Zen blue 2

HEK-293T cells or TREx BCBL1-Rta cells expressing ORF11-FLAG were grown on sterilised glass coverslips treated with Poly-L-Lysine (Sigma-Aldrich). After 24 h cells were washed in PBS and fixed in PBS containing 4% (v/v) paraformaldehyde for 10 min, washed twice in PBS and permeabilised using PBS containing 1% Triton X-100 for 10 min^{58 (link),59 (link)}. Coverslips were then incubated with anti-FLAG or anti-LANA and Alexa Flour 546 (Invitrogen), goat anti-Rat IgG or goat anti-Rabbit IgG respectively, for 1 h each at 37 °C before being mounted onto microscope slides using Vectashield® with DAPI. Slides were visualised on a Zeiss LSM 700 laser scanning confocal microscope and images analysed using Zen® blue 2.0 (Zeiss).

Immunofluorescent (IF) staining for insulin was performed on human pancreatic sections adjacent to those used for in vitro autoradiography binding studies. Briefly, sections were stained using Insulin A SC-7839 (Santa Cruz, Dallas, TX, USA; goat-polyclonal 1:1000). The sections were then incubated with secondary antibody Alexa fluor 488 (Invitrogen, Carlsbad, CA, USA; donkey anti-goat; dilution 1:100). Tile scan images were acquired with a Zeiss LSM780 confocal microscope.
Immunohistochemical (IHC) staining for insulin on NHP pancreatic sections was performed by primary antibody A0564 (Agilent, Santa Clara, CA, USA). The stains were developed by the Envision DAB system K4010 using an antirabbit secondary antibody. All sections were counterstained using Mayer’s Hematoxylin. The slides were captured digitally in bright-field mode at × 10/0.45 magnification, using an Axio Imager 2 microscope (Carl Zeiss Microscopy GmbH, Germany) mounted with an Axiocam MRc camera. Images were analyzed in Zen Blue 2.0 (Carl Zeiss Microscopy GmbH, Germany).

All C. elegans micrographs excluding electron micrographs are of lateral views of hermaphrodite animals. Nomarski DIC and epifluorescence micrographs were obtained using a Zeiss Axio Imager Z2 compound microscope and Zen Blue (Zeiss) software. Confocal microscopy was performed using the Zeiss LSM 800 instrument, and images were obtained using the Zen Blue 2.0 (Zeiss) software. The resulting images were prepared using ImageJ (National Institutes of Health) and Adobe Illustrator CS4 softwares. Image acquisition settings were calibrated to minimize the number of saturated pixels and were kept constant throughout each experiment. Graphs and indicated statistical analyses were performed using GraphPad Prism 6 software. For electron micrographs, animals were assayed by picking L4 males to plates, fixing 24 h later, and performing electron microscopic analysis, as previously described.

A co-culture of CHO and Lec2 (fluorescently labeled with actin-mCherry) was incubated for 1 h on ice (to prevent internalization) with a 10⁸ particles mL⁻¹ solution of UV-inactivated SARS-CoV-2 virions coupled with a Atto488 NHS ester dye (see above). The cells were then rinsed three times with PBS and fixed with formaldehyde (4% in PBS for 15 min) (Invitrogen, Thermo Fisher Scientific). After a final wash with PBS, cells were imaged with laser scanning confocal microscopy (Zeiss LSM 980) using a 40x water objective. Images were analyzed with the Zen blue 2.3 software (Zeiss). A maximum intensity projection was performed to obtain a single image from the z-stack.

Cellular senescence was tested using a β-galactosidase staining kit (Cell Signaling, Frankfurt, Germany) according to manufacturer’s instructions. Briefly, cells were seeded at 1 × 10⁵/well in a 6-well-plate (Cellstar^®, Greiner Bio-One GmbH, Frickenhausen, Germany) and cultivated to sub-confluence of approximately 70%. Cells were fixed, rinsed with PBS (Life Technologies), and incubated with β-galactosidase staining solution overnight at 37 °C in a dry incubator (FD-53, Binder GmbH, Tuttlingen, Germany). Plates were covered with 70% glycerol (Sigma Aldrich), photographed with an inverted microscope (Zeiss, Oberkocheln, Germany) connected to an Axiocam camera, and data were collected using the Zen Blue 2.3 software (Carl Zeiss Microscopy GmbH, Göttingen, Germany).

SMCs stably expressing GFP or RFP in the nucleus were seeded into 2-well chamber slides with glass coverslip bottoms (Thermo Fisher Scientific) and cultured in DMEM with 10% FBS (as described earlier). Dox was added to induce nuclear lamin expression and incubated for 24 hours before examination by microscopy. The cell culture chamber slide was mounted into a CO₂ and temperature-controlled stage on a Zeiss LSM 800 confocal laser-scanning microscope controlled by Zen Blue 2.3 software (all from Zeiss). The cells were visualized for 24 hours at 37°C and 5% CO₂ with a Plan-Apochromat 20×/0.8 NA objective. Images of a 3 × 3 tiled field (~1.3 mm²) were captured every 10 minutes. Differential interface contrast (DIC) was acquired with the transmitted light detector (T-PMT; Zeiss) at the same time as the GFP signal. Composite images of 10 z-sections (1 μm sections) were generated and analyzed for NM ruptures. The number of cells with a NM rupture, defined by the escape of GFP (or RFP) in the cytoplasm, was divided by the total number of cells in a field.