Methods for investigating STAT3 regulation of lysosomal function in mammary epithelial cells

The transcription factor STAT3 is activated by multiple cytokines and other extrinsic factors. 21 It plays a key role in immune and inflammatory responses and, when dysregulated, in 22 tumourigenesis. STAT3 is also an indispensable mediator of the cell death process that 23 occurs during post-lactational regression of the mammary gland, one of the most dramatic 24 examples of physiological cell death in adult mammals. During this involution of the gland, 25 STAT3 powerfully enhances the lysosomal system to efficiently remove superfluous milk 26 producing mammary epithelial cells via a lysosomal-mediated programmed cell death 27 pathway. The lysosome is a membrane-enclosed cytoplasmic organelle that digests and 28 recycles cellular waste, with an important role as a signalling centre that monitors cellular 29 metabolism. Here, we describe key strategies for investigating the role of STAT3 in 30 regulating lysosomal function using a mammary epithelial cell culture model system. These 31 include protocols for lysosome enrichment and enzyme activity assays, in addition to 32 microscopic analyses of the vesicular compartment in cell lines. Collectively, these 33 approaches provide the tools to investigate multiple aspects of lysosome biogenesis and 34 function, and to define both direct and indirect roles for STAT3.