The mechanical microenvironment of the cell is defined by the spatial confinement volume and elastic modulus of the substrate. As the PDMS membrane is approximately 5 mm thick for each device, a large damping of the applied force to lower the chamber is expected such that the total effective force at the chamber cell interface is dependent on the elasticity of the polymer matrix with minimal dependence on the force used to lower the chamber. In the case of a thin PDMS membrane the applied force would have a significant impact on the total effective force on the cell, however this is not the case in this system. In figure S2, we further investigate this by examining whether the load used to compress the cells affects the cell’s ability to deform the PDMS substrate. The numerical simulations do not show a difference between load and no-load conditions. Analysis Before extracting quantitative data on cell morphology the completeness of confinement was confirmed for each cell video by observing that no cells in the FOV transited under posts. In this way we ensured an expected and repeatable height of confinement was achieved. ImageJ was used to perform analysis on the images obtained experimentally. Specifically, the area function was used to calculate the areas of daughter cells after cytokinesis by manually outlining each daughter cell, after a complete cleavage furrow and separation was observed. Each mitosis event was manually identified and characterized. The onset of mitosis was defined as the point at which the cell lost its spindled morphology and became completely rounded. It should be noted that in highly confined cases, mitosis onset was identified by the appearance of a well defined circular cell edge, as most of the cells did not adopt the classical spindled morphology under high confinement. For statistical analysis, the population number (n) used corresponds to the total number of events observed in a single category of abnormality (e.g. multi-daughter divisions, uneven divisions, etc). These sample sizes were created by repeating each experimental condition two times, meaning two separate devices. The standard errors of the ‘per division’ metrics were found using the standard error of the estimate of a proportion. Confidence intervals between the mitotic duration times were performed with the Welch t-test, as the distributions did not have the same standard deviations. The confidence intervals in comparing References 1. Duncan AW, Taylor MH, Hickey RD, Hanlon Newell AE, Lenzi ML, et al. (2010) The ploidy conveyor of mature hepatocytes as a source of genetic variation. Nature 467: 707–710. doi:10.1038/nature09414. 2. Erenpreisa J, Cragg MS (2010) MOS, aneuploidy and the ploidy cycle of cancer cells. Oncogene 29: 5447–5451. doi:10.1038/onc.2010.310. 3. 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