Spinning disk microscope

To visualize cell morphology over time, 10⁵ BMDMs per well were seeded onto a poly-L-lysine (Sigma)-coated eight-well slide chamber (ibidi). The next day, the cells were infected with UV-inactivated or live MNV (MOI 5), followed by incubation at 37°C and 5% CO2. After 1 hour of infection, the medium was removed, cells were washed with PBS and 1 ml medium with fluorescent dye was added. To visualize membrane integrity, the non-permeable DNA stain Propidium iodide (BD Bioscience) was added to the medium (50 ng/ml). Cells were imaged every 30 minutes over a time-period of 24 hours on a Zeiss Spinning Disk microscope using a 25x objective.

Cells were visualized using the 63 × water objective at 37 °C on the LSM SP8 (Leica) and on a spinning disk microscope (Zeiss). For the analysis of mitochondrial fission events, GFP-Drp1 stable HeLa cells were seeded at 1 × 10⁴ cells in 4-well LabTek dishes two days before imaging. Twenty-four hours before imaging, these cells were transfected as described with the mito-mNeptune plasmid. The cells were then imaged using a spinning disk microscope in live imaging medium (LIM, Molecular Probes). Time-lapse images were collected every 1 second for 5 min in a single focal plane. For FCS measurements on the LSM SP8, HeLa cells were likewise seeded in 4-well LabTek dishes at 1 × 10⁴ cells per well. For niclosamide (Sigma-Aldrich) treatment, GFP-Drp1 HeLa Kyoto cells were seeded at 5 × 10³ cells in 8-well LabTek dishes two days before imaging. Live cells were visualized in LIM under an LSM SP8 confocal microscope. Four or five Zstacks were taken under every condition (before niclosamide addition; just after addition; 10, 20 and 30 min after addition; 30 min after addition in control cells (without niclosamide addition) and in cells treated only with DMSO - data not shown). Niclosamide was used at a final concentration of 10 μM. Each Zstack step was 0.8 μm.

SABG activity was assessed using the SABG Staining kit (Cell Signaling Technology) following the manufacturer’s instructions. Images were taken using an optical microscope and analysed using ImageJ software. Staining of lipid droplets was performed as previously described^{72 (link)}. In brief, cells seeded in 24-well plates were fixed with 4% PFA for 15 min, then permeabilized and blocked for 45 min in 200 mM glycine, 3% BSA, 0.01% saponin and 1× PBS. After washing with PBS 1×, cells were incubated for 30 min with LipidTox Red (Thermo Fisher) diluted 1:200 in 0.1% BSA, 0.01% saponin and 1× PBS. All steps were carried out at room temperature. The coverslips were mounted using a mounting medium containing DAPI. Cells were imaged using a Spinning Disk microscope (Zeiss, Zen software) and analysed using ImageJ software.

HeLa cells were transfected with indicated cDNAs 36 h prior live recording and seeded in an eight-wells IBIDI cell chamber (80826; IBIDI). Imaging was performed with Zeiss Spinning Disk Microscope using a 63× oil-immersion objective. Tubule’s quantification was performed manually using Icy software (http://icy.bioimageanalysis.org) on z-projections. 4D images were taken with 3 μm depth and 10 s interval for 90 s.

The images of GFP-Drp1 stable HeLa cell lines (24c, 22p, 25p, 28p) transfected with mito-mNeptune for staining of the mitochondria were acquired using a Zeiss spinning disk microscope. For every cell line five fields of view were analyzed, what gave in total about 20 cells per cell line. The images obtained were analyzed using ImageJ to estimate the cytoplasmic and mitochondrial fractions of Drp1. The mitochondrial fraction of Drp1 was taken as the GFP-Drp1 signal limited to the area covered by the mitochondrial mask (Fig. 7A2). The mitochondrial mask was generated by single dilation of the binary image of the mitochondrial network (Fig. 7A1) obtained using the ImageJ Ridge detection plugin. The mask of the whole cell (Fig. 7A4) was generated based on the mito-mNeptune signal (Fig. 7B1) using ImageJ (“MinError” thresholding” combined with dilation and median filtering operations). The mask for the generation of the cytoplasmic Drp1 fraction was generated by subtracting the mitochondrial mask from the whole cell mask.