Journal: F1000Research

Article Title: An assay to measure poly(ADP ribose) glycohydrolase (PARG) activity in cells

doi: 10.12688/f1000research.8463.2

Figure Lengend Snippet: Using a high content imaging system the Hoechst stained nuclei (left-hand panels) are used to create a nuclear mask (green circle in Analysis). Anti-PAR antibody (FITC) detects the increase in PAR chains (centre panels) that is then quantified using the nuclear mask from the Hoechst signal (right-hand panels). Fluorescence intensity is shown as red dots within the nuclear mask.

Article Snippet: Cells were then permeabilized using PBS/Triton 0.1% for 20 min, and washed once in PBS before adding anti-PAR antibody (10H (#AM80), Merck Millipore) at 1:4000 in antibody blocking buffer (ADB; 5% Fetal bovine serum, 0.1% Tween20 in PBS) and incubated overnight at 4°C.

Techniques: Imaging, Staining, Fluorescence

Journal:

Article Title: Synthetic Retinoid AM80 Inhibits Th17 Cells and Ameliorates Experimental Autoimmune Encephalomyelitis

doi: 10.2353/ajpath.2009.081084

Figure Lengend Snippet: AM80 is a potent inhibitor for Th17 cell differentiation in vitro. Whole splenocytes were stimulated with soluble anti-CD3 with retinoids added at a range of concentrations. A: Cells were cultured in the presence of IL-23 (broken line), TGF-β and IL-6 (solid line), or without the addition of cytokines (closed triangle) for 3 days and IL-17 production assessed by enzyme-linked immunosorbent assay (ELISA). B: Intracellular cytokine staining of IL-17 and IFN-γ among TcR+CD4+ cells with or without 10 nmol/L retinoid treatment assessed after 3 days of culture. Data depicted in A and B are representative of three independent experiments. In C, CD4+CD44−CD25−CD62Lhigh naïve T cells were stimulated under Th17-priming conditions with retinoids at a range of concentrations for 96 hours. Cells were rested for 48 hours before restimulation and IL-17 production was measured in culture supernatants after further 96 hours of stimulation. *P < 0.001. RNA from these cells was analyzed by quantitative RT-PCR for the transcription factors RORγt and Foxp3 (D). Data depicted in C and D are representative of four similar experiments.

Article Snippet: Some groups of mice also received 3 mg/kg AM80 in 0.5% carboxymethylcellulose (CMC) solution (WAKO Chemicals, Osaka, Japan) by oral gavage.

Techniques: Cell Differentiation, In Vitro, Cell Culture, Enzyme-linked Immunosorbent Assay, Staining, Quantitative RT-PCR

Journal:

Article Title: Synthetic Retinoid AM80 Inhibits Th17 Cells and Ameliorates Experimental Autoimmune Encephalomyelitis

doi: 10.2353/ajpath.2009.081084

Figure Lengend Snippet: AM80 treatment ameliorates EAE with reduction of IL-17 production in vivo. EAE was induced in B6 mice by immunization with MOG35–55. Groups of mice received either CMC or 3 mg/kg AM80 in CMC orally every other day from day 0. Mice were scored daily for clinical disease (A) analysis of variance for repeated measures shows significant differences from day 11 to 18 (*P < 0.001). On day 15 after immunization, groups of mice were sacrificed and spinal cords were examined histologically. Representative sections are shown in B. H&E staining (upper panels), Luxol fast blue staining (lower panels). Scale bar = 200 μm. Leukocytes and T cells were purified from the CNS at day 15 after immunization and cell numbers evaluated by hemocytometer (C). Quantitative RT-PCR was used to measure an array of transcription factors and cytokines (D, upper row) in which error bars represent duplicated PCR of the same samples. In the lower row, CNS-infiltrating T cells were restimulated with immobilized anti-CD3 antibody and supernatants were measured after 72 hours for the presence of a range of cytokines and chemokines using ELISA or CBA. Error bars represent measurements from duplicate wells. Data are representative of at least two independent experiments.

Article Snippet: Some groups of mice also received 3 mg/kg AM80 in 0.5% carboxymethylcellulose (CMC) solution (WAKO Chemicals, Osaka, Japan) by oral gavage.

Techniques: In Vivo, Staining, Purification, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Synthetic Retinoid AM80 Inhibits Th17 Cells and Ameliorates Experimental Autoimmune Encephalomyelitis

doi: 10.2353/ajpath.2009.081084

Figure Lengend Snippet: RAR agonist AM80 suppresses Th17 differentiation without inhibiting typical proliferative responses in vivo. B6 mice were immunized with MOG35–55 in CFA and vehicle (CMC) or AM80 (3 mg/kg in CMC) were administered orally every other day starting from day 0 until day 8. Draining lymph node cells were isolated at day 10 and restimulated with MOG peptide at various concentrations. After 72 hours, antigen-specific IL-17 production was assessed by ELISA (A). In B, TNF- α and IFN-γ production after restimulation with 100 μmol/L MOG were assessed by CBA. C: Intracellular cytokine staining of draining lymph node cells in the presence of 10 μmol/L MOG35–55 after 96 hours of culture shows reduced percentages of IL-17+ and IL-17+/IFN-γ+ double producing cells. Plots are gated on TCR+CD4+ lymphocytes. D: Expression of RORγt in T cells obtained from C was assessed by quantitative RT-PCR. E: Cell proliferation was assessed either by [3H]thymidine incorporation or cell number evaluation by fluorescence-activated cell sorting. Data in A, B, and E are representative of four independent experiments with three to five mice per group and C and D depict results from two experiments with three mice per group.

