Apodetect

Cells were seeded in a 10 cm plate and transfected with siRNA the following day. 48 hr after transfection, cells were harvested, washed twice in 1X PBS and slowly resuspended in cold 70% ethanol and left at -20°C overnight. The pelleted cells were washed, resuspended in PBS containing 500 ng/mL RNAse A and 40 ng/mL propidium iodide and incubating for 30 min at 37°C. Flow cytometry analysis was done within an hour after incubation. For apoptosis assay, ApoDETECT^™ (Invitrogen), transfected cells were harvested, washed with PBS and resuspended in 1X binding buffer (10 mM Hepes/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2). 10 μL of Annexin V-FITC was added to 190 μL of cell suspension and incubated for 10 min at room temperature. The Annexin V-FITC was washed out using 1X binding buffer and resuspended in 190 μL of 1X binding buffer before adding 10 μL of 20 μg/mL propidium iodide. The stained cells were analyzed immediately after.

To determine the effect of NKX2-1-AS1 levels on cell growth, H441 cells were transfected with the mix of three siRNAs or control as described above, cultured, trypsinized and counted at 0 h, 24 h, 48 h, and 72 h after seeding. Proliferation was determined with Click-iT® EdU Flow Cytometry Assay Kit (Invitrogen) coupled to Alexa Fluor®488 dye and analyzed by standard flow cytometry methods at the Boston University Flow Cytometry Core to determine the percentage of cells in each cell cycle phase. Apoptosis was determined using ApoDETECT™ (Invitrogen) that uses Annexin V-FITC and propidium iodide to detect cell surface exposure of phosphatidylserine in viable cells by flow cytometry following the manufacturer’s protocol.