H14 wa14

Human embryonic stem cell lines H9 (WA09), H14 (WA14) and WA22 were obtained from WiCell Research Institute. hES cells were maintained on hES cell qualified matrigel in mTeSR1 medium. Human normal skin fibroblasts CCD-1079Sk were acquired from ATCC and maintained in DMEM with 10% Fetal Bovine Serum and MEM non-essential amino acids. Cells were maintained at 37℃ in 5% CO2.
For cell death assays, cells were seeded at a density of 600,000 cells per well in 6-well plates.
Approximately after 24 hours, cells were treated with etoposide or tunicamycin. When MCL1 inhibitors (S63845 and AZD5991) were used, cells were treated with inhibitors in presence or absence of etoposide. Cells were stained with Nuclear Blue to label cell nuclei. Images were captured using Leica DMi8 microscope. Cell death was quantified on the basis of cellular and nuclear morphology, average of more than 800 cells were analyzed per condition. For siRNA transfections, cells were seeded at 350,000 cells per well in 6-well. Approximately after 24 hours, control siRNA (MISSION siRNA Fluorescent Universal Negative Control #1, Cyanine 3) and MCL1 siRNA (GGACUUUUAUACCUGUUAUtt) were transfected using lipofectamine 3000 reagent at 50 nM concentration. Approximately 24 hours post transfection cells, cells were treated with DMSO or etoposide and cell death was quantified as described above.