Mitopy1

300,000 HEK293T cells/well or 125,000 HepG2 cells/well were plated in four-well chamber slides (D35C4-20-1.5-N, Cellvis) precoated with 5 μg Poly-D-lysine (30-70 KDa, Alfa Aesar). After 20-24 hr, cells were pretreated with 10 μM PalmB, 5 μM ML348, or 2.5 μM mitoFP for 30 min at 37 °C. Control cells were pretreated with vehicle (DMSO). Hoechst 33342 and MitoTracker Deep Red were included for respective nuclear and mitochondrial visualization, as was 2 μM of the mitochondrial-targeted H₂O₂ probe, mitoPY1 (Sigma). After pretreatment, cells were briefly washed with DPBS, and treated with 100 μM H₂O₂ in fresh DPBS (400 μL) for 10 min at 37 °C. Control cells were untreated. Cells were then imaged on an inverted epifluorescence microscope. Analyses were performed in ImageJ (Wayne Rasband, NIH). For data analysis, the average fluorescence intensity per image in each experimental condition was obtained by gating cells using the brightfield image and applying that mask in the corresponding mitoPY1 image. All data was normalized to the average fluorescence intensity of the DMSO-pretreated control that was not exposed to H₂O₂. Each experiment was repeated in at least two biological replicates with identical results.

Total intracellular Cellular ROS was determined by staining the cells with carboxy-H₂DCFDA (Invitrogen) and mitochondrial ROS level was determined by using a specific mitochondrial H₂O₂ probe, MitoPY1 (Sigma) as previously described (Dickinson and Chang, 2008 (link); Dickinson et al., 2013 ). 2×10⁵ cells were incubated with 10 µM carboxy-H₂DCFDA or 10 µM of MitoPY1 for 30 min at 37°C. Cells were washed and analyzed by flow cytometry (BD FACSCanto). Intracellular NADPH level and GSH/GSSG ratio were determined using fluorimetric SensoLyte NADP/NADPH Assay (AnaSpec) and GSH/GSSG-Glo™ (Promega), respectively.