Anti il 8

Protein was extracted from 50-mg samples with a cell lysis kit (Bio-Rad Laboratories, Hercules, CA, USA). The samples were incubated in buffer (246 μL lysis buffer, 1.25 μL phosphatase inhibitor, 0.25 μL protease inhibitor, and 2.5 μL PMSF) on ice for 10 min and then centrifuged. For Western blotting, equal amounts of protein supernatant (60 μg) were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes for immunoblotting. The membranes were blocked with blocking buffer (Li-cor, Lincoln, NE, USA) for 2 h and then incubated with either anti-CKLF-1 (1:100, Peking University Center for Human Disease Genomics, Peking University, Beijing, China), anti-IL-6 (1:500, Abcam, Cambridge, UK), anti-IL-8 (1:500, Abcam), anti-IL-18 (1:500, Abcam), anti-TGF-β (1:500, Abcam), or anti-β-actin (1:2000, Santa Cruz Biotechnology, Dallas, TX, USA) antibodies for 12 h at 4 °C. The membranes were incubated with secondary antibodies (Li-cor, Lincoln, NE) at a 1:10,000 dilution in the dark for 1 h at room temperature and then detected with a double colour infrared laser imaging system (Odyssey, Li-cor, Lincoln, NE, USA).

Lipopolysaccharide (LPS; from Escherichia coli 0127:B8) was purchased from Sigma-Aldrich. Recombinant human cytokine IL-27 was purchased from R&D Systems. Antibody against IL-27RA (Novus Cat# NBP1-02708, RRID:AB_2125066) was purchased from Novus. Anti-phospho-STAT1(Y701) (Cell Signaling Technology Cat# 9171, RRID:AB_331591), anti-phospho-STAT3(Y705) (Cell Signaling Technology Cat# 4113, RRID:AB_2198588), anti-STAT1 (Cell Signaling Technology Cat# 9172, RRID:AB_2198300), and anti-STAT3 (Cell Signaling Technology Cat# 9132, RRID:AB_331588) antibodies were purchased from Cell Signaling Technology. Anti-TLR4 (Abcam Cat# ab13556, RRID:AB_300457), anti-IL-6 (Abcam Cat# 1457-1, RRID:AB_562150), and anti-IL-8 (Abcam Cat# ab34100, RRID:AB_775629) antibodies were purchased from Abcam. Beta(β)-actin (Millipore Cat# MABT825, RRID:AB_2571580) and horseradish peroxidase linked anti-rabbit (Millipore Cat# AP307P, RRID:AB_92641), anti-mouse (Millipore Cat# AP308P, RRID:AB_92635) and anti-goat (Millipore Cat# AP106P, RRID:AB_92411) secondary antibodies were purchased from Millipore. Secondary antibodies for IHC were from Dako.

Human IL-8 / CXCL8 protein (aa 23–99) was purchased from Sino Biological (Beijing, China). Antibodies used in the study are listed as follows: primary antibodies include anti-E-cadherin, anti-β-catenin, and anti-γ-catenin (BD Biosciences, Franklin Lakes, NJ, USA), anti-P-cadherin, anti-Akt1, anti-p-Akt (Ser473), and anti-pMLC (Cell Signaling Technology, Danvers, MA, USA), anti-α-tubulin (Proteintech, Wuhan, Hubei, China), anti-IL-8 (Abcam, Cambridge, UK). Secondary antibodies include AlexaFluor 568 anti-rabbit and Alexa Fluor 488 anti-mouse (Invitrogen, California, CA, USA) for immunostaining, HRP labeled anti-rabbit and anti-mouse (CWBIO, Beijing, China) for immunoblotting. Fluorescent dyes including DAPI and Phalloidin 647 were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

EmCaMSC2 and EmCaMSC3 were seeded at a cell density of 1 × 10⁵. After attaching CaMSCs, PBMCs were added and the cells were cocultured at different ratios (1:1, 1:2, 1:4, and 1:8), with or without IFNγ (100 ng/ml) treatment for 3 days. The cell viability was measured by using 2,3-bis-(2-methoxy-4-nitro-5-sulphenyl)(2H)-tetrazolium-5-carboxanilide (Biological Industries Ltd., Beit-Haemek, Israel). Briefly, the optical density was measured at 450 nm, and the absorbance at the reference wavelength of 650 nm was subtracted and adjusted with that of the blank control (wells without cells). All of the wells contained anti-CD3 (5 µg/ml) and anti-CD28 (5 µg/ml) antibodies to promote T-cell proliferation, and all experiments were conducted in triplicate. PBMC cells alone (0:1, 0:2, 0:4, and 0:8) were used as the positive control. For blocking or activating experiments, nivolumab (3,000 ng/ml; Opdivo), anti-PD-L1 (3,000 ng/ml; Abcam), recombinant IL-8 (300 ng/ml; ProSpect), anti-IL-8 (3,000 ng/ml; Abcam), anti-IGFBP2 (3,000 ng/ml; Santa Cruz Biotechnology, Dallas, TX, USA), and anti-insulin-like growth factor–binding protein 6 (IGFBP6) (3,000 ng/ml; Santa Cruz Biotechnology) were used.