Fig. 5

Lysosomal alkalization increases cytotoxic potential of nintedanib. a Impact of 1 h bafilomycin A1 pretreatment on the fluorescence activity of nintedanib in NCI-H1703 cells was measured after 1 h drug exposure by flow cytometry using the 405 nm and 488 nm lasers. ns, non-significant, *** p < 0.001, 2-way ANOVA, Tukey’s post-test. b Effect of 1 h bafilomycin A1 pretreatment on total intracellular nintedanib levels was determined at the indicated time-points by HPLC. <LOD, below limit of detection. *** p < 0.001, 2-way ANOVA, Tukey post-test. ns, non-significant. c Impact of 1 h bafilomycin A1 pretreatment on intralysosomal accumulation of 10 μM nintedanib was investigated at the indicated time-points by live cell microscopy. LysoTracker® Red served as marker for the lysosomal compartment. The scale bar indicates 10 μm. d Viability of NCI-H1703 lung cancer cells in the presence or absence of 10 and 25 nM bafilomycin A1 was analyzed by MTT assay after 72 h exposure to the indicated concentrations of nintedanib. *** p < 0.001, 2-way ANOVA, Tukey’s post-test. Asterisks indicate significance of difference at the respective nintedanib concentration points between control and both 10 nM and 25 nM bafilomycin A1. ns, non-significant; e Synergism of nintedanib/bafilomaycin A1 cotreatment of NCI-H1703 cells shown in (d) was determined calculating combination indices (CI) using CalcuSyn software. CI values below 0.9, between 0.9–1.1 or above 1.1 indicated synergism, additivity, and antagonism, respectively