Fig. 5

Effects of the combined treatment of osimertinib and T-DM1 on cell growth, cell death and acquisition of osimertinib resistance in H1975 cells. a HER-2 protein levels on cell surface were evaluated by flow-cytometry in H1975 cells treated for 48 h with the indicated concentrations of osimertinib, quantified as MEF, and expressed as fold increase versus control. Mean values of three independent measurements (±SD) are shown (**p < 0.01, ***p < 0.001, ***p < 0.0001 versus control; one-way analysis of variance followed by Bonferroni’s post-test). b H1975 cells were treated with increasing concentrations of osimertinib in absence or presence of T-DM1 2.5 μg/ml. After 72 h cell proliferation was assessed by MTT assay and the effect of drug combination was evaluated using the Bliss interaction model. Data are expressed as percent inhibition of cell proliferation versus control cells and are means ±SD of three separate experiments. c Cells were treated with the drugs (osimertinib 100 nM, T-DM1 5 μg/ml) for 72 h and then cell death was quantitated by fluorescence microscopy analysis on Hoechst 33342 and propidium iodide-stained cells. Data, expressed as percent values, are means ±SD of three independent experiments (***p < 0.001 vs ctrl; ###p < 0.001 vs osimertinib; $$ < 0.01 vs T-DM1; one-way ANOVA followed by Bonferroni’s post-test). d Following the schedules indicated in Fig. 4 a H1975 cells were plated in 96-well plates at a density of 350 cells/well and treated with osimertinib 100 nM and/or 5 μg/ml T-DM1. H1975 cells were scored as resistant when they had reached >80% confluence. Results are representative of two independent experiments