Uncoated control inserts

Lipofectamine® 2000 (Life Technologies) was used for all plasmid transfections. RIP assays were performed as previously published, using HEK293T cells transfected with 10 μg of FLAG-AR expressing plasmid, and 10 μg of lncRNA-expressing plasmid (Schmidt et al., 2016 (link)). Immunoprecipitation of FLAG-tagged AR was performed as previously published (Schmidt et al., 2017 ). For invasion assays, A375 cells were seeded at 25 × 10⁴ cells in a 6-well plate and transfected with 2,500ng of the indicated plasmid 24 hours later. For assays using FANA-modified oligos, media was changed 7–8 hours post-transfection to media containing 1 μM final concentration of the indicated oligo. Approximately 28 hours post-transfection, 2.5 × 10⁴ cells in serum-free media were plated in either BD BioCoat matrigel inserts or uncoated control inserts (Corning), placed into DMEM with 30% FBS (fetal bovine serum), and incubated for 16 hours. Cells that did not migrate or invade were removed using a cotton tipped swap, chambers were rinsed twice with PBS, and stained using Fisher HealthCare PROTOCOL Hema 3 Fixative and Solutions. Cells were imaged on 20x magnification in 8 fields of view for 3 independent replicates.

Cells were plated in either BD BioCoat™ matrigel inserts or uncoated control inserts (Corning) in serum-free media and placed into DMEM with 30% FBS. The number of invaded or migrant cells were imaged on 20x magnification in 8 fields of view for 3 independent replicates.