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Fig. 3 | Journal of Experimental & Clinical Cancer Research

Fig. 3

From: Trastuzumab emtansine delays and overcomes resistance to the third-generation EGFR-TKI osimertinib in NSCLC EGFR mutated cell lines

Fig. 3

Therapeutic potential of combining osimertinib and T-DM1 using different schedules of administration. a PC9 and PC9-T790M cells were treated with osimertinib (30 nM) (O) or T-DM1 (5 μg/ml in PC9 cells and 2.5 μg/ml in PC9-T790M cells) (T) on the basis of the following schedules: 72 h O and then 72 h O plus T (O → O + T); 72 h O and then 72 h T (O → T); 72 h O plus T and then 72 h O (O + T→O). Cell proliferation was assessed by crystal violet assay expressed as % versus control cells (###p < 0.001 vs osimertinib; $$$ < 0.001, vs T-DM1; one-way ANOVA followed by Bonferroni’s post-test). b cells were treated with increasing concentrations of T-DM1 for 72 h or with osimertinib for 72 h and then with T-DM1 for 72 h. Cell proliferation was assessed using crystal violet staining. Data are expressed as percent inhibition of cell proliferation versus control cells and are means ±SD of three separate experiments (*p < 0.05, ** < 0.01, ***p < 0.001, vs T-DM1; Student’s t test). c cells were treated with osimertinib for 72 h, T-DM1 for 48 h, or osimertinib for 72 h and then T-DM1 for 48 h, and analyzed by flow cytometry for cell cycle phase distribution. d cells were treated with osimertinib for 72 h and then with T-DM1, or with T-DM1 alone and at the indicated times cell death was quantitated by fluorescence microscopy analysis on Hoechst 33,342 and propidium iodide-stained cells. Data are expressed as percent values. (*p < 0.05, ** < 0.01, ***p < 0.001, vs T-DM1; Student’s t test). e PC9-T790M cells were treated with osimertinib for 72 h, or with T-DM1 for 48 h, or with osimertinib for 72 h and then with T-DM1 for 48 h, cells were lysed and Western blot analysis was performed on lysate proteins by using monoclonal antibodies directed to the indicated proteins. The results in a, c, d, e are representative of at least two separate experiments

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