Proliferation assay of GC with CRISPR-Cas9-induced ASB9 inhibition.

Bovine GC collected from large follicles were cultured in DMEM/F12 as described in materials and methods. GC were seeded in 96-well plates and incubated at 37°C/5% CO2 and proliferation was determined using the CellTiter assay kit (Promega). The CellTiter substrate was added for 3 hours before measuring absorbance at 490 nm with the SpectraMax i3 (Molecular Devices). Inhibition of ASB9 significantly increased GC proliferation (P<0.05).