Additional file 1:
Supplementary Figure S1. Synchronization procedure
a Schematic representation of the synchronization procedure. Cells were incubated with nocodazole for 20h, and MG132 was added to the medium for the last 30 min. Thereafter, the culture medium was replaced by medium containing only MG132 for 1.5h before mitotic shake-off and start of the aggregation assay of cells arrested in mitosis to monitor their clustering.
b 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.
c Representative fluorescence microscopy images (DAPI and alpha-tubulin) of cells blocked in mitosis (nocodazole/MG132) and used for the clustering assays. Scale bar: 10µm.
Additional file 2:
Supplementary Figure S2. 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: 650µm, inner diameter: 450µm, and height: 200µm) that are
(c) glued on the bottom of the compartments of CELLviewTM cell culture dishes for monitoring by time-lapse video-microscopy.
Additional file 3:
Supplementary Movie 1. Cell clustering in the microdevice
Time-lapse image acquisition of MCF-7 cells that express the LifeAct-mCherry fluorescent reporter during clustering. Transmitted and mCherry fluorescence images are merged. Movie duration: 3 hours. Scale bar: 50µm.
Additional file 4:
Supplementary Movie 2: Kinetics of one MCF-7 cell during clustering in a PDMS micro-well.
Fluorescence images from time-lapse acquisition of one control MCF-7 cell that expresses the LifeAct-mCherry fusion protein. On the right panel, the white line corresponds to the ROI used for morphometric parameter determination. Scale bar: 10µm.
Additional file 5:
Supplementary Movie 3: Cell kinetics of one MCF-7 cell incubated with CK666 during clustering in a PDMS micro-well.
Fluorescence images from time-lapse monitoring of one CK666-treated MCF-7 cell that expresses the LifeAct-mCherry fusion protein. On the right panel, the white line corresponds to the ROI used for morphometric parameter determination. Scale bar: 10µm.
Additional file 6:
Supplementary Movie 4: Cell kinetics of one latrunculin A-treated MCF-7 cell during clustering in a PDMS micro-well.
Fluorescence images from time-lapse monitoring of one latrunculin A-treated MCF- 7 cell that expresses the LifeAct-mCherry fusion protein. On the right panel, the white line corresponds to the ROI used for morphometric parameter determination. Scale bar: 10µm.
Additional file 7:
Supplementary Figure S3. Characterization of clusters in control and experimental conditions.
a, b Graphs showing the aspect ratio (a) and circularity (b) analysis results for the larger clusters formed in micro-wells in MCF-7 cells incubated or not (untreated, UNT) with nocodazole and MG132 (i.e., metaphase-synchronized/blocked, Met- sync), or with MG132 (MG) or nocodazole (Noco) alone after 3 hours of clustering. Each dot corresponds to the values in one micro-well from 5 independent experiments and bars correspond to the mean ± SD. c,d Determination of the average (µ) (c) and standard deviation (s) (d) aspect ratio in single MCF-7 cells incubated or not (untreated, UNT) with nocodazole and MG132 (i.e., metaphase- synchronized/blocked, Met-sync), or with MG132 (MG) or nocodazole (Noco) alone after 1 hour of aggregation. Each dot corresponds to one cell and the bars correspond to the mean±SD. Data are from 5 independent experiments with 5-6 cells analyzed per experiment. *, P<0.05; ** P, <0.01; ****, P <0.0001 (Mann Whitney non-parametric test).