Figure 3
Cisplatin-resistant cells had a high MMP and increased migration and invasion. (a) A549 cells were treated with the indicated concentrations of cisplatin for 3 days. The half-maximal inhibitory concentration (IC50) of cisplatin for A549 cells was determined by SRB assay. (b) Schematic diagram of cisplatin treatment. (c) A small population of lung adenocarcinoma cells was resistant to cisplatin treatment. A549 cells treated with cisplatin (10 μM) as indicated in b were imaged at 2-h intervals for 5 days using IncuCyte ZOOM (Essen Bioscience); a representative image is shown. Full time-course images are shown in Supplementary Movie 2. (d) After withdrawal of cisplatin, A549R cells remained viable but did not proliferate significantly. The majority existed as single cells and were viable at 10 days after cisplatin withdrawal. (e) The proliferation rates of A549 and A549R cells were determined by cell counting using Cytation 3 (Bioteck). (f) A549R cells survived a further three cycles of cisplatin treatment. (g) Cisplatin-resistant cells had a higher MMP than parental cells. The MMPs of parental (A549 and H1650) and cisplatin-resistant (A549R and H1650R) cells were investigated by tetramethylrhodamine, ethyl ester (TMRE) staining followed by flow cytometry (FACSVerse; BD Biosciences). 7-Amino actinomycin D (7-AAD) staining was used to evaluate cell viability. (h) Cisplatin-resistant cells had a greater number of mitochondria than parental cells. Parental and cisplatin-resistant cells were stained with Mitotracker and analyzed by flow cytometry. (i) Cisplatin-resistant cells had a higher cellular ATP level than parental cells, as determined using an ATP assay kit. (j, k) Cisplatin-resistant cells exhibited increased migration and invasion, as determined by Transwell migration and invasion assays. Data are means of at least three independent experiments. *P<0.05, **P<0.01, ***P<0.001 relative to parental cells.
