## 3 Results
### SA-MIPs Affect Cell Cycle Progression  
To further study the effect on proliferation seen at the cell population level, individual longitudinal tracking of RAW 264.7 cells was performed. Cells were cultured in the presence or absence of SA-MIPs, and individual cells were tracked for 48 h. Cells in the first image of every capture position, timepoint 0 h, were identified visually (in total, 109 control cell families and 91 cell families cultured with SA-MIPs) and tracked until the next division. Supplementary Figure S1 shows an example of cell division and segmentation from App Suite. The resulting daughter cells of each cell division were identified and tracking continued, which made it possible to construct cell family trees and to calculate the time between cell divisions for individual cells. A schematic representation of different family trees is shown for RAW 264.7 cells grown in 0 \(\mu\)g/mL SA-MIPs (control, Figure 7a) and 40 \(\mu\)g/mL SA-MIPs (Figure 7b). The complete set of cell family trees can be found in Figure S2. Notable is that the number of cell divisions during the 48 h time-lapse differed between individual cells with the same treatment, and it was also apparent that the cell cycle time varied (Figure 8). Figure 7: Schematic cell family trees for RAW-264.7 cells. RAW 264.7 cells were incubated with culture medium only (**a**) or with 40 \(\mu\)g/mL SA-MIP (**b**) for 48 h. Holographic images were captured every 15 min at several positions, thus creating 48 h time-lapse movies. For every capture position, cells in the first image (0 h) were selected and tracked until division. In the schematic cell family trees, each forking of the line indicates a cell division. The resulting daughter cells of each cell division were identified, and tracking continued throughout the 48 h time-lapse. In total 109 control cell family trees and 91 SA-MIPs treated cell family trees were analyzed. SA-MIPs treatment decreased the number of dividing cells in the cell culture as well as causing cells to divide fewer times. For RAW 264.7 cells cultured in the presence of SA-MIPs, only 10% of the tracked cell families managed to divide three times, while 20% of the cells never divided, and 45% divided only once. For control cells, three divisions were seen for 46% of the control cell families and even four divisions for 17% of the control cell families (Figure 8a). Only 2% of the cells never divided. The mean time between cell divisions was 22.6 \(\pm\) 11.8 h, 16.1 \(\pm\) 8.5 h and 9.6 \(\pm\) 3.4 h, for the respective first, second and third cell cycles of cells cultured in the presence of SA-MIPs (Figure 8b). For cells cultured in a culture medium, the mean time between divisions was 14.3 \(\pm\) 8.9 h, 12.6 \(\pm\) 5.5 h and 22.3 \(\pm\) 5.0 h, respectively. The difference in cell cycle time was only significant for cell cycle 1 with \(p\) = 0.02, while no significant difference was observed for either cell cycle 2 or 3. This indicates that although only a minor number of cells were dividing upon incubation with SA-MIPs, these dividing cells show the same cell division time as the cells incubated with a culture medium (Figure 8b). Figure 8: Number of cell divisions and length of individual cell divisions in the presence or absence of SA-MIP. Cells were grown with 0 or 40 \(\upmu\)g/mL SA-MIPs for 48 h. Images were captured using DHC time-lapse. Cell families were tracked longitudinally using App Suite. Data obtained from longitudinal tracking of cells are presented in supplementary data Figure S1. (**a**) shows the relative numbers of cell families undergoing none, one, two, three or four divisions observed during 48 h for all tracked cells in the first timepoint of the experiment are presented. (**b**) shows the length (h) of individual cell cycles for RAW 264.7 cells with 0 or 40 \(\upmu\)g/mL SA-MIPs. In total 109 untreated cell families and 91 SA-MIPs-treated cell families were longitudinally tracked.