P62-mediated inducer of mitophagy (10 μM; 0, 1, 3, 6, 24 hours) stabilizes Nrf2, and increases P62 expression (10 μM; 9 hours) to initiate mitophagy [1]. In MEFs, PINK1/Parkin signaling pathway is downstreamly acted upon by P62-mediated mitophagy (10 μM; 24 hours) [1]. Polyubiquitination and conjugation within the mitochondria are positively impacted by P62-mediated mitophagy inducers [1].

P62-mediated inducer of mitophagy (10 μM; 0, 1, 3, 6, 24 hours) stabilizes Nrf2, and increases P62 expression (10 μM; 9 hours) to initiate mitophagy [1]. In MEFs, PINK1/Parkin signaling pathway is downstreamly acted upon by P62-mediated mitophagy (10 μM; 24 hours) [1]. Polyubiquitination and conjugation within the mitochondria are positively impacted by P62-mediated mitophagy inducers [1].

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PMI (10 µM) stabilized Nrf2 protein levels in mouse embryonic fibroblasts (MEFs), with maximal levels observed at 6 hours post-treatment, which remained elevated at 24 hours. [1]
PMI (10 µM) increased the expression of Nrf2-dependent gene products heme oxygenase-1 (HO-1) and NAD(P)H quinone dehydrogenase 1 (NQO1) in mouse Hepatic1c7 cells over time, with peak cytoplasmic HO-1 at 6 hours and NQO1 at 24 hours. [1]
PMI induced NQO1 enzymatic activity with a CD (concentration causing a 2-fold induction) of 0.6 µM and a maximal 3.7-fold induction at 10 µM. [1]
PMI (10 µM) significantly increased p62 mRNA levels in MEFs after 9 hours of treatment, as measured by quantitative RT-PCR. [1]
PMI (10 µM, 24 hours) increased cytosolic P62 protein levels by 1.8-fold in MEFs compared to untreated controls, as shown by western blot. [1]
PMI (10 µM, 24 hours) did not increase the conversion of LC3B-I to LC3B-II in the cytoplasmic fraction of MEFs, either in the absence or presence of the autophagy inhibitor bafilomycin A1, indicating it does not trigger general macroautophagy. [1]
PMI (10 µM, 24 hours) reduced the size of the mitochondrial network in MEFs, as visualized by immunofluorescence staining of the mitochondrial F1-FO-ATP synthase β-subunit. [1]
PMI (10 µM, 24 hours) decreased the levels of the mitochondrial inner membrane protein MTCO1 (cytochrome c oxidase subunit I) in MEFs, as shown by western blot. [1]
PMI (10 µM, 24 hours) increased the level of LC3-II in the mitochondrial fraction of wild-type (WT) MEFs, but not in p62^-/- MEFs, as shown by western blot. [1]
PMI (10 µM, 24 hours) dramatically increased the colocalization of LC3B with mitochondria in WT MEFs under basal conditions, as measured by high-resolution confocal imaging. This increase was abolished in Nrf2^-/- MEFs. [1]
PMI (10 µM, 24 hours) substantially increased the colocalization of P62 with the mitochondrial network in WT MEFs under basal conditions, as measured by confocal imaging. [1]
PMI did not induce mitochondrial translocation of Parkin in MEFs, in contrast to the mitochondrial uncoupler FCCP. [1]
PMI (10 µM, 24 hours) increased mitochondrial recruitment of LC3 in MEFs with transient Parkin knockdown and in SH-SY5Y cells devoid of PINK1 (pink1 knockout), indicating its action is independent of a fully functional PINK1/Parkin pathway. [1]
PMI (10 µM, 24 hours) increased the level of poly-ubiquitination in mitochondrial fractions from MEFs. [1]
PMI (10 µM, 24 hours) increased the resting mitochondrial membrane potential (ΔΨm) in MEFs, as measured by TMRM fluorescence. The rate of FCCP-induced depolarization was not affected. [1]
PMI (10 µM, 24 hours) moderately increased mitochondrial superoxide production in MEFs (1.24-fold vs control), as measured by mitoSOX fluorescence, but did not alter cytosolic ROS levels measured by dihydroethidium (DHE). [1]

NQO1 Enzymatic Activity Assay: Hepatic1c7 cells were seeded in 96-well plates. After 12 hours, cells were treated with PMI or vehicle (final DMSO 0.1%) and incubated for 24 hours. The culture medium was aspirated and cells were lysed with a buffer containing 0.1% Tween-20 and 2 mM EDTA (pH 7.5). An enzyme reaction mixture containing Tris buffer, BSA, Tween-20, FAD, glucose-6-phosphate (G6P), NADP, G6P dehydrogenase, MTT, and menadione was added to each well. After 5 minutes at room temperature, a stop solution (SDS) was added. The absorbance at 595 nm was measured. Background was corrected using wells without cells. The ratio of optical densities (compound-treated/control) was calculated to determine induction of NQO1 activity. The concentration causing a doubling (CD) of control activity was determined. [1]

RT-PCR[1]
Cell Types: MEF
Tested Concentrations: 10 µM
Incubation Duration: 9 hrs (hours)
Experimental Results: Significant increase in p62 mRNA levels.

