Airyscan

Cells were plated on a glass-bottom cell culture Petri dish (NEST,801001) and grew for 24 h followed by fixation using 4% paraformaldehyde (Pierce™ 16% Formaldehyde ((w/v)), Methanol-free, ThermoFisher, 28906) in 1 × PBS for 20 min at room temperature. After washing three times in 1 × PBS, cells were blocked with 4% IgG-free Bovine Serum Albumin for 20 min at room temperature. Permeabilization of cells was performed with 0.2% Triton X-100 (Sigma Aldrich, X100) in 1 × PBS for 10 min, followed by adding primary antibody 1:500 in 1 × PBS and incubating at 37 °C for 2 h. Cells were washed with 1 × PBS three times, followed by incubation with a secondary antibody 1:1000 in 1 × PBS for one hour. After washing three times, the nuclei were stained with 10 µg/ml DAPI (Beyotime). Images were acquired by a confocal microscope (Carl Zeiss, LSM 880 with Airyscan or Nikon A1 R) with a 100× objective. At least three different dishes were quantified per treatment type.

The mitochondrial structural network was detected by 200 nM Mitotracker Red and ER tracker Green (Cell Signaling Technology) in a culture medium at 37 °C for 30 min. Then, the cells were fixed with 4% paraformaldehyde, permeabilized in 0.2% Triton X-100 and then stained with other antibodies. Cells were imaged using a Zeiss 880 Laser Scanning Confocal Microscope with Airyscan and Nikon A1R N-SIM N-STORM Microscope. Acquired images for mitochondria morphology were analyzed^{16 (link)}. The colocalization indexes Pearson’s and Manders’ coefficients were calculated with the ImageJ colocalization analysis plugin according to previous articles^{86 (link),87 (link)}. MERCs were also reconstructed and analyzed via surface-surface contact site area tool by Imaris (Bitplane) followed the manufacturer instructions.