Data includes ET tilt series and reconstructions from samples prepared on 1.2.2029 and 9.10.2019. Sara Wickström, Kate Miroshnikova Primary human epidermal progenitor cells were grown on elastomere membranes exposed for 0 min, 30 min, 180 min or 360 min 40% stretch. Samples were fixed with 2.5% GA in 5 mM CaCl2, 0.1 M sodium cacodylate buffer, pH 7.4 at RT for 5 min, following 1 h fixation on ice. Following fixation all the steps were performed on ice. Samples were first washed 5 times with 0.1 M sodium cacodylate buffer and blocked for 15 min in 10 mM glycine/10 mM potassium cyanide in 0.1 M sodium cacodylate buffer (blocking buffer). Following blocking samples were rinsed 1X with 0.1 M sodium cacodylate buffer and then stained with 10 μM DRAQ5 in 0.1% saponin in 0.1 M sodium cacodylate buffer for 10 min, followed by washing 3x for 5 min with above-described blocking buffer. Samples were then incubated with 2.5 mM diaminobezidine tetrahydrochloride in 0.1 M sodium cacodylate buffer and placed onto a glass-bottom dish, monolayer side down, then mounted onto Zeiss confocal microscope surrounded by ice-packs to maintain a near 4°C temperature and imaged using a 63X oil immersion objective lens and 633 nm filter set to continuously illuminate the membranes for 10 min. Samples were then rinsed 3x for 5 min with 0.1 M sodium cacodylate buffer, following a 1 h stain with 1% osmium tetroxide, 2 mM CaCl2, 1.5% potassium ferrocyanide in 0.15 M sodium cacodylate buffer. Post staining, samples were washed twice for 2 min with double-distilled water, dehydrated in ethanol series prior to gradual infiltration into epoxy (TAAB 812). After polymerization in Epon overnight at 60°C, a pyramid was made using a razor blade on the area of cells of darker, photo-oxidized nucleus. 230-nm-thick sections were cut with a 35° diamond knife and collected on Pioloform-coated single slot copper grids. Dual axis tilt series were recorded from a semi-thick section using SerialEM software running on a Tecnai FEG 20 TEM operated at 200 kV, high tilt specimen holder and a 4k by 4k Ultrascan CCD camera. The tilt series were acquired at one-degree intervals between ± 62° at nominal magnification of 14,500x and 10-nm gold particles placed on both grid faces served as fiducial markers for alignment. Prior alignment and reconstruction with IMOD software package the images were binned by two providing a pixel size of 1.5 nm. The tomograms were reconstructed with a simultaneous iterative reconstruction technique using 14 iterations. Nava MM, Miroshnikova YA, Biggs LC, Whitefield DB, Metge F, Boucas J, Vihinen H, Jokitalo E, Li X, García Arcos JM, Hoffmann B, Merkel R, Niessen CM, Dahl KN, Wickström SA. Heterochromatin-Driven Nuclear Softening Protects the Genome against Mechanical Stress-Induced Damage. Cell. 2020 May 14;181(4):800-817.e22. doi: 10.1016/j.cell.2020.03.052. Epub 2020 Apr 16. PMID: 32302590; PMCID: PMC7237863. Fig S3E, videos S1, S2 and S3.

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