## 3 Results
### Longitudinal Tracking of Effects of Salinomycin on Motility
After time-lapse imaging, the cells were fixed and stained for the expression of CD24 and E-cadherin and fluorescence images were acquired at the same field of view as the time-lapse. The tracked cells from the time-lapse were characterized as being either CD24\({}^{+}\) or CD24\({}^{-}\) and the E-cadherin expression was characterized as low, medium, or high, resulting in six groups. The cells that were tracked but that were not present in the last frame of the DHM time-lapse are called unknown (UN). Max motility speed (**A**,**B**) is the accumulated distance over the time of treatment (max motility) divided by the time of tracking. Average migration directness (**C**,**D**) is the migration, i.e., the shortest distance between the starting point of the tracking and the end point of the tracking, divided by motility. **(A)** Significant differences for the max motility speed was found between the CD24\({}^{+}\) and CD24\({}^{-}\) populations (\(p\) = 0.0008) of the control, and between cells with low or high expression of E-cadherin (\(p\) = 0.007). There was also a significant difference between CD24\({}^{+}\)E-cad\({}^{\text{mid}}\) cells and CD24\({}^{-}\)E-cad\({}^{\text{mid}}\) cells (\(p\) = 0.002). **(B)** Significant differences for max motility speed for the salinomycin-treated sample were found between cells with low or medium expression of E-cadherin (\(p\) < 0.0001), as well as those with high expression (\(p\) < 0.0001). There was also a significant difference between CD24\({}^{-}\)E-cad\({}^{\text{low}}\) cells and CD24\({}^{-}\)E-cad\({}^{\text{mid}}\) cells (\(p\) < 0.0001). **(C)** For average migration directness, significant differences were found comparing cells with low E-cadherin expression compared to medium expression (\(p\)-value = 0.008), and for CD24\({}^{+}\)E-cad\({}^{\text{low}}\) cells compared to CD24\({}^{+}\)E-cad\({}^{\text{high}}\) (\(p\) = 0.004). **(D)** No significant differences in average migration directness were found for the different groups of salinomycin-treated cells. **(A,B)** Salinomycin treatment significantly increases the max motility speed in all three E-cadherin groups (low: \(p\) < 0.0001, mid: \(p\) < 0.01, and high: \(p\) < 0.0001) compared to control. **(C,D)** Salinomycin treatment significantly decreased the average migration directness for cells with low E-cadherin expression (\(p\) < 0.0001) and mid E-cadherin expression (\(p\) < 0.001). All statistics were evaluated using a one-way ANOVA and Tukey honest significant differences. In the control (Figure 3A), there was a significantly higher max motility speed for cells with low expression of E-cadherin compared to cells with high E-cadherin expression (\(p\) = 0.007). The data also show that the max motility speed was higher in CD24\({}^{+}\) cells compared to CD24\({}^{-}\) cells (\(p\) = 0.0008) independent of E-cadherin expression. In cultures treated with 0.5 \(\upmu\)M salinomycin for 48 h (Figure 3B), the max motility speed for cells with low expression of E-cadherin was significantly higher than the max motility speed for cells with mid E-cadherin (\(p\) < 0.0001) and high E-cadherin (\(p\) < 0.0001) expression (Figure 3B). The CD24\({}^{-}\)E-cadlow cells had a significantly higher motility speed than CD24\({}^{-}\)E-cadmid (\(p\)-value < 0.0001). Altogether, in general, cells with low expression of E-cadherin had a higher max motility speed than cells with a higher expression of E-cadherin. Average migration directness is the migration, i.e., the shortest distance between the starting point of the tracking and the end point of the tracking, divided by motility (Figure 3C,D). As with motility speed, there was no difference between average migration directness comparing control and salinomycin-treated cells with unknown identity (Figure 3C,D).