The co-culture assay was established using transwell membranes (pores 0.4 μm, Merck Millipore, USA) in a 12-well format. OS cells were pretreated with GW4869 (Selleck, USA; 20 μM) or DMSO for 24 h in advance. The co-culture was performed for 3-6 days whereas OS (5 × 104 cells) on the permeable membranes were treated with GW4869 (20 μM) or DMSO, and then MRC5 cells (5 × 104 cells at the beginning) below the membranes were ready for further cytological experiments.

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