Pd l1

The Magna RIPTM RNA-binding protein immunoprecipitation kit (Millipore, USA) was used to carry out the RIP assay in accordance with the protocol. Magnetic beads were coated with antibodies against IgG (Millipore, MA, USA), HOXC8 (Proteintech, Wuhan, China), AGO2 (Abcam, Burlingame, USA), PD-L1 (Abcam, Burlingame, USA), METTL3, and YTHDC1 (Cell Signalling Technology, MA, USA) for 30 minutes at room temperature. To capture circSLCO1B3, the cell lysate from 2×10⁷ cells was incubated with magnetic beads coated with antibodies. TRIzol was utilized to extract the entire RNA for MeRIP. In a manner similar to RIP, magnetic beads from Millipore in Massachusetts, USA were coated with 5 μg of an anti-IgG or anti-m6A antibody and incubated at room temperature for 30 minutes. Following this, 50μg of overall RNA was introduced to the beads coated with antibodies, and then incubated in immunoprecipitation buffer containing RNase inhibitor at a temperature of 4℃ for the duration of the night. Following the digestion of proteinase K, the m6A-bound RNA precipitation was completed using the RNA Clean & Concentrator kit (ZYMO research, Hangzhou). The m6A enrichment of circSLCO1B3 was assessed using qPCR and normalized to the input.

The total proteins of the cells and clinical tissues were extracted by using the RIPA lysis buffer solution (Beyotime, China) according to the manufacturer’s protocol. Based on the protocols provided by the previous study [38 ], Western Blot was used to determine the expression levels of proteins involved in this study. The primary antibodies against PD-L1 (1:1000, Abcam, UK), β-actin (1:2000, Abcam, UK), Cyclin D1 (1:1500, Abcam, UK), Bax (1:1000, Abcam, UK), Bcl-2 (1:2000, Abcam, UK), N-cadherin (1:1500, Abcam, UK), Vimentin (1:1000, Abcam, UK) and TSG101 (1:1500, Abcam, UK) were purchased. The horseradish peroxidase-conjectured goat anti-rabbit secondary antibody (1:5000, Abcam, UK) was also obtained. Finally, the protein bands were visualized by using a electrochemiluminescence (ECL) system and the grey values were measured by Image J software to evaluate relative protein levels, and normalized to β-actin.

Activated T cells were treated with either PBS, recombinant human PD-L1 protein (10 ug/mL; R&D Systems, Minneapolis, MN, USA), PD-L1 combined with goat anti-human IgG (10 ug/mL; R&D Systems, Minneapolis, MN, USA), or PD-L1 combined with PD-L1 neutralizing antibody (10 ug/mL; Abcam, Cambridge, UK), and the supernatants were subjected to ELISA after a 72-hour culture to detect the secretion of IFN-γ, IL-1β, TNF-α, and IL-2. Lymphocytes isolated from peripheral venous blood of TAO patients were treated with either PBS, recombinant human PD-L1 protein (10 ug/mL), PD-L1 combined with goat anti-human IgG (10 ug/mL), or PD-L1 combined with PD-L1 neutralizing antibody (10 ug/mL) for 48 hours, and the proportion of CD3+CD40L+ cells were determined by FCM. OFs from patients with TAO (TAO-OFs) were treated with either PBS, PD-L1 (10 ug/mL), autologous activated T cells (OFs: T cells = 1: 10), or PD-L1 combined with T cells for 24 hours. TAO-OFs were subjected to flow cytometry (FCM) and immunofluorescence staining (IF) for CD40 expression, and the total protein extracted from TAO-OF layers were subjected to WB to detect the expression levels of p38, ERK1/2, JNK, and NF-κB in TAO-OFs. After a 48-hour co-culturing, the supernatants were subjected to analysis of sICAM-1, IL-6, IL-8, and HA production by ELISA.

IHC staining was performed on formalin-fixed, paraffin-embedded tumor samples and the method was as described in the previous literature^{19 (link)}. Briefly, paraffin sections were taken and dewaxed to water. Antigen repair solutions were dripped on the sections and washed with PBS 3 times. The first antibodies were added to the sections and washed with PBS (PH7.4) 3 times at 4 °C overnight. Secondary antibodies were added and rinsed with PBS 3 times again. Immunostaining was performed with DAB. The sections were counterstained with hematoxylin. The antibodies Ki67 (1:200, Abcam), CD31 (1:400, DAKO, USA), α-SMA (1:500, Themo Fisher), PD-L1 (1:600, Abcam), CD8 (1:2000, NOVUS, USA), Ly6G (1:800, Servicebio), MMP-2 (matrix metalloproteinase-2, 1:1500, Servicebio) and MMP-9 (1:800, Servicebio) were used. Visualize staining of tissue under a microscope, acquisitive and analysis image (Nikon DS-U3, Japan). The results of IHC staining were analyzed by Image J software 1.8.0 (Media Cybernetics, Rockville, MD, USA). The Ki67 and CD8 antibody staining results were evaluated by the percentage of positive cells and the positive cells density respectively, and the percentage of positive staining areas evaluated the α-SMA, CD31, PD-L1, Ly6G, MMP-2 and MMP-9 antibody staining results. Five random visual fields were counted for each sample and the average was determined.