DOI: 10.5061/dryad.ns1rn8pvv
title: Data for: SUN2 regulates mitotic progression in response to extracellular matrix rigidity

description: How cells adjust their growth to the spatial and mechanical constraints of
 their surrounding environment is central to many aspects of biology. Here
 we examined how ECM rigidity affects cell division. We found that cells
 divide more rapidly when cultured on rigid substrate. While we observed no
 effect of ECM rigidity on rounding or post-mitotic spreading duration, we
 found that changes in matrix stiffness impact mitosis progression. We
 noticed that ECM elasticity up-regulates the expression of the LINC
 complex component SUN2, which in turn promotes metaphase-to-anaphase
 transition by acting on mitotic spindle formation. Whereas when cells
 adhere on soft ECM, low levels of SUN2 expression perturb astral
 microtubule organization and delay the onset of anaphase.
description: <strong>Raw data corresponding to Figure 3A and 3B
 (quantifications are provided as Data and Videos as
 </strong><strong>Supplemental
 Information</strong><strong>)</strong>
 Quantification of the duration of mitosis phases in HeLa Kyoto
 EGFP-LaminA/H2B-mCherry cells transfected with control siRNA or siRNA
 targetting SUN2. Mitosis duration was calculated as the time from the
 first image showing the Nuclear Envelope Breakdown (NEBD) to the first
 image showing nuclear envelope reformation. Prometaphase duration was
 calculated as the time from the first image showing NEBD to the last image
 showing all chromosomes congression at the metaphase plate. Metaphase was
 calculated as the time from chromosomes congression at the metaphase plate
 to the image just before chromosomes segregation. Anaphase was calculated
 as the time from the first image showing chromosomes segregation to the
 first image showing nuclear envelope reformation. Data were collected from
 3 independent experiments (biological replicates). Table provides:
 biological replicate (n=1 to 3), condition name (control or SUN2 siRNA),
 first frame number and last frame number for each phase, number of frames
 for each phase and duration (min) of each phase, calculated as number of
 frames * 2.5 (2.5=frame rate, as one frame every 2.5 min)
 Live imaging was performed using spinning disk confocal
 microscope Andromeda TILL-FEI fitted with a Plan-Apochromat 20x/0.75, WD
 610 objective and coupled to a EMCCD camera iXon 897. The microscope was
 equipped with a temperature- and CO<sub>2</sub>-controlled
 incubator box (37°C and 5% CO<sub>2</sub>) and images were
 taken every 2.5 minutes for 15 hours minimum. Acquisition was performed
 with LA, FEI software and analysis with OA, FEI software and with
 ImageJ.<strong> </strong>4 avi videos: 2 control siRNA and 2
 SUN2 siRNA videos are provided as Supplemental
 Information.<strong><br></strong>
 <strong>Raw data corresponding to Figure 2B (western blot
 </strong><strong>as Supplemental
 Information).</strong>  A7R5 were cultured on 1
 or 50 kPa matrix for 6h, 24h and 5 days. Cells were lysed in Laemli and
 processed for western blot (SUN2 and GAPDH) to compare SUN2 expression.
 Images of uncropped gels for 2 replicates are provided (Western_Blot_1 and
 Western_Blot_2) for both SUN2 and GAPDH<strong> </strong>as
 Supplemental Information.