 + cells in IELs of DSS-treated Bmal1 -/- and WT mice. ( E ) Gross appearance of colorectum, ( F ) body weight during DSS induction, ( G ) colorectal length, ( H ) histologic score of colon, ( I ) H&E of colon from DSS-treated WT mice, WT mice with IL33 antagonist antibody, Bmal1 -/- mice, and Bmal1 -/- mice with IL33 antagonist antibody. ( J ) FACS graphs and ( K ) proportion of CD5 + CD1d + cells from B220 + cells in IELs. ( L ) Fluorescence-activated cell sorter (FACS) graphs and ( M ) proportion of B220 + PDL1 + , ( N ) CD1d + PDL1 + , and ( O ) CD5 + PDL1 + cells in IELs. ( P ) PDL1 expressions in B cells isolated from WT and Bmal1 -/- spleen treated with IL21, TGFβ, or IL33 neutralizing antibodies by Western blot. ( Q ) PDL1 expression in B cells isolated from WT and Bmal1 -/- spleen treated with IL33 by Western blot. ( R ) The effects of Bmal1 and clock complexes on luciferase activities of IL33 promoter reporter with or without E-box site mutation. ( S ) ChIP assay to detect the binding of IL33 gene promoter with Bmal1 in SPLs and B cells. ∗ P < .05. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: For others, 5 ng/mL TGFβ (7666-MB; RD) was added in DMEM medium for 72 hours, 50 ng/mL IL21 (210-21; Peprotech) for 72 hours, 30 ng/mL anti-IL-33 neutralizing antibody (AF3626; RD) for 48 hours, and 10 ng/mL IL33 (3626-ML/CF; RD) for an hour, respectively.

Techniques: Fluorescence, Isolation, Western Blot, Expressing, Luciferase, Mutagenesis, Binding Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Circadian Clock Disruption Suppresses PDL1 + Intraepithelial B Cells in Experimental Colitis and Colitis-Associated Colorectal Cancer

doi: 10.1016/j.jcmgh.2021.02.008

Figure Lengend Snippet: Primers Used for qRT-PCR, ChIP, and Plasmid Construction

Article Snippet: For others, 5 ng/mL TGFβ (7666-MB; RD) was added in DMEM medium for 72 hours, 50 ng/mL IL21 (210-21; Peprotech) for 72 hours, 30 ng/mL anti-IL-33 neutralizing antibody (AF3626; RD) for 48 hours, and 10 ng/mL IL33 (3626-ML/CF; RD) for an hour, respectively.

Techniques: Plasmid Preparation, Expressing