Mitotracker deep red

Live cells were washed twice in PBS and incubated with 2 μg/ml BODIPY^493/503 (Life Technologies, Cat D3922) in PBS for 15 min at 37°C. For MitoTracker Deep Red staining, live cells were incubated with 0.5 μg/ml MitoTracker Deep Red (Invitrogen, Cat M22426) in PBS for 30 min at 37°C. After staining, the cells were washed in PBS and fixed in 3.5% PFA for 10 min. Then, the cells were washed and counterstained with Hoechst 33342 (Sigma, Cat B2261) for 10 min before being covered on glass slides for imaging. Images were observed and photographs were captured under an optical inverted fluorescence microscope (Nikon, TE2000-S).

For Confocal imaging, U2OS BAX^−/−BAK^−/− cells seeded on coverslips were transfected with (Halo-BOK, GFP-BOK or GFPBOK∆C) and (GFPSEC61 or mCherry-SEC61) and then treated with 10 µM QVD. 16 h after transfection, the cells were incubated with 150 nM MitoTracker Deep Red (Thermo Fisher) and HaloTag TMR Ligand (Promega) (when transfecting with Halo-BOK) for 20 min at 37 °C. The cells were then washed 3 times with fresh media and were fixed using Paraformaldehyde. Imaging was performed on a TCS SP8 confocal laser scanning microscope (Leica Microsystems) equipped with a PL Apo 63x/1.40 Oil CS2 objective and a tunable white light laser (470–670 nm). The signal was acquired with sensitive HyD detectors (Leica Microsystems).
For STED imaging, U2OS BAX^−/−BAK^−/− cells seeded on coverslips were transfected with Halo-BOK and Smac-GFP and then treated with 10 µM QVD. 16 h after transfection, the cells were incubated with 0.3 μM Janelia Fluor 549 HaloTag Ligand (Promega) and 150 nM MitoTracker Deep Red (Thermo Fisher) for 20 min at 37 °C. The cells were then washed 3 times with fresh media and were fixed using Paraformaldehyde. Images were acquired using TCS SP8 gSTED microscope (Leica Microsystems) equiped with HL PL APO 100x/1.40 Oil STED, a tunable white light laser (470–670 nm) and 750 nm depletion laser. The signal was acquired with sensitive HyD detectors (Leica Microsystems).

HT29 cells were seeded on µ-Slide 8 well glass bottom (ibidi, Fitchburg, WI, USA) at 3 × 10⁴ cells/well and cultured for 24–36 h before each experiment. Fluorescently labeled compounds TRZ2-PEG-Fluo and TRZ7-Fluo, and their modified versions without Ru atom, or vehicle control, were added to cells at 1 µM in Dulbecco’s phosphate-buffered saline (DPBS) (Thermo Fisher Scientific) for 15 min. Then, a washing step with DPBS was included and cells were subsequently labeled with MitoTracker™ Deep Red and Hoechst 33342 dyes (both from Thermo Fisher Scientific), according to supplier’s instructions. Unbounded dyes were removed by washing with DPBS. Cells were then imaged using a laser scanning confocal microscope (Leica TCS-SP5; Leica, Germany) and a two-photon–excited fluorescence microscope (Ti:sapphire laser; Spectra-Physics Mai Tai BB). Images were collected with 512 × 512 pixels at a scan rate of 100 Hz with the 488 nm Ar+ laser line for fluorescent compounds, the 633 nm He-Ne laser line (for MitoTracker™ Deep Red; Thermo Fisher Scientific), and the 780 nm with the Ti:sapphire laser (for Hoechst 33342). A 63 × 1.2 N.A. water immersion objective was used (HCX PL APO CS 63.0 × 1.20 WATER UV; Leica).

Quantitative analysis of mitochondrial membrane potential (ΔΨm), and mitochondrial superoxide production (mtROS) was carried out using 100 nM MitoTracker Deep Red and 5 μM MitoSOX Red reagent (both from Thermo Fisher Scientific, Waltham, MA, USA), respectively. Cytosolic ROS production was measured with 20 μM dihydroethidium (DHE) (Invitrogen Molecular Probes, Carlsbad, CA, USA). Sytox Red (500 nM, Thermo Fisher Scientific, Waltham, MA, USA) and PI (1.5 μg/mL) were used for exclusion of dead cells dyed with MitoSOX Red or DHE, and MitoTracker Deep Red, respectively. The samples were analyzed using a FACS Calibur flow cytometer as described above.

DDC and 1,10-phenanthroline (PTL) were purchased from MPbiomedicals (Irvine, CA, USA); Hoechst 33342 (H342), LysoTracker^® Green DND-26, MitoTracker Green, MitoTracker Deep Red, and ER-Tracker Red were from Molecular Probes Inc. (Carlsbad, CA, USA); and fetal bovine serum was from Gibco (Carlsbad, CA, USA). Other chemicals were from Sigma (Milwaukee, WI, USA).

Cell culture medium and supplements were purchased from Life Technologies (Grand Island, NY, USA). Antibodies against phosphorylated AKT (Ser473) (p-AKT) (#4060S), AKT (#2920S), phosphorylated GSK3β (Ser9) (p-GSK3β) (#9336S), GSK3β (#9832S), and β-actin (#3700) were obtained from Cell Signaling Technology (Beverly, MA, USA), and antibody against OPA1 (#612606) was obtained from BD Biosciences (San Jose, CA, USA). Chamber slides, goat anti-rabbit (#656120) and anti-mouse (#626520) secondary antibodies, Lipofectamine 3000 (#L3000015), and 4′,6-diamidino-2-phenylindole (DAPI) (#P36931) were obtained from Invitrogen (Carlsbad, CA, USA). MitoSOX Red (#M36008), tetramethylrhodamine methyl ester (TMRM) (#T668), MitoTracker Green (MT Green) (#M7514), and MitoTracker Deep Red (#M22426) (all from Molecular Probes, USA) were purchased from Life Technologies. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay kits (#11684795910) were obtained from Roche (Mannheim, Germany). HT (#H4291), H₂O₂ (#88597), 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8) (#T8325), LY294002 (#L9908), N-acetyl-l-cysteine (NAC) (#A7250), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (#M2128) were obtained from Sigma-Aldrich (St. Louis, MO, USA). ATP assay kit (#S0026) was obtained from Beyotime Institute of Biotechnology (Shanghai, China).