Mitopedpp

3‐[4‐(Perylenylphenylphosphino) phenoxy] propyltriphenylphosphonium iodide (MitoPeDPP) (M466, Dojindo Molecular Technologies, Inc.) is a cell‐membrane‐permeable probe that can penetrate the cell membrane and aggregate in mitochondria, and used to assess lipid peroxidation in mitochondria. After IL‐1β inducement and AB4 treatment, the cells were washed thrice with PBS, stained with 0.3 μmol/L MitoPeDPP and 10 μmol/L DAPI in the dark at 37°C for 15 min and detected under fluorescence microscope.

In AMCMs or NMCMs, cellular or mitochondrial iron levels were determined by FerroOrange staining (1 µm, DOJINDO, Japan) or Mito‐FerroGreen staining (5 µm, DOJINDO, Japan); cellular or mitochondrial lipid peroxides were determined by Liperfluo staining (5 µm, DOJINDO, Japan) or MitoPeDPP staining (0.5 µm, DOJINDO, Japan); cell ROS level was determined by CellROS staining (1 µm, Sigma‐Aldrich, USA) and mitochondrial superoxide level was determined by MitoSox staining (1 µm, Invitrogen, USA). Mitochondrial membrane potential was measured using JC‐1 staining (1 µm, Invitrogen, USA) for NMCMs and TMRM staining (1 µm, Invitrogen, USA) for AMCMs. Hoechst 33 342 were used for nuclei staining. All living cell dyes were used per manufacturer's instructions. Fluorescence intensities were captured and quantified using an Operetta CLS High Content Imaging System (Perkin Elmer, USA) or BD FACS Celesta flow cytometry (BD Biosciences, USA). All results were presented as relative intensity using vehicle group for normalization.

Mitochondrial lipid peroxidation was assessed using mitoPeDPP (Dojindo), a fluorescence probe that specifically detects lipid peroxides in the mitochondrial inner membrane. Cells were incubated with 0.5 μM mitoPeDPP solution for 30 min at 37 °C. After three washes with PBS, mitochondrial lipid peroxidation was fluorometrically measured using a BioTek Synergy 2 microplate reader at an excitation wavelength of 452 nm and an emission wavelength of 470 nm.

The H9c2 cells were incubated with Mito-FerroGreen (M489 Dojindo Laboratories, Kumamoto Prefecture, Japan) or MitoPeDPP (M466, Dojindo Laboratories, Kumamoto Prefecture, Japan) at 37 °C for 30 min out of the light. After incubation, the cells were washed with Hanks’ HEPES buffer, and the fluorescence was measured with an Olympus FV1000 LSCM under excitation and emission wavelengths of 470 nm and 525 nm.

The MitoPeDPP (Dojindo), a cell-membrane-permeable probe that specifically localizes in mitochondria due to the triphenylphosphonium moiety, will be used to assess lipid peroxidation in mitochondria. Cells were treated as indicated, then washed twice with PBS and stained with 1 μM MitoPeDPP and 10 μM Hoechst nuclear stain in certain experiments. Following that, images of cells were captured at random by a fluorescence microscope with 100 m scale bars.

Primary antibodies used for western blot include: ARA70 Polyclonal Antibody (Immunoway Biotechnology, YT0302), Anti-Glutathione Peroxidase 4 antibody (Abcom, ab125066); Anti-Ferritin Heavy Chain antibody (Abclonal, A1144); Anti-Nrf2 antibody (Abclonal, A0674); Anti-Mitochondrial Ferritin antibody (Immunoway Biotechnology, YN4227); Anti-β-Actin Mouse Monoclonal Antibody (Invitrogen); Anti-p62 Polyclonal antibody (Proteintech Group, 18420-1-AP); anti-LC3B Antibody (Merck-Sigma-Aldrich, L7543); Anti-GAPDH antibody (Abcom, ab8245); Anti-VDAC1/Porin Polyclonal antibody (Proteintech Group, 55259-1-AP); Anti-MFN2 Polyclonal antibody (Proteintech Group, 12186-1-AP); Anti-HA Rabbit mAb (Cell Signaling Technology, 3724); Anti-Parkin antibody (Abcam,ab77924). Secondary antibodies: horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Merck-Sigma-Aldrich, A9917), HRP-conjugated goat anti-rabbit IgG (Merck-Sigma-Aldrich, A0545).
Chemicals and reagents: MitoPeDPP (Dojindo, M466), Liperfluo (Dojindo, L248), FerroOrange (Dojindo, F374), DCFH-DA (Beyotime, S0033S), Mito-Tracker Red CMXRos (Beyotime, C1035), Mito-Tracker Green (Beyotime, C1048), JC-1 (Beyotime, C2003S), Ferrostatin-1 (Topscience, T6500), Erastin (Topscience, T1765), 3-methyladenine (MedChemExpress, HY-19312), carbonyl cyanide 3-chlorophenylhydrazone (CCCP, Merck-Sigma-Aldrich, C2759).