ATR mediates cisplatin resistance in 3D-cultured breast cancer cells via translesion DNA synthesis modulation

a Cellular viability analysis of cells exposure to different doses of cisplatin for 72？h, by XTT assays. b, c Cell death assessment in cells treated with cisplatin for 72 and 120？h, by fluorescence microscopy. A combination of dyes (fluorescein di-acetate, propidium iodide, and Hoechst) was used to distinguish viable cells from those in necrosis or apoptosis. c Cells were counted, and the percentages were plotted on a graph. d Cellular viability assay, in response to 72？h of cisplatin treatment, in MCF-7 cells cultured under 2D, 3D, or non-adherent 3D (with or without media supplemented with 4% Matrigel？) conditions. e Immunodetection of cisplatin–DNA adducts, by slot-blot assays, upon 1？h of treatment. f, g γ-H2AX detection, followed by flow cytometry, for evaluation of DNA damage induced by cisplatin (4？h). In all graphs, the results are presented as mean？±？SEM from at least two independent experiments performed in duplicate. Two-way analysis of variance and Bonferroni post hoc test were used for statistical analysis and the differences were considered significant for *P？≤？0.05 and ***P？≤？0.001. ns highlights non-significant changes. b The red arrows indicate necrotic cells (type of cell death mostly detected in MCF-7 cells) whose standard staining is characterized by the red nuclei and cytoplasm homogeneously stained with propidium iodide. The white scale bar correspond to 10？μ