C2210000

Human lung cancer cell lines A549 (ATCC Cat#: CCL-185), NCI-H23 (ATCC Cat#: CRL-5800), NCI-H1299 (ATCC Cat#: CRL-5803), NCI-H441 (ATCC Cat#: HTB-174), HCC827 (ATCC Cat#: CRL-2868), NCI-H1975 (ATCC Cat#: CRL-5908) and NCI-H2172 (ATCC Cat#: CRL-5930) were obtained from American Type Culture Collection (ATCC) and cultured as instructed. The human lung cancer PC14 cell line was kindly provided by Mr. Yu (The Shanghai Cancer Institute, China). The cancer cells were maintained in Roswell Park Memorial Institute (RPMI)1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin (all from Invitrogen). Cells were maintained under a standard gas atmosphere of humidified air/5% CO₂.
Cell growth was measured using CellTiter-Glo (Promega). The cells were seeded in 96-well plates in 100 μl of medium with different drug doses. Cisplatin (C2210000, Sigma Aldrich) and panobinostat (EPI009, Sigma Aldrich) were dissolved in dimethyl sulfoxide (DMSO). After the cells were cultured for 48 hours, 100 μl of CellTiter-Glo reagent was added to each well to measure cell growth according to the manufacturer's instructions.

The stock solutions of cisplatin (Sigma, C2210000) and GSK2730371 (Sigma, SML1048) were prepared in water, and etoposide (Sigma, E1383) and doxorubicin (Sigma, D1515) were prepared in DMSO. Isogenic PPM1D WT and mutant clones generated from MOLM13, OCI-AML2, and OCI-AML3 were seeded at 5000 cells per well in 96-well flat-bottom plates. An 8-point, 3-fold serial dilution of the drugs was added to the plates, to a final volume of 100uL per well (n = 3 replicates for each concentration). Cell viability was measured after 48 hours by the addition of Cell Counting Kit-8 (WST-8) reagent (Dojindo), 10uL per well, followed by incubation at 37°C for 4 h. Absorbance values of the wells were recorded with microplate reader (Perkin Elmer) at 450 nm. The viability readings were normalized to that of cells treated with vehicle alone. The resulting data were analyzed by using the dose-response function in Prism 6 (Graphpad Software, San Diego, CA). Dose-response curves were created and the concentration corresponding to the IC₅₀ was determined.