Fig. 2

Dose- and time-dependent detection of intracellular nintedanib accumulation by flow cytometry and live cell microscopy. a-c Time-dependent intracellular fluorescence activity of indicated concentrations of nintedanib in NCI-H1703 (a), DMS114 (b) and NCI-H520 cells (c) was measured by flow cytometry. Nintedanib was detected using the 488 nm laser. Signals are plotted as arbitrary units. Mean autofluorescence values were 6.3, 7.9 and 6.6 for NCI-H1703, DMS114 and NCI-H520, respectively. * p < 0.05, *** p < 0.001, 2-way ANOVA, Tukey’s post-test. ns, non-significant. Statistical significance is indicated by the asterisks and includes testing of all time-points between each drug concentration. d Intracellular accumulation of 10 μM nintedanib in NCI-H1703 cells over time was analyzed by live cell microscopy. The scale bar indicates 10 μm. e Quantification of nintedanib pixel intensities of representative micrographs from (D). Signals derived from individual cells are plotted as arbitrary units. The mean value for the 0 min control was 0.003. *** p < 0.001, 1-way ANOVA, Tukey’s post-test