Mccoys 5a media

shCT and shDAB2IP HCT116 cells were evenly plated in McCoy's 5A media (Corning, catalog no. 10–050-CV) containing 10% FBS and allowed to grow to 90% confluency (approximately 2 days in culture) to generate conditioned media. The conditioned media was collected and centrifuged at 1,000 × g for 5 minutes to remove dead cells. For each well, 600 μL of the appropriate media was added to the bottom well, and each condition was plated in triplicate. A total of 2 × 10⁵ RAW264.7 cells in 100 μL DMEM were added to the top well of the plate. Cells were allowed to migrate for 4 to 6 hours at 37°C and 5% CO₂. Following migration, cells remaining on the upper surface of the membrane were removed with cotton swabs, and inserts were incubated in 100% methanol for 10 minutes at −20°C to fix the migrated cells on the lower surface. Inserts were then stained in 0.2% crystal violet for 10 minutes at room temperature, washed 3 times in deionized water, and allowed to dry. Membranes were removed from the transwell insert and mounted on slides using Permount Mounting Medium (Fisher Scientific, catalog no. SP15–100). Membranes were imaged at ×10 magnification, with 5 random ROI selected for each membrane, and the migrating cells were manually counted. Three biological replicates were performed. One representative experiment with appropriate statistics is shown.

U-2 OS were obtained from ATCC (Manassas, VA, USA). HeLa, Hep3B, and HEK293T cells were obtained from Amgen Discovery Research (South San Francisco, CA, USA). U-2 OS cells were maintained in McCoy’s 5A media (Corning) containing 10% fetal bovine serum (FBS) (Sigma) and 1% penicillin-streptomycin (PS) solution (Corning). HeLa cells were maintained in DMEM (Corning) containing 10% FBS (Sigma) and 1% PS solution (Corning). Hep3B cells were maintained in Eagle’s minimum essential medium (EMEM from ATCC 30-2003) with 10% FBS. Hep3B-Cas9 cells were also maintained in 10 μg/mL blasticidin. Suspension-adapted HEK293 cells were maintained in FreeStyle 293 expression medium containing GlutaMAX-I (Gibco), 2% FBS (Gibco), and G418 (Gibco). HeLa cells were maintained in DMEM (Corning) containing 10% FBS (Sigma) and 1% PS solution (Corning). All cells were cultured at 37°C with 5% CO₂. Viruses were generated in a suspension of 293T cells by the triple transfection method using PEI MAX (Polysciences). Viruses were purified by iodixanol gradient or affinity columns using POROS CaptureSelect AAV8, AAV9, or AAVX. Virus titer was determined using a QuickTiter AAV quantitation kit (Cell Biolabs) according to the manufacturer’s instructions. AAV2-mCherry was made as previously described.⁴² (link) The plasmid ratio of VP1-mCherry/VP2+VP3 was 1:1.