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Immunofluorescence[1]
Cell Types: MEF
Tested Concentrations: 10 µM
Incubation Duration: 24 hrs (hours)
Experimental Results: Demonstration that induction of P62 mitochondrial recruitment is Parkin-independent.

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Western Blot Analysis[1]
Cell Types: MEF
Tested Concentrations: 10 µM
Incubation Duration: 0, 1, 3, 6, 24 hrs (hours)
Experimental Results: Nrf2 levels reached maximum after 6 hrs (hours) and remained elevated at 24 hrs (hours).

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Mitochondrial Membrane Potential (ΔΨm) Measurement: MEFs were loaded with 100 nM TMRM in recording medium for 30 minutes at 37°C. Cells were washed and imaged using a confocal microscope. After recording basal fluorescence, 1 µM FCCP was added to induce depolarization. Mitochondrial regions of interest were selected, and TMRM fluorescence intensities were calculated. [1]
Reactive Oxygen Species (ROS) Analysis: For cytosolic ROS, cells were incubated with 5 µM dihydroethidium (DHE) in recording medium for 30 minutes at 37°C. For mitochondrial superoxide, cells were incubated with 5 µM MitoSOX Red under the same conditions. Cells were washed and fluorescence intensity was measured by continuous recording for at least 10 minutes using a confocal microscope. Mitochondrial regions of interest were selected for fluorescence quantification. [1]
Subcellular Fractionation (Mitochondrial Isolation): Cells were lysed in cold isotonic sucrose buffer by passing through a needle. Unbroken cells and nuclei were removed by centrifugation at 800 x g for 5 minutes at 4°C. The supernatant was centrifuged at 10,000 x g for 10 minutes at 4°C to pellet mitochondria. The resulting supernatant was collected as the cytosolic fraction. The mitochondrial pellet was washed once in isotonic buffer, recentrifuged, and then lysed in lysis buffer containing Triton X-100. [1]
Western Blotting: Protein concentrations were quantified. Equal amounts of protein were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked and incubated with primary antibodies (e.g., anti-LC3, anti-P62, anti-ubiquitin, anti-parkin, anti-MTCO1, anti-β-actin/tubulin as loading controls) overnight at 4°C. After washing, membranes were incubated with peroxidase-conjugated secondary antibodies. Blots were developed using enhanced chemiluminescence, and band densities were analyzed using ImageJ software. [1]
Immunofluorescence and Colocalization Analysis: Cells grown on coverslips were fixed, permeabilized, and blocked. They were incubated overnight at 4°C with primary antibodies (e.g., anti-β-subunit for mitochondria, anti-P62, anti-LC3, anti-parkin) in blocking solution. After washing, cells were incubated with fluorophore-conjugated secondary antibodies. Cells were mounted with DAPI-containing medium. High-resolution confocal images were acquired. The degree of colocalization between markers (e.g., LC3 and mitochondria) was quantified using appropriate software, calculating colocalization coefficients or normalized fluorescence values. [1]
Quantitative Real-Time PCR (qRT-PCR): Total RNA was extracted from cultured cells and purified. cDNA was synthesized from 1 µg of total RNA. Gene transcripts (e.g., p62/Sqstm1) were amplified using SYBR Green detection and gene-specific primers on a real-time PCR system. An absolute quantification method was used with a standard curve generated from known amounts of PCR product. The level of gene expression was expressed as a copy number. [1]

No significant cytotoxic effects were observed at the concentrations used in the experiment (e.g., 10 µM). [1]
The compound was designed to lack covalently binding motifs, thereby reducing its cytotoxicity to below that of covalent Nrf2 inducers (such as sulforaphane, which is cytotoxic at concentrations above 10 µM in MEF cells). [1]

[1]. PMI: a ΔΨm independent pharmacological regulator of mitophagy. Chem Biol. 2014 Nov 20;21(11):1585-96.

PMI is a pharmacological inducer of mitophagy. Its mechanism of action is to upregulate the autophagy adaptor protein P62 by stabilizing the transcription factor Nrf2, rather than by disrupting the mitochondrial membrane potential. [1]
Its mechanism of action is downstream of the classical PINK1/Parkin pathway and can function independently of this pathway. [1]
PMI is a prototype tool compound that can be used to study the mechanism of mitophagy, avoiding the confounding nonspecific effects of mitochondrial depolarizers such as FCCP. [1]
The chemical name of PMI is 1-(3-iodophenyl)-4-(3-nitrophenyl)-1,2,3-triazole. [1]

C14H9IN4O2

392.151334524155

391.977

1809031-84-2

122190591

Light yellow to yellow solid powder

3.5

0

4

2

21

379

0

N1(C2=CC=CC(I)=C2)C=C(C2=CC=CC([N+]([O-])=O)=C2)N=N1

LSVWEYNSNZJEGB-UHFFFAOYSA-N

InChI=1S/C14H9IN4O2/c15-11-4-2-5-12(8-11)18-9-14(16-17-18)10-3-1-6-13(7-10)19(20)21/h1-9H

1-(3-iodophenyl)-4-(3-nitrophenyl)triazole

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~8.33 mg/mL (~21.24 mM)

Solubility in Formulation 1: ≥ 1.25 mg/mL (3.19 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 1.25 mg/mL (3.19 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)