Azd6244

AZD6244, recombinant human (rh)TRAIL and z-VAD-FMK were purchased from AstraZeneca (Macclesfield, UK), Calbiochem (Hertfordshire, UK) and Sigma-Aldrich (Gillingham, Dorset, UK), respectively. Isoleucine-zipper TRAIL (iz-TRAIL) was expressed and purified in-house^{14 (link)}. iz-TRAIL is a modified form of rhTRAIL that comprises an isoleucine zipper motif fused to its N-terminus, which enhances its ability to trimerise — a process required for its apoptotic activity, consequently increasing its potency. siRNA sequences targeting RALA (_8 SI03101133), RALB (_1 S100045199; _4 SI00045178; _6 SI03054793; _7 SI03117492), RALGDS (_5 SI03231424), Caspase-8 (_11 SI02661946) and Caspase-9 (_5 SI00299600) were purchased from Qiagen (Crawley, UK). siRNA sequences targeting DR4 and DR5 were purchased from Dharmacon. TRAIL neutralising antibody, Chloroquine and Bafilomycin A1 were purchased from R&D systems (Abingdon, UK), Invivogen (San Diego, CA) and Merck Millipore (Darmstadt, Germany), respectively.

Cell viability was determined by using CCK8 (Dojindo, Kumamoto, Japan) according to the instruction of manufacture. Cells were seeded in 96-well plates for 24 hours. Then cells were treated with serials concentration (0, 1, 2, 3 and 4 μM) of AZD6244 (AstraZeneca, London, UK) for 24 or 48 hours. A 10 μL of CCK8 solution was then added to each well, and the plates were incubated at 37°C for 2 hours. The optical density (OD) of each well was measured at 450 nm.

The human lung adenocarcinoma cell lines A549, PC9 (EGFR mutant) and the human colon cancer cell lines SW480 and SKCO1 (KRAS mutant) were obtained from the China Center for Type Culture Collection (Wuhan, China). A Cisplatin-resistant subline of A549 and a gefitinib-resistant subline of PC9 were established by repeated subculturing with gradual increasing in the Cisplatin (2, 4, 6, 8 and 10 μM) or gefitinib concentration (4, 8, 16, 20 and 40 μM) over a 3-month period.^{52 (link)} In addition, A549, PC9, SW480 and SKCO1 cell lines with stable overexpression or knockdown of ZNF32 were constructed as described previously.^{35 (link)}BALB/c male nude mice, 6 weeks of age, were maintained under standard conditions in the animal facility of Sichuan University. All experiments in this study were performed in accordance with the nation's relevant laws and animal welfare requirements.
Cisplatin and 5-FU were purchased from Sigma-Aldrich (St Louis, MO, USA), and gefitinib and AZD6244 were obtained from AstraZeneca (Cheshire, UK) and dissolved in DMSO to obtain a stock solution of 10 mM. These drugs were diluted with culture medium for use in the experiments. Recombinant human TGF-β1 (TGF-β) was purchased from R&D Systems and used at a concentration of 10 ng/ml. LY2157299 (Selleck), an inhibitor of TGF-β signaling, was used at a concentration of 1 μM in the experiments.

AZD9291 and AZD6244 were provided by AstraZeneca Pharmaceuticals. Erlotinib, gefitinib, afatinib, CO1686, crizotinib, capmatinib, cabozantinib, MGCD-265, and merestinib were purchased from Selleckchem. All drugs were dissolved in dimethyl sulfoxide (DMSO) at a 10 mM concentration and stored in small aliquots at -20°C until further use. Antibodies specific for p-EGFR (Tyr1068), EGFR, p-AkT (Ser473), AkT, p-ERK1/2 (Thr202/Tyr204), ERK1/2, p-MET (Tyr1234/1235), MET, and Ki-67 were obtained from Cell Signaling Technologies. HRAS, Maspin, and β-actin antibodies were obtained from Santa Cruz Biotechnology. HRAS siRNA, Maspin siRNA and control siRNA were purchased from Santa Cruz Biotechnology.