Mitochondrial Morphology Quantification in Infected Cells

Urothelial cells were intracellularly infected at an MOI 5–10 or mock infected as described above. Cells were washed in PBS and fixed in 4% paraformaldehyde for 20 minutes at room temperature followed by permeabilization in 1% Triton-X-100 for 5 minutes at room temperature. After blocking in 10% BSA, immunostaining was performed with anti-HSP60 (1:200; Cell Signaling Technologies; 12165S) and anti-rabbit IgG Alexa Fluor 546 (1:500; Thermo Fisher Scientific; A10040). Super-resolution mitochondrial images were acquired using a Nikon SIM microscope equipped with a 1.49 NA 100x Oil objective and Andor DU-897 EMCCD camera. Quantification of mitochondrial morphology was performed in NIS-Elements (Nikon) as described previously^{59 (link)}. Mitochondria were segmented in 3D and skeletonized using the resulting binary 3D mask. All mitochondria within each cell were averaged resulting in one data point per cell. Acquisition and analysis of mitochondrial imaging data was performed by a blinded experimenter.