The following compounds were used in this study: gefitinib; dasatinib [LC Laboratories (MA, USA)]; thioridazine (10-[21-methyl-2-piperidyl) ethyl]-2-methylthio-phenothiazine) [Sigma Aldrich (Singapore)]. All reagents were dissolved in dimethyl sulfoxide (DMSO) and the final concentration of DMSO never exceeded 0.1% in culture. Antibodies: Akt [36 (link)], phospho-Akt (Ser473), caspase-3, 8, 9, cleaved caspase-3, 8, 9, ERK, phospho-ERK, IκBα, phospho-IκBα, p70S6K, phospho-p70S6K (Thr421 / Ser424), PKD2, PRKCSH, PARP, cleaved-PARP, RelA, phospho-RelA, Src, phospho-Src, GAPDH were purchased from Cell Signaling (MA, USA).
The human NSCLC cell lines H1975 and PC14 were purchased from the American Type Culture Collection (Manassas, VA). These cells were cultured in RPMI 1640 supplemented with 10% fetal calf serum, 100 U/ml of penicillin, and 100 μg/ml of streptomycin. The H1975 cell line has an EGFR T790M mutations in exon 20, associated with gefitinib and erlotinib resistance. The PC14 cell line has an EGFR delE746-A750 mutation in exon 19, associated with TKI sensitivity.
The human NSCLC cell lines H1975 and PC14 were purchased from the American Type Culture Collection (Manassas, VA). These cells were cultured in RPMI 1640 supplemented with 10% fetal calf serum, 100 U/ml of penicillin, and 100 μg/ml of streptomycin. The H1975 cell line has an EGFR T790M mutations in exon 20, associated with gefitinib and erlotinib resistance. The PC14 cell line has an EGFR delE746-A750 mutation in exon 19, associated with TKI sensitivity.