Anoikis Induction and Apoptosis Analysis

Cells (5 × 10⁵) were seeded in the absence or presence of compound into 6-well plates with an Ultra-Low Attachment Surface (Corning, NY, USA) for 48 h to induce anoikis, as described previously^{25 (link)}. Cells were washed and stained with 5 μl of annexin V and 5 μl of propidium iodide (PI) per 1 × 10⁵ cells for 15 min at room temperature in the dark, and the percentage of apoptotic cells was analyzed using flow cytometry. The cells were harvested after the induction of anoikis, washed with PBS, and lysed for immunoblot analysis. Where indicated, annexin V or PI-positive cells relative to the total cells were counted to determine the percentage of apoptotic cells.