Quantitative Analysis of Apoptosis Markers

All immunostained sections were digitally imaged using a Nikon CoolScope (Nikon). Immunohistochemistry of mouse liver was performed as described previously^{78 (link)} using (a) anti-phospho-IRE1α (Abcam), (b) anti-phospho-JNK (ThermoFisher Scientific), and (c) anti-cleaved lamin (small subunit, Cell Signaling Technology) antibodies and 3,3′-diaminobenzidine (DAB) as the chromogen. Phospho-IRE1α, phospho-JNK, and cleaved lamin A indices were calculated by dividing the DAB-positive area by the region of interest (ROI), and results are expressed as A.U. The cleavage of lamin is a well-characterized event in apoptosis^{79 (link)}. TUNEL staining was performed as previously described^{17 (link), 22 (link)} using the FragEL^TM DNA Fragmentation Detection Kit (EMD Millipore-Calbiochem) in accordance with the manufacturer’s instructions. TUNEL-positive cells within 0.145 mm² of the ROI were counted, and TUNEL indices were calculated as the number of TUNEL-positive cells per unit area (in mm²); results are expressed as A.U.