Immunofluorescence microscopy

WT and KO MEFs cells were cultured on cover slips coated with 0.1% gelatin in 6-well plates prior to the experiments. After starvation, the MEFs were fixed with 4% paraformaldehyde (PFA) in PBS for 15 min and blocked with 5% BSA in PBS containing 0.5% Triton X-100 for 20 min at room temperature and incubated overnight at 4°C with the primary antibody indicated in the figure legends. The cells were then washed with PBS for 3 times and incubated with secondary antibody for 1 h at room temperature. The images were obtained using a BZ-X700 deconvolutional microscope (Keyence, Osaka, Japan) or a CSU-W1 spinning disc confocal microscope (Nikon, Tokyo, Japan) with 2 Hamamatsu Flash 4 cameras at the Northwestern University Center for Advanced Microscopy facility, and data were processed with ImageJ 1.51w software. For hepatic mitochondria staining, neonatal livers from both WT and KO pups were harvested shortly after birth (~6 h) and stained with anti-Tom20 antibody. And images were acquired with CSU-W1 spinning disc confocal microscope as described above. For mitochondrial length quantification, images from both WT and KO (n = 15 for each group) with mitochondrial staining were analyzed for average mitochondrial length in ImageJ with a marco kindly provided by the Microscopy Core Facility at the University of Chicago. Intact whole mitochondrial length from 5 random areas in each image were manually measured to further validate the results by the marco. For mitochondrial quantification of elongation and fragmentation, both WT and KO cell images (n = 25 for each group, unless specified elsewhere) were subjected to a blind-scoring based on the mitochondrial morphology by 3 different viewers. The percentage for each sub-type of mitochondria with different categories (fragmented, intermedium or elongated) was calculated based on the scoring results.