Tetramethylrhodamine methyl ester perchlorate tmrm

The HDAC8 activator TM-2-51 (1-Benzoyl-3-phenyl-2-thiourea), the DRP1 inhibitor Mdivi-1 and cobalt chloride were purchased from Sigma-Aldrich (Oakville, Canada). The HDAC8-specific inhibitor PCI-34051 was purchased from Cayman Chemical (Burlington, Canada). Tetramethylrhodamine methyl ester perchlorate (TMRM) and Mito-Tracker^® Red CM-H2X ROS were obtained from Sigma-Aldrich and Invitrogen (Molecular Probes), respectively. Antibodies for mitochondrial voltage-dependent anion channel (VDAC) and p38 mitogen-activated protein kinases (p38) were purchased from Cell Signaling Technologies (Danvers, MA). Antibodies for H3K27ac, pan-histone H3 and HIF-1α were obtained from Active Motif (Carlsbad, CA), Bio Vision (Milpitas, CA) and Novus Biologicals (Littleton, CO), respectively. Antibodies for cytochrome C and DRP1 were obtained from Santa Cruz Biotechnology (Dallas, TX). The HDAC8-EGFP plasmid was prepared as previously described^{38 (link)}. The HDAC8-H180R-EGFP plasmid was constructed after cloning the mutant HDAC8 from hHDAC8-6His-pET20b-H180R^{62 (link)}, using the PCR primers: 5′-AGATTCTCGAGATGGAAGAACCGGAAGAACC-3′ and 5′-TTCAAGAATTCGAGAACCACGACCTTCGATAACA-3′ and inserting into the pEGFP-N1 vector using the XhoI and EcoR1 restriction enzymes.

Cells were seeded on 35 mm glass-bottom dishes (MatTek) coated with poly-d-lysine (0.1 mg ml⁻¹) (Sigma-Aldrich). The next day, cells were stained for 30 min with 100 nM mitotracker green (MTG) (Invitrogen) and 50 nM tetramethylrhodamine methyl ester perchlorate (TMRM) (Sigma-Aldrich), and then washed three times with corresponding cell culture media followed by imaging of the respective samples. The measurement of mitochondrial membrane potential was performed using a Perkin Elmer Spinning Disc microscope equipped with a 60× oil-immersion objective (numerical aperture 1.49) and a Hamamatsu C9100 camera using its standard software. Cells were maintained at 37 °C and 5% CO₂ while imaging. For each field of view, 20 optical sections were imaged where each optical section had a step size of 0.6 µm, which was sufficient to cover the thickness of the whole cell. TMRM and MTG were excited using 560 and 488 nm laser lines, respectively. The corresponding emission filters, used for collecting TMRM and MTG intensities, were 615 ± 35 nm and 527 ± 27.5 nm. Image analysis was performed using Fiji software. After background subtraction and maximum-intensity Z projection, regions of interest encompassing individual cells were marked using the polygon tool in Fiji, followed by determining the ratio of mean TMRM to MTG signal intensities per cell.