Mitotracker deep red fm

MTA1 and ATP5A colocalization were assessed using immunofluorescence. Briefly, cells were cultured on sterilized coverslips. MitoTracker Deep Red FM (#8778, Cell Signaling Technology) was added to the growth medium at a final concentration of 500 nm, and the cells were incubated for 30 min at 37 °C. After incubation, the cells were fixed with 4% paraformaldehyde at room temperature for 15 min, rinsed with PBS three times for 5 min each, permeabilized with 0.25% Triton X‐100 at room temperature for 10 min; and then, blocked with 0.5% bovine serum albumin for 30 min. The coverslips with cells were subsequently incubated overnight with a mouse monoclonal MTA1 primary antibody (ab51266, Abcam) and rabbit antibody against ATP5A (14676‐1‐AP, Proteintech); and then, incubated with corresponding fluorescence‐conjugated secondary antibodies diluted in blocking buffer for 1 h. The coverslips with cells were finally mounted with mounting medium containing DAPI. Images were acquired using confocal laser scanning microscopy (GE, DeltaVision OMX V4/SR).

HeLa cells (8.0 × 10⁴ cells/well) were incubated in EMEM (+10% FBS, supplemented with penicillin/streptomycin) in six-well plates with a glass bottom for imaging (Chemglass, Cat. No. CLS-1812-006). Culture media was aspirated, cells were washed twice with PBS, EMEM lacking FBS was added to the wells, and cells were incubated at 37 °C for 16 h. Culture media was aspirated, and cells were washed twice with PBS. Wells were treated with CysOx1 or CysOx 2 (50 µM final concentration) and an organelle-selective dye (Invitrogen, ER-tracker red, Cat. No. E34250 or Cell Signaling Technology, Mitotracker Deep Red FM, Cat. No. 8778 S) in EMEM with 0.1% DMSO for 1 h at 37 °C. Selected wells were also treated with H₂O₂ (300 µM final concentration). After incubation, culture media was aspirated, cells were washed twice with PBS, fresh PBS was added, and analyzed using an Olympus FluoView IX81 confocal microscope using FV10-ASW software v3.0.

To check whether PDC-E2/p-STAT3 interaction occurs in mitochondria, NHC and Hep-G2 cells were treated with 100 µM and 250 µM GCDC, respectively. Then cells were incubated with 500 nM MitoTracker-Deep Red FM (#8778, Cell Signaling) for 30 min at 37 °C and 5% CO₂ followed by fixation, permeabilization and blocking processes as we described previously for PLA staining. Incubation with PDC-E2 (Santa Cruz, #271534) and p-STAT3 (S727) (Abcam, #30647) antibodies diluted 1:500 were carried out overnight at 4 °C. After washing with PBS, the incubation with secondary donkey anti-rabbit antibody conjugated with FITC (#711-095-152 Jackson ImmunoResearch, 1:500 dilution) and anti-mouse antibody conjugated with Rhodamine (#T15-295-150 Jackson ImmunoResearch, 1:500 dilution) was conducted for 1 h at RT. Then cells were mounted on slides using the Vectashield mounting medium with DAPI (#H-1200, Vector Laboratories, Burlingame, USA) to visualized cell nuclei and analyzed using Olympus FV 1000 confocal system with IX81 inverted microscope.

HeLa cells were seeded on 4-well chamber slides and infected with NDV at MOI of 1. Cells were fixed with 4% paraformaldehyde for 10 min, washed thrice with PBS, permeabilized with 0.2% Triton X-100 for 10 min, and washed thrice with PBS. Cells were then incubated with antibody (1:200) diluted in PBS (5% BSA) for 2 h, washed thrice with PBS, and then incubated with secondary antibody conjugating with FITC or TRITC (DAKO) for 2 h (1:200 diluted in PBS, 5% BSA), followed by PBS washing. Cells were next incubated with 0.1 μg/ml DAPI diluted in PBS for 10 min and rinsed with PBS. Finally, the specimen was mounted with glass cover slips using fluorescent mounting medium (DAKO) containing 15 mM NaN₃. Images were collected with a META 510 confocal laser-scanning microscope (Zeiss).
MitoTracker^® Deep Red FM (Cell signaling technology #8778) was diluted directly in DMEM to a final concentration of 0.5 μM and incubated with cells for 30 min at 37°C. After incubation, cells were fixed in ice-cold, 100% methanol for 15 min and rinse 3 times with PBS, followed by immunostaining.

Cultured cells, fibers, and muscle sections were fixed with acetone or 4% formaldehyde solution and blocked with 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) containing 5% goat serum. After blocking, they were stained with the following primary antibodies: anti-MyHC (clone MF20; eBioscience), cleaved caspase-3 (clone 5A1E; Cell Signaling Technology, Danvers, MA, USA), antilaminin-a2 (clone 4H8-2; Sigma), and anti-GFP (EMD Millipore, Billerica, MA, USA). After staining, they were incubated with a secondary antibody conjugated with Alexa-488 and -568 (Molecular Probes). After primary and secondary staining, EdU staining was performed using the Click-iT EdU Imaging Kit (Invitrogen), according to the manufacturer’s instructions. Nuclei were stained with 4,6′-diamidino-2-phenylindole (DAPI). Stained cells or sections were analyzed using a BZ-X810 fluorescence microscope (Keyence, Osaka, Japan). For mitochondrial analysis, MBs were incubated for 1 h with the MitoTracker® Deep Red FM (#8778; Cell Signaling), a dye that stains the mitochondria in live cells. Stained cells were analyzed using a confocal laser scanning microscope (SPF5; Leica).

HeLa FlpIn TRex cells with doxycycline inducible MTS-EGFP and PRKN were treated with RNAi for 96 h, while 0.25 µg/ml doxycycline was added 24 h prior to microscopy. For GTPP treated cells, doxycycline was added only during the 6 h treatment. The cells were then stained by 50 nM Mitotracker Deep Red FM (Cell signaling 8778) for 20 min in pre-warmed RPMI 10% FBS medium. Cells were washed with PBS and incubated in RPMI 10% FBS during measurements. The Yokogawa CQ-1 with 60x magnification and automated focus was used to take live-cell images with 488 nm excitation 525/50 nm emission for EGFP and 640 nm excitation 685/40 nm emission for Mitotracker Deep Red FM. 8 images with minimum 100 cells per biological replicate in total were analyzed by JACoP ImageJ plugin^{59 (link)}. The co-localization between MTS-EGFP and Mitotracker Deep Red FM was determined by thresholded M2 (tM2) Manders coefficient and gave an estimate to the amount of protein import into the matrix. The tM2 value was used as the inducible MTS-EGFP cell line contained also cells without detectable EGFP fluorescence.

Whole blood was removed by retroorbital bleed and stained for flow cytometry (BD FACSCanto II) using the following antibodies or fluorescent dyes: Biotin anti-mouse CD71 REAfinity (130-109-572, Miltenyi), anti-biotin-PE (130-113-291, Miltenyi), 0.0025 µg/µL PE-Cyanine7 Anti-Mouse TER-119/Erythroid Cells (116222, BioLegend), 0.0025 µg/µL APC-Cyanine7 Anti-Mouse CD45 (103116, BioLegend), 100 nM Thiazol orange (390062, Sigma Aldrich), 500 nM MitoTracker Deep Red FM (8778, Cell Signaling), and 2.5 µg/mL 7-AAD live-dead viability staining (420404, BioLegend).