Mitotracker red cmxros

For the mitochondrial transfer study, MitoTracker fluorescent probes were used to label active mitochondria in cells. hUC‐MSCs were grown to 80% confluence, washed with PBS to remove any residual platelet lysate, before staining and pre‐staining with 200 nm of MitoTracker Red CMXRos (40741ES50, Yeasen, China) in 10 mL of serum‐free α‐MEM; this was then incubated in darkened conditions for 45 min. Then, cells were washed five times with PBS, replaced with fresh serum‐free α‐MEM, and MSC‐EVs were extracted, as described previously.
The mitochondria of rPACs were stained with 200 nm MitoTracker Green FM (40742ES50, Yeasen, China) for 45 min. Cells were twice with PBS to remove excess dye; then, the MSC‐EVs (50 µg mL⁻¹) labeled with MitoTracker Red CMXRos were added and incubated for 6 h to allow EVs to internalized. After two washes with ice‐cold PBS, mitochondrial transfer was observed and photographed using an inverted confocal microscope (LSM780, Carl Zeiss, Germany) in the same Z‐axis plane (63×, oil lens).

Opti-MEM (31985088) was purchased from Gibco. MitoTracker Red CMXRos (40740ES50) was purchased from YEASEN. DAPI (C1002) and N-acetylcysteine (S0077) were purchased from Beyotime. 3-methyladenine (3-MA) (S2767) and bafilomycin A1 (Baf-A1) (S1413) were purchased from Selleck Chemicals. CCCP (C2759) (SAB5701328) was purchased from Sigma-Aldrich. Anti-Occludin (91131S), Claudin-1 (13255S), GAPDH (5174S), Parkin(2132S), and β-actin (3700S) were purchased from Cell Signaling Technology. Anti-P62 (T55546), ATG5 (T55766), and LC3B primary antibodies were purchased from Abmart.

Mitochondrial staining was conducted with the mitochondrial probe MitoTracker Red CMXRos (Yeasen, Shanghai, China) according to the manufacturer's protocols. Briefly, cells were incubated with CMXRos (500 nM) at 37°C in an incubator for 30 min. After washing with PBS, the cells were counterstained with DAPI for 10 minutes and imaged with an Olympus BX63 microscope (Olympus, Japan).

4T1 cells were treated by PBS, OxPt [40 µm], Mito‐FFa [8 µm], and FF [80 µm] for 6 h. The treated 4T1 cells were incubated with MitoTracker Red CMXRos (Yeasen, China) at 37 °C for 45 min and cells were next fixed with cold methanol at ‐20 °C for 10 min and permeabilized with 0.1% Triton X‐100. Then, 4% paraformaldehyde was used to refix cells at RT for 10 min, and NaOH solution ([50 mm] in 50% ethanol) was added to denature DNA. After washed by PBS three times, cells were blocked with 1% BSA/PBS, and incubated with an FITC‐8‐hydroxyguanine antibody (bs‐1278R‐FITC, Bioss) at 4 °C for 1 h. Then, nuclei were stained with DAPI (Beyotime, China). Finally, cells were observed with Olympus FV3000 LSCM.

SUVECs were seeded on the glass coverslips of the 35 mm diameter cell culture dishes and cultured to 20–30% confluency. Then, SUVECs were transfected with pEGFP-C1 or pEGFP-NS4A. The cells were further cultured for 36 h. Then, the cells were washed three times and were incubated with 1 μM ER-tracker Red (Beyotime, China) for 30 min at 37°C or incubated with 200 nM Mito-Tracker Red CMXRos (Yeasen, China) for 45 min at 37°C and washed with PBS for three times. Then, the cells were fixed with 4% paraformaldehyde for 20 min at room temperature and incubated with Hoechst 33342 for 10 min at room temperature. After three washes with PBS, cells were observed by laser scanning confocal microscopy (LSM510 META; Zeiss, Germany). ER-tracker Red is a red fluorescent probe of ER and used for marking the location of intracellular ER. Mito-Tracker Red CMXRos is a red oxidized fluorescent dye of mitochondria and used for marking the location of intracellular mitochondria. Hoechst 33342 is a blue fluorescent dye of nuclear DNA and used for marking the location of cell nucleus.

Cells were cultured and treated with tyrosol as described above. Mitochondrial membrane potential and ROS were stained using MitoTracker Red CMXRos and MitoSOX Red Mitochondrial Superoxide Indicator (both from Yeasen Biotech, China), respectively. Cells were labeled with MitoTracker Red CMXRos (final concentration, 100 nM) or MitoSOX Red (final concentration, 3 μM) at 37°C for 30 min. The cells were then fixed with 4% paraformaldehyde for 15 min, followed by nuclei staining using DAPI (Beyotime). Images were taken with DMI6000B (Leica) and fluorescence intensity was measured using ImageJ software. The results were expressed as the mean of relative fluorescence intensity per cell.