Article Snippet: Some groups of mice also received 3 mg/kg AM80 in 0.5% carboxymethylcellulose (CMC) solution (WAKO Chemicals, Osaka, Japan) by oral gavage.

Techniques: In Vivo, Isolation, Enzyme-linked Immunosorbent Assay, Staining, Expressing, Quantitative RT-PCR, Fluorescence, FACS

Journal:

Article Title: Synthetic Retinoid AM80 Inhibits Th17 Cells and Ameliorates Experimental Autoimmune Encephalomyelitis

doi: 10.2353/ajpath.2009.081084

Figure Lengend Snippet: AM80 suppresses IL-17 production by differentiated Th17 cells and ameliorates EAE in therapeutic intervention. A: CD4+CD44+CD25−CD62Llow memory T cells were stimulated by plate-bound anti-CD3 antibody for 4 days in the presence of IL-23. Dose-dependent decrease of IL-17 production by RAR agonists was shown in left panel. Intracellular cytokine staining of TCR+CD4+ cells shows decreased percentage of IL-17+ memory T cells after treatment with 100 nmol/L AM80 (B). Graphs are representative of two independent experiments. C: CNS-infiltrating T cells from mice with severe EAE (score 3–4) were isolated and restimulated with immobilized anti-CD3 in the presence of RAR agonists (100 nmol/L) for 3 days. Supernatants were analyzed for IL-17 production by ELISA and cells were subjected to quantitative RT-PCR for RORγt expression. Data are a representative of four similar experiments. D: Clinical EAE scores of animals treated daily from day 13 on (arrow) either with vehicle (CMC) or AM80. Displayed scores are representative of two experiments with n = 6.

Article Snippet: Some groups of mice also received 3 mg/kg AM80 in 0.5% carboxymethylcellulose (CMC) solution (WAKO Chemicals, Osaka, Japan) by oral gavage.

Techniques: Staining, Isolation, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing

Journal:

Article Title: Synthetic Retinoid AM80 Inhibits Th17 Cells and Ameliorates Experimental Autoimmune Encephalomyelitis

doi: 10.2353/ajpath.2009.081084

Figure Lengend Snippet: Continuous AM80 treatment is less effective on EAE suppression with a differentially modulated cytokine profile. A: The H&E-stained sections and the Luxol fast blue-stained sections in EAE mice treated with or without AM80 are shown. The control animal (score 2) and AM80-treated animal (score 2) at day 25 were subjected to histological examination. Scale bar = 200 μm. B: Total leukocyte numbers and isolated T cell numbers from spinal cords were evaluated in animals that had received CMC or AM80 at day 25 after immunization. The upper row of C depicts purified T cells from B that were subjected to quantitative RT-PCR. Error bars represent duplicated PCR of the same samples. In the lower row of C, CNS-infiltrating T cells were restimulated with immobilized anti-CD3 mAb and cytokine or chemokine production in culture supernatants were assessed by ELISA or CBA after 72 hours. Error bars represent measurements from duplicate wells (*P < 0.001). D: IL-10 production by stimulated CNS-infiltrating T cells derived from CMC-treated or AM80-treated animals were assessed by either quantitative RT-PCR or CBA. CNS-infiltrating T cells were isolated at day 15 or day 25 after EAE induction. B is a representative of four independent experiments; A, C, and D of two with six animals per group.

Article Snippet: Some groups of mice also received 3 mg/kg AM80 in 0.5% carboxymethylcellulose (CMC) solution (WAKO Chemicals, Osaka, Japan) by oral gavage.

Techniques: Staining, Isolation, Purification, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal:

Article Title: Synthetic Retinoid AM80 Inhibits Th17 Cells and Ameliorates Experimental Autoimmune Encephalomyelitis

