Journal: Oncotarget

Article Title: Arctigenin functions as a selective agonist of estrogen receptor β to restrict mTORC1 activation and consequent Th17 differentiation

doi: 10.18632/oncotarget.13338

Figure Lengend Snippet: A. and B. Serum-starved EL4 T cells were treated with arctigenin (10 μM) or E2 (1 μM) in the presence or absence of MPP (0.1 μM), PHTPP (0.1 μM) and G15 (0.1 μM) for 24 h. Cells were harvested and lysed, and the levels of p-mTOR, mTOR, p-RPS6, RPS6 and GAPDH were analyzed by immunoblotting. C. Serum-starved EL4 cells were treated with arctigenin (10 μM) for 0-24 h, and D. EL4 cells were treated with either control siRNA or ERβ siRNA followed by treatment with arctigenin or E2 for 24 h, and the levels of p-mTOR, mTOR, p-RPS6, RPS6 and GAPDH were analyzed by immunoblotting. E. Serum-starved Jurkat cells were treated with or without arctigenin (10 μM) or E2 (1 μM) or PHTPP (0.1 μM) for 24 h. mTOR was immunoprecipitated from whole-cell lysates with antibody to mTOR and the immunoprecipitates were used in mTORC1 in vitro kinase assay using recombinant p70S6K as a substrate. The kinase assay products were subjected to immunoblotting. Densitometry analysis of immunoblotting was also shown. Data shown are representative of at least three experiments. * P < 0.05, ** P < 0.01 vs . control group; # P < 0.05, ## P < 0.01 vs . indicated group (Dunnett's test). ARCG: arctigenin.

Article Snippet: Antibodies against p-RPS6 and RPS6 (Sangon Biotech, Shanghai, China), and against mTOR (Abcam, Cambridge, UK) as well as against regulatory-associated protein of mTOR (raptor) (Proteintech, Wuhan, China) were used.

Techniques: Western Blot, Control, Immunoprecipitation, In Vitro, Kinase Assay, Recombinant

Journal: Oncotarget

Article Title: Arctigenin functions as a selective agonist of estrogen receptor β to restrict mTORC1 activation and consequent Th17 differentiation

doi: 10.18632/oncotarget.13338

Figure Lengend Snippet: Naïve CD4 + T cells were cultured under Th17-polarizing conditions in the presence of arctigenin (10 μM) or E2 (1 μM) with or without MPP (0.1 μM), PHTPP (0.1 μM), G15 (0.1 μM) and ICI (0.1 μM). A . and B . After treatment for 72 h, IL-17A production in CD4 + T cells was assessed by flow cytometry. C. After treatment for 48 h, the mRNA expressions of ERβ, CAV1 and ENPP2 were detected by quantitative PCR. D. Purified CD4 + T cells were pretreated with or without arctigenin (10 μM) or E2 (1 μM) or PHTPP (0.1 μM) for 24 h, followed with Th17 cell polarizing cocktails for 3 h. Cells were harvested and lysed, and the levels of p-mTOR, mTOR, p-RPS6 and RPS6 were analyzed by immunoblotting. E. Naïve CD4 + T cells were transfected with scrambled siRNA or ERβ siRNA and treated under Th17-polarization condition as indicated. After treatment for 72 h, IL-17A production in CD4 + T cells was assessed by flow cytometry. F. EL4 cells were treated with either control siRNA or ERβ siRNA followed by treatment with arctigenin or E2 for 24 h, the mRNA expressions of IL-17A and RORγt were detected by quantitative PCR. Results are representative of three independent experiments. * P < 0.05, ** P < 0.01 vs . control group; # P < 0.05, ## P < 0.01 vs . indicated group; $$ P < 0.01 vs . normal group (Dunnett's test). ARCG: arctigenin.

Article Snippet: Antibodies against p-RPS6 and RPS6 (Sangon Biotech, Shanghai, China), and against mTOR (Abcam, Cambridge, UK) as well as against regulatory-associated protein of mTOR (raptor) (Proteintech, Wuhan, China) were used.

Techniques: Cell Culture, Flow Cytometry, Real-time Polymerase Chain Reaction, Purification, Western Blot, Transfection, Control

Journal: Oncotarget

Article Title: Arctigenin functions as a selective agonist of estrogen receptor β to restrict mTORC1 activation and consequent Th17 differentiation

doi: 10.18632/oncotarget.13338

Figure Lengend Snippet: Mice were treated with 2.5% DSS in drinking water for 7 days, followed by normal drinking water for 3 days to induce colitis. Arctigenin (50 mg/kg) was oral administered daily. PHTPP (1 mg/kg) and E2 (1 mg/kg) were injected intraperitoneal daily. Mice were sacrificed on day 10 after colitis induction. A. The mRNA expressions of ERα, TFF1, BRCA1, ERβ, CAV1, ENPP2 and GPER in colons of colitis mice (without OVX) induced by drinking DSS ( n = 6). B. The mRNA expressions of CAV1 and ENPP2 in colons of each group ( n = 6). C. The levels of p-mTOR, mTOR, p-RPS6 and RPS6 in colon tissues were detected by Western blot analysis ( n = 6). D. Intracellular IL-17A production of CD4 + T cells in MLNs was measured by flow cytometry, and the percentage of IL-17A + CD4 + T was evaluated ( n = 6). E. The mRNA expression of RORγt in colons of each group ( n = 6). F. Disease activity index of each group ( n = 10). G. The colon length of each group ( n = 10). H. The mRNA expressions of pro-inflammatory cytokines TNF-α and IL-6 in colons of each group ( n = 6). I. Histological scores of colon from each group ( n = 6). J. Representive histological changes of colon, characterized by distinct infiltration of inflammatory cells and crypt destruction (Magnification × 200). $ P < 0.05, $$ P < 0.01 vs . control group; * P < 0.05, ** P < 0.01 vs . model group; # P < 0.05, ## P < 0.01 vs . indicated group (Dunnett's test). ARCG: arctigenin.

Article Snippet: Antibodies against p-RPS6 and RPS6 (Sangon Biotech, Shanghai, China), and against mTOR (Abcam, Cambridge, UK) as well as against regulatory-associated protein of mTOR (raptor) (Proteintech, Wuhan, China) were used.

Techniques: Injection, Western Blot, Flow Cytometry, Expressing, Activity Assay, Control