Crl 5800

TC1 cells (ATCC, Manassas, VA, USA) derived from primary lung epithelial cell tumors of C57/B6 mice were cultured at 37°C, 95% air, 5% CO₂ incubator in full tumor medium as recommended by the American Type Culture Collection (ATCC). TC1 cells were cultured in RPMI-1640 medium with 2 mmol/L L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 10 mmol/L HEPES, and 1.0 mmol/L sodium pyruvate supplemented with 2 mmol/L nonessential amino acids, penicillin and streptomycin, and 10% FBS (Life Technologies, Foster City, CA).
Mice exposed to either control sleep conditions (SC; n = 12) or SF (n = 12) were inoculated with TC1 cells (1 × 10⁵ cells in 0.2 mL PBS) by subcutaneous injection into the right lower flank. Tumors volumes were estimated every 3 days by externally measuring its length and width with an electronic caliper. After 28 days from tumor injection during which the sleep-related exposures were continued, mice were sacrificed, blood collected, and tumors excised and weighed.
The human adenocarcinoma, non-small cell lung cancer cell line was obtained from ATCC (CRL-5800, ATCC, Manassas, VA) and used for experiments with OSA patients-derived plasma exosomes (see below).

Plasmids or siRNA were transiently transfected into HeLa cells (ATCC® CCL-2™, American Type Culture Collection (ATCC), Manassas, VA, USA), CRL5800 (ATCC® CRL-5800™, ATCC) and MDA-MB-231 (ATCC® HTB-26™, ATCC) using Lipofectamine 3000 (Invitrogen, Grand Island, NY, USA). Stable cell lines expressing YFP or RCC2-YFP were also established by selections with G418 (400 μg/ml) for 3 weeks, and the YFP and RCC2-YFP expression were monitored using an inverted fluorescence microscope.