Journal: Journal for Immunotherapy of Cancer

Article Title: N-cadherin inhibitor creates a microenvironment that protect TILs from immune checkpoints and Treg cells

doi: 10.1136/jitc-2020-002138

Figure Lengend Snippet: Evaluation of whether N-cadherin knockout impacts the production of Treg cells. (A) CXCL10 and CXCL11 expression levels in cell lines were examined by real-time qRT-PCR. Fold changes are shown. (B) The concentrations of CXCL10 and CXCL11 in the culture of cell lines were analysed by ELISA. Cells were cultured with RPMI medium containing 10% FBS. Then, 48 h later, the concentrations of CXCL10 and CXCL11 were examined. (C) IRF1 expression in cell lines and mouse tumour cells was evaluated by real-time qRT-PCR. Fold changes are shown. (D) The protein expression of members of the AKT-mTOR signalling pathways in cell lines was examined by western blotting. (E) The concentrations of CCL1, CCL17, and CCL22 in the culture medium were analysed by ELISA. Cells were cultured with RPMI medium containing 10% FBS. Then, 48 h later, the concentrations of CCL1, CCL17, and CCL22 were examined. (F) Fatty acid metabolism-related genes were compared using RNA-sequencing data. (G) FASN/FAS was examined by real-time qRT-PCR and western blotting. FFA concentrations in tumour samples (H) were evaluated using the Free Fatty Acid Quantification Kit and in culture medium (I) using LC-MS. Cell lines were cultured with FBS-free RPMI medium containing 10% lipids. Then, 48 h later, the concentrations of FFAs were examined.

Article Snippet: Human cytokines (IFN-α, TNF-α, IL8, IL6, CCL5 and IL10) in the mouse serum samples were assayed by ELISA (Human cytokine ELISA kit, Biocompare, Newburyport, Massachusetts, USA).

Techniques: Knock-Out, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Cell Culture, Western Blot, RNA Sequencing, Liquid Chromatography with Mass Spectroscopy