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. 2016 Jun 29;7(31):50161–50179. doi: 10.18632/oncotarget.10324

Figure 3. DMS114/NIN cells develop a MDR phenotype upon nintedanib selection due to upregulation of ABCB1.

Figure 3

A. Sensitivities of DMS114 and DMS114/NIN cells towards different anticancer drugs were tested in a large-scale cytotoxicity screen evaluated by CellTiter Glo assay after 72 hours drug exposure. Sensitivities of DMS114 and DMS114/NIN cells are plotted as area under the curve (AUC) values determined from 8-point dose-response curves to individual drugs. Green symbols represent drugs less active in DMS114, red symbols represent drugs less active in DMS114/NIN cells. Triangles indicate compounds that were highly active in DMS114 cells with IC50 values below the lowest tested concentrations. Vertical error bars indicate SD of AUC of DMS114/NIN, horizontal error bars show SD of AUC of parental cells, respectively. Known ABCB1 substrate drugs are indicated by the numbers: 1) carfilzomib; 2) vinblastine; 3) vincristine; 4) mitoxantrone; 5) docetaxel; 6) CUDC-101; B. Whole-genome gene expression patterns of DMS114 and DMS114/NIN cells were established by microarray analysis and data were processed using GeneSpring software (Agilent Technologies). Raw and normalized mRNA expression values are depicted for ABCB1. C. ABCB1 mRNA expression was analyzed by quantitative RT-PCR and data are given normalized to ACTB mRNA expression. *** p<0.001, unpaired t-test. ABCB1-overexpressing SW480Tria cells served as positive control and were set as 100 (dashed line). n.d., not detected; D. ABCB1 protein expression levels of DMS114 (SCLC), NCI-H1703 and NCI-H520 (both NSCLC SCC) and its respective nintedanib-selected sublines were determined by Western blot. ß-actin was used as loading control. E. Impact of short-term (24 and 48 hours) nintedanib exposure on ABCB1 expression in DMS114 and DMS114/NIN cells was analyzed by Western blot analysis. ß-actin was used as loading control. F. Impact of removal of nintedanib selection pressure on ABCB1 expression levels in DMS114/NIN cells was analyzed by Western blot analysis over 40 days. ß-actin was used as loading control.