Fig. 2

Anchorage-independent aggregation is inhibited in metaphase-blocked MCF-7 breast cancer cells. a Schematic representation of the synchronization procedure. Cells were incubated with nocodazole for 20 h, and MG132 was added to the medium for the last 30 min. Then, the culture medium was replaced by medium containing only MG132 for 1.5 h before mitotic shake-off and initiation of the aggregation assay of cells arrested in mitosis to monitor their clustering. b Control (untreated) and metaphase-synchronized MCF-7 cells were seeded in 96-well low-attachment plates and monitored by video-microscopy for 5 h. Representative transmitted light microscopy images of cell aggregation at the indicated time points. Segmentation (red line) was performed using a dedicated MATLAB software. Green lines correspond to the excluded holes, and blue to isolated cells. c Using the automated image processing data, the aggregate area was measured over time. The graph corresponds to the percentage of compaction calculated from the normalized area variation relative to the initial time point. Data correspond to the mean ± SD of 48 aggregates for each condition from 3 independent experiments. *P < 0.001 (Mann–Whitney non-parametric test)