Celltiter fluor reagent

For FACS, cells were seeded in ULA 10‐cm dish (#3263; Corning) at a density of 500 000 cells per dish. After incubated at 37 °C, 5% CO₂ for 48–72 h, cells were collected, trypsinized to get single cell suspension, and stained with propidium iodide (PI) and Annexin V (#V13242; Sigma) for 15 min at room temperature in dark. LSRII FACS analyzer was used to do the FACS with proper gating.
For the caspase 3/7 activity assay, cells were seeded in ULA 96‐well plates (#7007; Corning) at a density of 10 000 (OV17R, OVCA429, and OV7 clones) or 5000 (CH1 clones) cells per well. After 72‐h or 96‐h incubation, 20 μL of CellTiter‐Fluor reagent (for cell viability, #TB371; Promega (Madison, WI, USA)) was added to all the wells and the fluorescence was measured using a Tecan plate reader (Tecan, Männedorf, Switzerland; infinite 200) after 1‐h incubation at 37 °C. One hundred microlitee of caspase‐Glo 3/7 reagent (#TB323; Promega) was then added to all the wells, and the luminescence was measured after 1–2 h incubation at room temperature. The caspase 3/7 activities were divided by the cell viabilities and then normalized to their respective controls.

ATP level was measured using CellTiter-Fluor Cell Viability Assay which was multiplexed with CellTiter-Glo Luminescent Assay, according to manufacturer’s instructions. Briefly, approximately 3000 cells were dispensed into each well of a white 96-well plate in the absence or presence of p32-I, grown for 72 h in the 37 °C incubator under 5% CO₂ then added 20 µl of CellTiter-Fluor reagent (Promega). The plate was incubated for another hour and the fluorescent intensity was measured (390 nm-_Ex/505 nm-_Em). Then 100 µl of CellTiter-Glo (Promega) reagent was added to the cells and incubated for 10 min at RT. Both the fluorescence and the luminescence levels were measured with the EnSpire multimode microplate reader (PerkinElmer). The ATP level was normalized to the corresponding cell numbers of the same group.