Journal: Atherosclerosis

Article Title: IL-22 affects smooth muscle cell phenotype and plaque formation in apolipoprotein E knockout mice.

doi: 10.1016/j.atherosclerosis.2015.08.006

Figure Lengend Snippet: Fig. 1. Expression of the IL-22 receptor in the vascular wall. Sections from the aortic root of 12 week old (A) and 24 week old (B) Apoe/ mice were stained for the IL-22RA1 subunit of the IL-22 receptor and a-actin. Positive staining is shown in brown (DAB). (C) Carotid arteries from wild type C57Bl6 mice, with our without vascular injury were stained for IL- 22RA1 or myosin heavy chain (MyHC11) using immunofluorescence. IL-22RA1 and MyHC11 are shown in red, DAPI in blue, and elastic membranes in green autofluoresence. Exclusion of primary antibody served as negative control. Scale bars represent 100 mm.

Article Snippet: For analysis of aortic root plaques, frozen tissue sections were cut from the aortic root, frozen and stained using the following primary antibodies: rat anti-mouse IL-22RA1 antibody (R&D systems, Abingdon, UK), rabbit anti-mouse SMC a-actin antibody (Abcam, Cambridge, UK), rat anti-mouse MOMA-2 antibody (BMA Biomedicals, Augst, Switzerland), rabbit-anti-mouse Caldesmon (Abcam), rabbit-anti-mouse CD68 (Abcam).

Techniques: Expressing, Staining, Negative Control

Journal: NPJ Regenerative medicine

Article Title: Aryl hydrocarbon receptor regulates IL-22 receptor expression on thymic epithelial cell and accelerates thymus regeneration.

doi: 10.1038/s41536-023-00339-7

Figure Lengend Snippet: Fig. 2 Knockout of AHR in TECs decreased IL-22RA1 expression. a B6-Ahrfl/flmice and B6-Ahrfl/fl;Foxn1-cre mice, pretreated by 5.5 Gy TBI, were intraperitoneally injected with vehicle or rmIL-22 (200 ng) thrice weekly for 2 weeks. At day 14, thymus cells were counted as described (n = 5). b, c Thymii were collected from irradiated mice. qPCR was performed to detect mRNA levels of IL-22 and IL-22RA1 (n = 4). The values of control groups were set as 1, and the values of other groups were 2–ΔΔCT relative to controls. d Thymic stromal cells were isolated from irradiated mice (n = 3). Western blot was performed with indicated antibodies. Band intensity was analyzed using ImageJ. e Cryosections of thymii were stained by immunofluorescence with anti-IL-22RA1 (n = 4). Fluorescence intensity was analyzed using ImageJ. Scale bar: 50 μm. Data are mean ± SD, compared using one-way ANOVA test or Student’s t test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s., not significant.

Article Snippet: IL-22RA1 (13462-1- AP), AHR (67785-1-Ig) and β-actin (20536-1-AP) were from Proteintech (Carlsbad, CA, USA). β-actin antibody was used at the dilution of 1:2000, and the other antibodies were used at 1:1000.

Techniques: Knock-Out, Expressing, Injection, Irradiation, Control, Isolation, Western Blot, Staining, Fluorescence

Journal: NPJ Regenerative medicine

Article Title: Aryl hydrocarbon receptor regulates IL-22 receptor expression on thymic epithelial cell and accelerates thymus regeneration.

doi: 10.1038/s41536-023-00339-7

Figure Lengend Snippet: Fig. 3 Targeting AHR had an impact on IL-22RA1 expression in mTEC1 cells. a CH-223191 was used to treat mTEC1 cells for 24 h. b, c mTEC1 cells, stably transduced with control shRNA or Ahr-shRNA lentivirus, were treated with vehicle or FICZ (100 nM) for 24 h. d Total cell proteins were extracted from the mTEC1 cells as described above. Western blot was performed with indicated antibodies (n = 3). e FICZ was used to treat mTEC1 cells for 24 h. qPCR was performed to detect mRNA levels in cells as described (n = 5). f mTEC1 cells were treated by vehicle, CH-223191 (10 μM) or FICZ (100 nM) for 24 h. ChIP assay was performed with anti-AHR. The precipitated DNA was detected by qPCR with primers of mouse IL-22RA1 gene promotor (n = 5). g mTEC1 cells stably transduced with control shRNA or Ahr-shRNA lentivirus, h mTEC1 cells stably transduced with control or Ahr-overexpression lentivirus, i mTEC1 cells, without genetic modification, were treated by vehicle, CH-223191 (10 μM) or FICZ (100 nM) for 24 h. g, h, i These cells were transfected with pGL4.10 plasmids (firefly, following mouse IL-22RA1 gene promoter sequence) and pRL-TK plasmids (renilla, normalizer) for 12 h. Luciferase activity was measured (n = 5). Data are mean ± SD, compared using one-way ANOVA test or Student’s t test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s., not significant.

Article Snippet: IL-22RA1 (13462-1- AP), AHR (67785-1-Ig) and β-actin (20536-1-AP) were from Proteintech (Carlsbad, CA, USA). β-actin antibody was used at the dilution of 1:2000, and the other antibodies were used at 1:1000.

