Mitochondrial Oxidative Stress Assay

SH-SY5Y control, Parkin O/E and Parkin O/E;SREBP2(−/−) cells were grown in galactose-supplemented media prior to staining with MitoSOX Red™ (Invitrogen, USA) and rotenone treatment. Cells were co-stained with 5 μM MitoSOX, Hoechst (1:1000; Enzo Life Sciences, USA) and BODIPY 493/503 (Invitrogen, USA) (1:1000) in culture media for 10 min at 37°C. Cells were rinsed and imaged in HBSS without phenol red (Gibco, USA) supplemented with Ca²⁺ and Mg²⁺. Live confocal fluorescence imaging was performed on a spinning disc confocal (SDC) setup built around a Nikon Ti2 inverted microscope equipped with a Yokogawa CSU-W1 confocal spinning head, a Plan-Apochromat objective (100×, 1.45 NA) and a back-illuminated sCMOS camera (Prime 95B; Photometrics, USA). All image acquisitions were carried out using MetaMorph (Molecular Device, USA) with exposure time 500 ms. Automated MitoSOX intensity measurements were done using MatLab algorithm (66 (link)) and is available upon request.