Super-resolution microscopy reveals LINC complex recruitment at nuclear indentation sites.

Microcontact printing Circular and rectangular (aspect ratio of 1:10) microfeatures of 1600 μm 2 were drawn with the Clewin software and generated to a silicon master by deep reactive-ion etching (FH Vorarlberg University of Applied Sciences, Microtechnology, Dornbirn, Austria). The silicon surface was passivated under vacuum with vapors of fluorosilane (tridecafluoro-1,1,2,2-tetrahydrooctyl-1-trichlorosilane) for 30 min, and then molded with polydimethylsiloxane, PDMS, (Sylgard 184 Silicone Elastomer Kit; Dow Corning, Midland, MI). After 4 h of curing at 60°C, the PDMS layer was peeled off and stamps of 1 cm 2 were cut manually. The structured surface of the PDMS stamps were first oxidized in an UV/O 3 cleaner for 8 min and then inked for 1 h at room temperature with a 25 μg.ml −1 fibronectin (FN) solution from human plasma. FN-coated stamps were dried under filtered nitrogen and gently deposited on a flat PDMS-coated glass coverslip for 15 seconds, as described previously 42 43 . Uncoated regions were blocked by incubating micropatterned coverslips for 5 min in a 1% solution of Pluronic F-127 44 .

Cell culture

Primary human umbilical vein endothelial cells, HUVECs (Cells Applications, San Diego, CA) were grown in complete endothelial cell growth medium (Cells Applications, San Diego, CA) supplemented with 1% antibiotics and antimycotics and maintained at 37°C in a humidified atmosphere with 5% CO 2 . Cells between passages 2 and 8 were cultured on microprinted PDMS coverslips at a concentration of about 15,000 cells ml −1 . Immunohistochemistry and labeling After 24 h in culture on micropatterned substrates, HUVECs were fixed and permeabilized with 4% paraformaldehyde, 0.05% Triton X-100 in Phosphate buffered saline (PBS) for 15 min at 37°C and then washed three times in PBS. Fixed cells were incubated for 30 min at room temperature in a blocking solution (FBS 5%, BSA 1%) and labeled for F-actin (Alexa Fluor 488 Phalloidin 1:200, Molecular Probes), nuclear lamina (anti-lamin A/C antibody produced in mouse 1:200, Sigma-Aldrich SAB4200236) and LINC-complex (anti Syne-2 antibody produced in rabbit 1:100, Sigma-Aldrich HPA003435) for 45 min at 37°C in PBS. After three successive washes in PBS, cells were incubated for 45 min at 37°C with an anti-mouse antibody produced in goat and labeled with tetramethylrhodamine (Molecular Probes, Invitrogen, T2762), an anti-mouse antibody produced in rabbit and labeled with fluorescein isothiocyanate (Sigma-Aldrich, F9137) or an anti-rabbit antibody produced in goat an labeled with tetramethylrhodamine (Sigma-Aldrich, T6778). All secondary antibodies were used in a 1:200 concentration in PBS. Slides were mounted in Slow Fade Gold Antifade (Molecular Probes). Living cell relaxation To observe nuclear DNA in living cells, HUVECs were transduced with the BacMam 2.0 CellLight Histone 2B-GFP according to the manufacturer's protocol (Molecular Probes). Routine culture flasks were incubated with approximately 20 BacMam particles/cell for at least 24 hours. Labeled cells were then detached and grown on rectangular FN micropatterns (1:10 aspect ratio) for 24 hours and finally transferred to an inverted confocal microscope equipped with a temperature and CO 2 level controller. The GFP fluorescence signal was recorded during the relaxation of the elongated cells, which was induced by the addition of 1 ml of accutase after a gentle PBS wash.

Structured illumination microscopy and confocal imaging

Immunostained preparations were observed in Structured Illumination Microscopy (SIM) performed on an Eclipse Ti inverted microscope equipped with a Nikon Plan Apo ×100 TIRF objective (NA 1.49, oil immersion) and an Andor DU-897X-5254 camera. Z-step size was set to 0.120 um, which is well within the Nyquist criterion. For each focal plane, 15 images (5 phases, 3 angles) were captured with the NIS-Elements software. SIM image processing, reconstruction and analysis were carried out using the N-SIM module of the NIS-Element Advanced Research software. Confocal imaging was performed with an inverted Nikon Eclipse Ti-E motorized microscope (Nikon C1 scanhead; Nikon, Japan) equipped with Plan Apo ×60 and ×100 (NA 1.45, oil immersion) objectives, two lasers (Ar ion 488 nm; HeNe, 543 nm) and a modulable diode (408 nm). Confocal images were acquired with NIS Elements Advanced Research 3.0 software (Nikon) by using small Z-depth increments between focal sections (0.150 μm).

Image analysis

Confocal and SIM images were acquired and analyzed with the NIS-Elements Advanced Research 3.0 software. Three-dimensional normal views were obtained in maximum intensity projection mode. Unless otherwise stated, orthogonal views were obtained in maximum intensity projection mode and cropped to represent a one micron-length of the nucleus. LINC complexes quantifications were obtained after the thresholding and the binarization of SIM slides or Extended Depth of Focus images for actin co-localization or quantification on the whole nuclear projected area, respectively. Nuclear compression experiments We used circular glass coverslips of 22 mm in diameter, 170 μm thick and 0.136 grams to apply a homogeneous pressure on the nucleus. After 20 hours in culture on FN-coated circular micropatterns, a glass coverslip was placed over HUVECs during 6 hours. The top side of glass coverslips was coated with a 1% solution of pluronic F-127 to avoid cellular adhesion and exerted a normal pressure proportional to its weight.

Data presentation and statistics

Results are presented as the means ± Standard Deviation (SD). Student's two-tailed t -test was performed in Origin 8.0 where two groups were compared. Values of p < 0.05 were considered statistically significant.

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