Journal: iScience

Article Title: Amitriptyline revitalizes ICB response via dually inhibiting Kyn/Indole and 5-HT pathways of tryptophan metabolism in ovarian cancer

doi: 10.1016/j.isci.2024.111488

Figure Lengend Snippet: Expression of MYD88, IDO1, and IL4-I1 in EOC cells after LPS-induced combination with AMI (A−C) RT-qPCR and Western blot verification of MYD88 and IDO1 expression in A2780, OVCAR3, and ID8 EOC cells after LPS-induction (1 μg/ml). (D−F) mRNA expression of MYD88 and IDO1 by the combination of LPS with AMI in A2780, OVCAR3 (AMI: 50, 100μm) and ID8 (AMI: 30, 60μm) EOC cells. (G−H) MYD88 and IDO1 expression was not detected in A2780 EOC cells according to the pre-WB results. Therefore, only the expression of MYD88 and ID8 protein levels in OVCAR3 and ID8 were performed in the combined treatment. (Protein expression of MyD88 and IDO1 relative to Tubulin in EOC cells treated with different concentrations of drugs). (I) mRNA expression of IL4I1 on A2780, OVCAR3, and ID8 EOC cells after LPS combined with AMI treatment was analyzed by RT-qPCR, in which IL4I1, as a secreted protein, was detected in the levels in the EOC cells culture supernatant with Elisa. Each assay was performed in triplicate, and data are presented as mean ± SD ( n = 3).

Article Snippet: ELISA kit- IFN-γ (Mouse) , Novus Biologicals , VAL607.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Amitriptyline revitalizes ICB response via dually inhibiting Kyn/Indole and 5-HT pathways of tryptophan metabolism in ovarian cancer

doi: 10.1016/j.isci.2024.111488

Figure Lengend Snippet: The killing activity of T lymphocytes against EOC cells and expression of CD39 after AMI stimulation (A) Expression of CD8 + CD39 + in T lymphocytes after exposure to different concentrations of AMI-treated (AMI concentrations refer to the preceding) EOC cells culture supernatants for 12 or 24 h as analyzed by FCM. (B) The killing activity of corresponding T lymphocytes (effector cells) to parental EOC cells (target cells) or AMI-treated EOC cells (target cells) shown are from the effector/target ratio at 1:1, 5:1, and 10:1. Supernatant 0h, 24h, and 48h represent the stimulation of T lymphocytes by supernatants collected at different times from EOC cells treated with AMI, respectively. (C) Measure the levels of TNF-α and IFN-γ in lymphocyte culture supernatants stimulated with AMI-treated EOC cell culture supernatants by Elisa assays. Each assay was performed in triplicate, and data are presented as mean ± SD ( n = 3). In Elisa results, ∗ indicates compared to the lymphocytes without any treatment (LN), # indicates compared to the lymphocytes stimulated with DMSO-treated EOC cell culture supernatant. #, ##, ### and #### indicates p < 0.05, p < 0.01, p < 0.001, and p < 0.0001 respectively.

Article Snippet: ELISA kit- IFN-γ (Mouse) , Novus Biologicals , VAL607.

Techniques: Activity Assay, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Amitriptyline revitalizes ICB response via dually inhibiting Kyn/Indole and 5-HT pathways of tryptophan metabolism in ovarian cancer

doi: 10.1016/j.isci.2024.111488

Figure Lengend Snippet: AMI reshapes TIME, the serum level of IL-4I1 and TRP metabolites peripheral blood of EOC-bearing mice (A and B) Tryptophan metabolites in the serum of unimplanted tumor-bearing mice (normal mice) and compared with implanted tumor-bearing mice (controls). (C and D) Tryptophan metabolites in the serum of normal mice (without implanted tumors) before and after the use of AMI. (E−H) EOC-bearing mice receiving PD-1 inhibitor - combined AMI therapy, LC-MS/MS was used to detect the amount of KYN, KYNA, TRP, and the ratio of KYN/TRP in the serum of EOC-bearing mice. (I) IL4I1 in EOC-bearing mice serum by Elisa. (J) LC-MS/MS was used to detect the amount of AHR ligands (I3P, IAA, I3A, and ILA) in the serum of EOC-bearing mice. Data are presented as mean ± SD ( n = 3).

Article Snippet: ELISA kit- IFN-γ (Mouse) , Novus Biologicals , VAL607.

Techniques: Liquid Chromatography with Mass Spectroscopy, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Amitriptyline revitalizes ICB response via dually inhibiting Kyn/Indole and 5-HT pathways of tryptophan metabolism in ovarian cancer

doi: 10.1016/j.isci.2024.111488

Figure Lengend Snippet:

Article Snippet: ELISA kit- IFN-γ (Mouse) , Novus Biologicals , VAL607.

Techniques: Recombinant, Bicinchoninic Acid Protein Assay, Membrane, Isolation, Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay, Software