doi: 10.2353/ajpath.2009.081084

Figure Lengend Snippet: Continuous AM80 treatment impairs IL-10 production by T cells, which are identified as RORγt+/FOXP3+ population. A: Naïve T cells were cultured under neutral (no priming) or Th17-priming conditions in the presence of retinoids (100 nmol/L). After 96 hours, expression of IL-10 mRNA was assessed by quantitative RT-PCR. For further analysis, cells were rested for 48 and restimulated with anti-CD3 antibody for another 96 hours and examined for expression of IL-10 transcript. B: Naive T cells stimulated under Th17 priming conditions without RAR agonists were then restimulated as described above. RAR agonists (100 nmol/L) were added during the secondary stimulation and assessed for their expression of IL-10 mRNA by quantitative RT-PCR. Results are representative of two independent experiments. C: CNS-infiltrating T cells were isolated from EAE animals (score 3) and restimulated with immobilized anti-CD3 mAb in the presence of AM80 or ATRA (100 nmol/L) for 72 hours. The amount of IL-10 in culture supernatants was examined by ELISA. D: Whole splenocytes were cultured for 96 hours with TGF-β and IL-6 and RAR agonists (10 nmol/L) and CBA analysis were performed for analyzing the levels of IL-10 production in the culture supernatants. Cells were then subjected to intracellular cytokine staining by fluorescence-activated cell sorting. The histogram gated on CD45highTCR+CD4+ lymphocytes displays the comparative population of IL-10+ cells by Foxp3+ (black line) and Foxp3− (gray line) cell populations. Broken line represents the data stained with isotype control antibody. E: CD45highTCR+CD4+ lymphocytes derived from D were analyzed for their expression of RORγt and Foxp3. Comparative dot plots were shown with or without AM80 treatment (10 nmol/L). Addition of AM80 decreased the number of RORγt+ cells, including the RORγt+FOXP3+ cells. F: The levels of IL-10 producing cells from the quadrants labeled in E are shown.

Article Snippet: Some groups of mice also received 3 mg/kg AM80 in 0.5% carboxymethylcellulose (CMC) solution (WAKO Chemicals, Osaka, Japan) by oral gavage.

Techniques: Cell Culture, Expressing, Quantitative RT-PCR, Isolation, Enzyme-linked Immunosorbent Assay, Staining, Fluorescence, FACS, Derivative Assay, Labeling

Journal: Scientific Reports

Article Title: A synthetic retinoic acid receptor agonist Am80 ameliorates renal fibrosis via inducing the production of alpha-1-acid glycoprotein

doi: 10.1038/s41598-020-68337-z

Figure Lengend Snippet: Am80 suppresses renal fibrosis and inflammation in UUO mice at a level similar to that for atRA. ( a ) atRA or Am80 were administered i.p. in corn oil at a dose of 20 mg/kg, each day (from day 0 to day 6) after the UUO treatment. ( b ) Treatment of Am80 or atRA reduced the mRNA expression of α-SMA and Col1a2 at day 7. ( c ) a-SMA was immunostained and the accumulation of collagen in kidney was detected by picrosirius red staining. Scale bar, 100 μm. ( d ) Treatment of Am80 or atRA reduced the mRNA expression of IL-6 and IL-1β at day 7 ( e ) The side effect of body weight loss was observed in atRA-treated UUO mice, but not in the Am80 treated UUO mice. *P < 0.05 compared with control; # P < 0.05 compared with corn oil; † P < 0.05 compared with atRA (n = 6 for control group; n = 10 for each UUO group). Data are presented as the mean ± SE.

Article Snippet: After the UUO treatment, atRA (Wako, 20 mg/kg) or Am80 (Nippon Shinyaku, Kyoto, Japan, 20 mg/kg) was intraperitoneally administered each day.

Techniques: Expressing, Staining, Control

Journal: Scientific Reports

Article Title: A synthetic retinoic acid receptor agonist Am80 ameliorates renal fibrosis via inducing the production of alpha-1-acid glycoprotein

doi: 10.1038/s41598-020-68337-z

Figure Lengend Snippet: Am80 treatment increases ORM1 expression both in vivo and in vitro . ( a ) Hepatic ORM1 expression in the UUO-treated mice at day 7. ( b ) Plasma AGP protein expression in the UUO-treated mice at day 7. *P < 0.05 compared with the control mice. The full-length gel and bands are included in the Supplementary Fig. 2. # P < 0.05 compared with the corn oil-treated UUO mice. † P < 0.05 compared with the atRA-treated UUO mice. (n = 4 for control group; n = 5 for each UUO group) ( c , d ) mRNA expression of ORM1 in HepG2 and THP-1 cells after incubation with atRA or Am80 (0.1 μM). *P < 0.05 compared with control; # P < 0.05 compared with atRA-treated group. Data are expressed as the mean ± SE (n = 3 for control; n = 4 for atRA or Am80 treatment).

Article Snippet: After the UUO treatment, atRA (Wako, 20 mg/kg) or Am80 (Nippon Shinyaku, Kyoto, Japan, 20 mg/kg) was intraperitoneally administered each day.

Techniques: Expressing, In Vivo, In Vitro, Clinical Proteomics, Control, Mouse Assay, Incubation

Journal: Scientific Reports

Article Title: A synthetic retinoic acid receptor agonist Am80 ameliorates renal fibrosis via inducing the production of alpha-1-acid glycoprotein

doi: 10.1038/s41598-020-68337-z

Figure Lengend Snippet: Am80 treatment fails to alleviate UUO-induced renal fibrosis in AGP-KO mice. The mRNA expression of α-SMA and Col1a2 in the UUO-treated AGP-KO mice were not significantly alleviated by Am80 administration (n = 4 for corn oil group; n = 5 for atRA or Am80 group).

Article Snippet: After the UUO treatment, atRA (Wako, 20 mg/kg) or Am80 (Nippon Shinyaku, Kyoto, Japan, 20 mg/kg) was intraperitoneally administered each day.

Techniques: Expressing