A CellProfiler computational pipeline to quantify localization of PPM1H on mitochondria

Here we present a CellProfiler (1) software pipeline to quantify the localization of PPM1H on mitochondria. In this work, we have fragmented the mitochondria by hypotonic swelling to facilitate localization and highlight membrane contacts (2); PPM1H-mApple labeled pixels that coincide with GFP-Mito labeled pixels are scored. Wild-type MEFs expressing PPM1H-mApple and GFP-Mito are treated with oligomycin/antimycin (4 hours) and then incubated for 2 minutes in a hypotonic medium. Images of live cells are acquired using a spinning disk confocal microscope. The multi-channel Z-stack images are maximum intensity projected and used for CellProfiler analysis.