Techniques: Expressing, Stable Transfection, Transduction, Control, shRNA, Western Blot, Over Expression, Transfection, Sequencing, Luciferase, Activity Assay

Journal: NPJ Regenerative medicine

Article Title: Aryl hydrocarbon receptor regulates IL-22 receptor expression on thymic epithelial cell and accelerates thymus regeneration.

doi: 10.1038/s41536-023-00339-7

Figure Lengend Snippet: Fig. 4 AHR transcriptionally increased IL-22RA1 expression in mTEC1 cells. a mTEC1 cells were treated by vehicle or colivelin (1 μM) for 24 h. Immunoprecipitation was performed with anti-STAT3 or anti-AHR (n = 3). Western blot was used to detect proteins in the precipitate. b mTEC1 cells were treated by vehicle, stattic (1 μM) or CH-223191 (10 μM) for 24 h. ChIP assay was performed with anti-STAT3 as described (n = 5). c, d mTEC1 cells were treated by vehicle, stattic or colivelin for 24 h with/without FICZ (100 nM). qPCR was performed to detect mRNA levels in cells as described (n = 5). e mTEC1 cells were treated by colivelin and/or FICZ for 24 h (n = 5). Luciferase reporter assay was performed as described. f, g Total proteins were extracted from thymic stromal cells isolated from irradiated wild type mice which were treated by vehicle or FICZ (n = 3). Whole-cell proteins were extracted from mTEC1 cells treated by vehicle, FICZ (100 nM) or colivelin (1 μM) for 24 h (n = 3). Western blot was used to detect proteins as indicated. h Wild type C57BL/6 mice, pretreated by 5.5 Gy TBI, were intraperitoneally injected with vehicle, FICZ (0.1 mg/kg) or combined FICZ (0.1 mg/kg) and stattic (10 mg/kg) thrice weekly for 2 weeks. At day 14, thymus cells were counted as described (n = 5). i mTEC1 cells were treated by vehicle or rmIL-22 (20 ng/ml) in the presence of different doses of CH-223191 for 24 h (n = 5). Cell viability was detected by CCK-8 assay. Data are mean ± SD, compared using one-way ANOVA test or Student’s t test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s., not significant.

Article Snippet: IL-22RA1 (13462-1- AP), AHR (67785-1-Ig) and β-actin (20536-1-AP) were from Proteintech (Carlsbad, CA, USA). β-actin antibody was used at the dilution of 1:2000, and the other antibodies were used at 1:1000.

Techniques: Expressing, Immunoprecipitation, Western Blot, Luciferase, Reporter Assay, Isolation, Irradiation, Injection, CCK-8 Assay

Journal: NPJ Regenerative medicine

Article Title: Aryl hydrocarbon receptor regulates IL-22 receptor expression on thymic epithelial cell and accelerates thymus regeneration.

doi: 10.1038/s41536-023-00339-7

Figure Lengend Snippet: Fig. 5 TECs-specific knockout of IL-22RA1 blocked thymus regeneration. a, b Thymic stromal cells were isolated from wild-type C57BL/6 mice treated by 5.5 Gy TBI. Day 0 indicates the samples were collected right before TBI. a qPCR was performed to detect mRNA levels (n = 5). The values of Day 0 were set as 1. b Western blot was performed with indicated antibodies. c, d B6-Il22ra1fl/flmice and B6-Il22ra1fl/fl;Foxn1-cre mice, pretreated by 5.5 Gy TBI, were intraperitoneally injected with vehicle, rmIL-22 (200 ng) or FICZ (0.1 mg/kg) thrice weekly for 2 weeks. At day 14, c thymus cells were counted as described (n = 5). d H&E staining was performed on slides of thymus. Scale bar: 500 μm. Data are mean ± SD, compared using one-way ANOVA test or Student’s t test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s., not significant.

Article Snippet: IL-22RA1 (13462-1- AP), AHR (67785-1-Ig) and β-actin (20536-1-AP) were from Proteintech (Carlsbad, CA, USA). β-actin antibody was used at the dilution of 1:2000, and the other antibodies were used at 1:1000.

Techniques: Knock-Out, Isolation, Western Blot, Injection, Staining

Journal: NPJ Regenerative medicine

Article Title: Aryl hydrocarbon receptor regulates IL-22 receptor expression on thymic epithelial cell and accelerates thymus regeneration.

doi: 10.1038/s41536-023-00339-7

Figure Lengend Snippet: Fig. 6 Targeting AHR or IL-22RA1 had an impact on thymus regeneration, T-cell reconstitution and pathogenesis of murine cGVHD following allo-HCT. In the cGVHD models, BALB/c mice were used as donors. Wild type C57BL/6 (a), B6-Il22ra1fl/fland B6-Il22ra1fl/fl;Foxn1-cre mice (b) were recipients. Wild type C57BL/6 recipients were treated by vehicle or FICZ thrice weekly for 2 weeks. a, b Survival (n = 15 in each group) and cGVHD score (Day 60) of recipient mice were monitored. c Histological analyses (Day 90): H&E staining, Masson staining and collagen immunofluorescence staining (n = 4). Ashcroft score was determined by inflammation and fibrosis. Area of collagenous and fluorescence intensity was analyzed using ImageJ. Scale bar: 50 μm. d, e At day 90, thymus cells were counted as described (n = 7 or 5). f At day 90, blood samples were collected and flow cytometry was used to analyze T-cell subpopulations (n = 7 or 5). g At day 90, T cells were isolated from spleen cells of recipients. Lymphocytes were isolated from spleen cells of normal C57BL/6 mice. The isolated recipients’ T cells were cultured alone (control) or co-cultured (MLR) with normal lymphocytes (1:1) for 72 h. Cell viability was detected by CCK-8 assay (n = 7 or 5). Survival is compared using log-rank test. Data are mean ± SD, compared using one-way ANOVA test or Student’s t test. *, p < 0.05; **, p < 0.01; n.s., not significant.

Article Snippet: IL-22RA1 (13462-1- AP), AHR (67785-1-Ig) and β-actin (20536-1-AP) were from Proteintech (Carlsbad, CA, USA). β-actin antibody was used at the dilution of 1:2000, and the other antibodies were used at 1:1000.

Techniques: Staining, Cytometry, Isolation, Cell Culture, Control, CCK-8 Assay