Spectramax i3x detection system

For cytokine analysis, serum was separated from the blood of each patient, and culture supernatants were collected at the indicated times after cell stimulation. The level of IL-17A in the serum and supernatant samples were detected using the LEGEND MAX™ Human IL-17A ELISA Kit (BioLegend, San Diego, USA) according to the manufacturer's protocols. Absorbance was measured at 450 nm using the SpectraMax i3x detection system (Molecular Devices, California, USA).

For the determination of mouse Gal-1 protein, serum and BALF of each mouse were collected and the level of Gal-1 was detected using Mouse Galectin-1/LGALS1 ELISA Kit (ORIGENE, Rockville, United States) according to the manufacturer’s protocols. For the analysis of human Gal-1 protein concentration in the medium of A549 cells, culture supernatants were collected at the indicated times and were detected using Human Galectin-1/LGALS1 ELISA Kit (Sino Biological, Beijing, China) according to the manufacturer’s instructions. Absorbance was measured at 450 nm using the SpectraMax i3x detection system (Molecular Devices, CA, United States).

Cytotoxic effect of Gal-1 on the A549 cells was determined by MTT assays using MTT Cell Proliferation and Cytotoxicity Assay Kit (Solarbio, Beijing, China), according to the manufacturer’s protocols. Briefly, A549 cells were seeded into 96-well plates with 3000–10000 cells per well. After being incubated at 37°C for 18 h, the cells were treated with mock or Gal-1 for the indicated time points (24 and 72 h). Control cells were treated only with medium. Then the culture medium was discarded and replaced with 90 μL fresh medium containing 10 μL MTT solution per well (provided in the kit) for another 4 h. Medium was discarded again and replaced with 110 μL provided formazan solution per well, incubating on the shaker at a low speed for 10 min to fully dissolve the crystals. Finally, the absorbance was determined at a wavelength of 490 nm using the SpectraMax i3x detection system (Molecular Devices, CA, United States). For each time point, the treated cells were compared with control cells and the percentage of cell viability was calculated as (mean OD of treated cells/mean OD of control cells) × 100.