Search

Additonal file 5: Figure S5. Microdevice to study clustering at the single-cell scale. a Mask used for the fabrication of the silicon wafer. b One array of 9 PDMS micro-wells (outer diameter: 650 µm, inner diameter: 450 µm, and height: 200 µm) that are (c) glued on the bottom of the compartments of CELLviewTM cell culture dishes for monitoring by time-lapse video-microscopy.

Additional file 4: Figure S4. Aggregation ability of cells accumulated in mitosis independently of microtubule cytoskeleton disruption is also altered. a Flow cytometry analysis of control (untreated) cells, cells incubated with 6 µM RO3306 (RO3306) for 20 h, or cells at the indicated time points after RO3306 removal from the culture medium. The upper panels show the histograms of the propidium iodide fluorescence intensity (DNA content) and the lower panels show the dot plots of DNA content versus intensity for the detection of the mitotic marker 3.12.I.22 (see “Methods” section). The percentage of mitotic cells (shown in green) is indicated for each condition. b Schematic representation of the experimental design for the aggregation assay. c The percentage of compaction was calculated at each time point of the clustering assay in control cells and in cells collected by shake-off at 2 h after RO3306 removal. Data correspond to the mean ± SD of 32 aggregates in control and 35 aggregates in treated cells from 3 independent experiments.

Additional file 1: Figure S1. Synchronization procedure. a Flow cytometry analysis of control (untreated) and mitosis-arrested cells (nocodazole/MG132). Mitotic cells were detected with the mitotic-specific monoclonal 3-12-I-22 antibody. b Representative fluorescence microscopy images (DAPI and α-tubulin) of cells blocked in mitosis (nocodazole/MG132) and used for the clustering assays. Scale bar: 10 µm.

Additional file 2: Figure S2. Impact of incubation with MG132 alone on MCF-7 cells clustering. a Control (untreated) and MG132-treated 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. b 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 time 0. Data correspond to the mean ± SD of 48 aggregates for each condition from 3 independent experiments. a and b The data of the Fig. 2 obtained with metaphase-synchronized cells are shown for comparison.

Additional file 3: Figure S3. MCF-7 cell clustering occurs independently of microtubules cytoskeleton disruption. a Immunostaining of -tubulin on control untreated MCF-7 cells (Control) and cells incubated with 10 µM nocodazole for 2 h. For both conditions, each row corresponds to two different fields of view. Inserts show the higher magnification of the region outlined with dotted lines in the corresponding image. *mitotic cells in control and nocodazole conditions. Scale bar: 10 µm. b Schematic representation of the experiment. To test the impact of microtubule depolymerization on aggregation dynamics, MCF-7 cells were pre-treated with 10 µM nocodazole for 1 h, then they were seeded in 96-well low-attachment plates in presence of 10 µM nocodazole, and monitored by video-microscopy for 5 h (clustering assay). c Percentage of compaction calculated from the normalized area at each time points (see “Methods” section) for control (untreated) cells and cells incubated with nocodazole, as described in b. For each time point, data correspond to the mean ± SD of 32 aggregates/condition from 3 independent experiments.