A549 human nsclc cell line

LLC murine NSCLC cancer cell line and A549 human NSCLC cell line were obtained from ATCC (Manassas, VA). They were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C in a humidified atmosphere with 5% CO2.
ITGA5 KO cell line was generated by using CRISPR technology. LLC cells were infected with the lentivirus packaged by ITGA5‐All‐in‐one lentiviral sgRNA‐CRISPR‐Cas9 plasmid encoding EGFP and puromycin resistance (Horizon Discovery Ltd., Cambridge, UK). The successfully knocked‐out cells were selected by cell sorting of EGFP+ and ITGA5 KO populations. Cells were further confirmed by western blot analysis for the lack of ITGA5 expression.
Female C57BL/6 mice (4–6 weeks) (Strain #:000664) were purchased from Jackson Laboratory (ME, U.S.A). All animals were housed under pathogen‐free conditions according to AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care) guidelines. All animal‐related experiments were performed in full compliance with institutional guidelines and approved by the Animal Use and Care Administrative Advisory Committee at the University of Pittsburgh. under Protocol #: 21099779. Mice were housed at an ambient temperature of 22 °C (22–24 °C) and humidity of 45%, with a 14/10 day/night cycle (on at 6:00, off at 20:00), and allowed access to food ad libitum.

The A549 human NSCLC cell line was purchased from ATCC (CCL-185, Rockville, MD, USA). For conventional passage, cells were cultured in RPMI 1640 medium, supplemented with 2 mM l-glutamine (Corning Life Science, Manassas, VA, USA), 10 mM HEPES, 100 U/mL penicillin, 100 U/mL streptomycin, and 10% (v/v) fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO, USA) and incubated in 5% CO₂ at 37 °C for 24 h. For cell starvation, cells were cultured in RPMI 1640 medium containing 0.5% (v/v) FBS for 24 h. After cell starvation, the medium was changed, cells were cultured in the same low serum containing RPMI 1640 medium for drug administration during the next 48 h: TGF-β (10 ng/mL), LPS (100 ng/mL and 500 ng/mL), and CSE (1% and 2%). The cells without stimulation were used as the control. During the stimulation, we captured pictures under an optical microscope (#OlympusCKX31, Olympus Corporation, Tokyo, Japan) and then harvested cells for Western blot analysis.

The human breast cancer cell lines, MDA-MB-231 and SK-BR-3 cells, and human pancreatic cancer cell line, Panc1, were supplied by American Type Culture Collection—ATCC (Manassas, VA, USA) and cultured in DMEM containing 10% FBS and 1% antibiotic–antimycotic (final concentrations of 100 U/mL penicillin and 100 μg/mL streptomycin) in an incubator at 37 °C with 5% CO₂ until 75–80% confluency, as described in previous studies [61 (link),66 (link),67 (link)].
The A549 human NSCLC cell line was acquired from American Type Culture Collection—ATCC (Manassas, VA, USA) and cultured in RPMI-1640 medium containing 10% FBS and 1% antibiotic–antimycotic (final concentrations of 100 U/mL penicillin and 100 μg/mL streptomycin) in an incubator at 37 °C with 5% CO₂ until 75–80% confluency, as published previously [68 (link)].