Methylcellulose based serum free medium

Tumor sphere formation assay was performed under non-adherent and serum-free conditions. Briefly, 5,000 cells were suspended in 0.8% methylcellulose-based serum-free medium (Stem Cell Technologies, Vancouver, Canada) supplemented with 20 ng/ml epidermal growth factor (BD Biosciences, San Jose, CA, USA), 10 ng/ml basic fibroblast growth factor and 5 μg/ml insulin (Sigma-Aldrich, St Louis, MO, USA) in ultra-low adherent 6-well plates (Corning Incorporated, Kennebunk, ME, USA). Cells were cultured for two weeks after which tumor spheres were examined under a light microscope. In order to assess self-renewal property of the cells, spheres were collected by gentle centrifugation, dissociated into single cell suspensions, filtered and cultured under the same conditions to form secondary spheres.

The formation of spheroids was performed under serum-free conditions in an ultralow attachment plate as previously reported [7 ]. NSCLC-derived H292 and A549 cells were pretreated with PNA-A15 and scramble (5 μM) for 48 h. Then, cells were detached using 1 mM EDTA and suspended into single cells. These cells were grown in a 24-well ultralow attachment plate at a density of 2.5 × 10³ cells/well in stem cell media (SCM) (i.e., 0.8% (w/v) methylcellulose-based serum-free medium (Stem Cell Technologies, Vancouver, BC, Canada) supplemented with 20 ng/ml epidermal growth factor (BD Biosciences, San Jose, CA, USA), 20 ng/ml basic fibroblast growth factor and 4 mg/ml insulin (Sigma) for 7 days to form primary spheroids. These primary spheroids were harvested, resuspended as single cells using 1 mM EDTA and cultured in SCM for 14 days in a 24-well ultralow attachment plate to form secondary spheroids.