## 3 Results  
Evaluating the Effect of Salinomycin on Proliferation Using a Combination of Longitudinal Tracking of Time-Lapses Obtained with DHM and Fluorescence Microscopy  
By using DHM, we have previously shown that salinomycin treatment affects cell migration and cell proliferation of JIMT-1 cells [30, 41]. Herein, we decided to combine DHM with fluorescence microscopy to further increase our understanding of how salinomycin affects different subpopulations. Figure 2: Longitudinal tracking combined with fluorescence imaging of JIMT-1 cells cultured in the absence or presence of 0.5 \(\upmu\)M salinomycin. (**A**) Longitudinal tracking of digital holographic microscopy (DHM) time-lapses displayed as representative cell family trees based on the original data in Figure S2. The dashed lines in each panel separate the CD24\({}^{+}\) and CD24\({}^{-}\) cell populations as well as the unknown cells (UN, no label). The expression of E-cadherin is represented as colors of the end-circle in the 0-48 h panels, red = low, blue = medium, and green = high. O: known expression of fluorescence, X: unknown expression due to division before end of the time-lapse, clumping of cells or leaving the frame before the end of the time-lapse. (**B1–B4**) Proportion of JIMT-1 cells designated into different groups depending on their expression of fluorescence at 48 h. The data are compiled from all data shown in Figure S2. They are derived from 3 independent experiments with 2 independent time-lapses in each. The experiments were set up in two different ways (Figure 1), in which both salinomycin was added 24 h after seeding. For time-lapses 1 and 2, DHM images were acquired in two 24-h intervals, one starting immediately after drug-addition and the other 24 h after drug-addition (Figure 1A). For Time-lapse 3, DHM images were acquired for 48 h, starting immediately after drug-addition (Figure 1A). After DHM imaging, the cells were fixed and labeled for the expression of CD44 and CD24 or E-cadherin and CD24 with fluorochrome-conjugated antibodies. The cells were tracked throughout the DHM time-lapses (Figure 1B). The cells in the last frame of the time-lapse were then identified in the respective fluorescence image and the expression of each cell was evaluated (Figure 1C). Since CD44 is expressed on all JIMT-1 cells [30], the CD44 expression was only used to identify the location of the cells. Representative images from a DHM time-lapse and the fluorescence images can be found in Figure S1. The longitudinal tracking data was used to create cell family trees where the tracked cells were matched to their expression of the fluorescent markers. The complete set of cell family trees are found in Figure S2 and schematic representations of family trees are shown in Figure 2A. Supplementary Table S1 shows the maximal number of cell divisions for each family tree for each experimental set up (i.e., control 0-24, control 24-48, salinomycin 0-24, salinomycin 24-48, control 0-48, and salinomycin 0-48). If a cell family had two branches with different numbers of divisions, the higher number was counted. The first observation is that siblings had the same CD24 expression in all cases in both control and salinomycin-treated cultures (Figure 2A). Thus, it is evident that salinomycin treatment did not result in daughter cells with different CD24 expression. The data show that salinomycin treatment resulted in a relative increase in the CD24\({}^{+}\) population by inhibiting the proliferation of the CD24\({}^{-}\) population. Figure 2(A,B1-B4) show that there was an increase in the CD24\({}^{-}\) population in control cell cultures between 24 and 48 h compared to 0-24 h. The increase seems to be the result of more cell divisions in the CD24\({}^{-}\) population compared to the CD24\({}^{+}\) population. Table S1 shows that in salinomycin-treated cultures, no cell families were found to have completed three cell divisions.