After different types of gene perturbations, the cells were cultured in 48-well plates, at the starting density of 20,000 cells per well. The IncuCyte live-cell imaging and analysis system (ESSEN Bioscience) was used to monitor real-time cell proliferation and morphology changes. Cell proliferation was quantified by measuring the occupied area (% confluence) in the cell images over time. The proliferation curves were made with confluence fold change (FC) at different time points in relative to the confluence at time 0.
For colony formation assays, 1000 cells were seeded in the 6-well plates and incubated with normal growth medium for 14 days. Then cells were fixed and stained with 0.5% crystal violet for 15 min. Lastly, the colonies were imaged via a camera or a microscope. For the anchorage-independent colony formation assay, the cells were collected, resuspended in culturing media containing 10% FBS and 0.3% agarose (Amresco), and then seeded on top of 0.6% agarose gel containing 10% FBS in six-well plates. The cells were cultured in regular media for 3–4 weeks, and the colonies were stained with crystal violet and photographed.
For colony formation assays, 1000 cells were seeded in the 6-well plates and incubated with normal growth medium for 14 days. Then cells were fixed and stained with 0.5% crystal violet for 15 min. Lastly, the colonies were imaged via a camera or a microscope. For the anchorage-independent colony formation assay, the cells were collected, resuspended in culturing media containing 10% FBS and 0.3% agarose (Amresco), and then seeded on top of 0.6% agarose gel containing 10% FBS in six-well plates. The cells were cultured in regular media for 3–4 weeks, and the colonies were stained with crystal violet and photographed.