Fig. 1
Motility and proliferation of BRAFV600E mutant MaMel cells. A Phase contrast micrographs of MaMel cells. Arrows indicate dendrite-like protrusions in the cells. B Flow cytometric analysis of MaMel cell proliferation rate, detected as a reduction of the fluorescence intensity of eFluor 670 dye. Representative histograms at different time points (T) are shown for each cell line. C Quantification of the median fluorescence intensity (MFI) of the eFluor 670 dye. Data from three independent experiments are shown (n = 3). D Determination of proliferative state of the cells. Left panel: representative flow cytometric analysis of Ki67 and DAPI intracellular co-staining, defining the cell cycle phases as indicated. Right panel: representative immunofluorescence images of Ki67 staining. Nuclei (DAPI) and cytoskeleton (tubulin) are stained for visualization of the cellular structure. E Quantification of the frequencies of cells in each cycle phase shown in D. M-phase was distinguished from the G2-phase by staining of histone H3 Ser28 phosphorylation (not shown). Data from five independent experiments are shown (n = 5). F Live cell tracking of MaMel cells. Representative polar plot showing ten randomly selected individual tracks for each cell line. G Speed (µm/h) and H net displacement (µm) of cells during 48 h, averaged from 80 individual cell tracks. I, J Representative real-time cell analysis (RTCA) of the I migratory and J invasive behavior of MaMel cells. K, L Quantification of the slopes (1/h) from curves of cell K migration and L invasion over time obtained by RTCA. Data from two independent experiments are shown (n = 2). All bar plots show the mean ± SD of the experiments. #p < 0.1, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bars: 200 µm