Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Impeding Nucleotide‐Binding Oligomerization Domain‐Like Receptor 3 Inflammasome Ameliorates Cardiac Remodeling and Dysfunction in Obesity‐Associated Cardiomyopathy

doi: 10.1161/JAHA.124.035234

Figure Lengend Snippet: A through H , NRVMs treated with PBS or MitoTEMPO were incubated with BSA or BSA+PA medium for 24 h. A , Schematic diagram showing the procedure of NRVMs. B , Intracellular total ROS using DCFH‐DA staining (upper) and mitochondrial ROS using MitoSOX staining (lower) were determined in NRVMs. C , Cell lysates of NRVMs were immunoprecipitated with TXNIP antibody, and immunoblot assays were performed using NLRP3, TXNIP, and TRX antibodies. NRVMs were treated with siTXNIP to knockdown of TXNIP. D , Triple immunofluorescence staining for TXNIP (red), NLRP3 (green), and nuclei (DAPI, blue) was performed in indicated NRVMs. E , Representative immunoblots of ASC, procapase‐1, cleaved caspase‐1 p20, IL‐1β, and IL‐18 protein in NRVMs from indicated groups. F , Oil Red O staining of NRVMs from indicated groups. Red indicates lipid droplets, blue indicates nuclei. G , qRT‐PCR detection of indicated genes related to fatty acid transport genes Cd36 and fatty acid oxidation genes Cpt1b , Acadl , and Acadvl in hearts from indicated mice (n=6 independent experiments). H , Expression level of hypertrophic marker gene BNP was determined by RT‐PCR and normalized to that of GAPDH (n=6 independent experiments). ASC indicates apoptosis‐associated speck like protein; BNP, B‐type natriuretic peptide; BSA, Bovine Serum Albumin; IL, interleukin; IP:TXNIP, immunoprecipitation:thioredoxin‐interacting protein; ns, not significant; NLRP3, nucleotide‐binding oligomerization domain‐like receptor 3; NRVM, neonatal rat ventricular myocyte; PA, palmitic acid; qRT‐PCR, quantitative real‐time polymerase chain reaction; and ROS, reactive oxygen species.

Article Snippet: Selective NLRP3 inhibitor MCC950 sodium (HY‐12815A) and mitochondria ROS scavenger MitoTEMPO (HY‐112879) were obtained from from Medchem Express (Shanghai, China).

Techniques: Incubation, Staining, Immunoprecipitation, Western Blot, Knockdown, Immunofluorescence, Quantitative RT-PCR, Expressing, Marker, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Real-time Polymerase Chain Reaction

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Impeding Nucleotide‐Binding Oligomerization Domain‐Like Receptor 3 Inflammasome Ameliorates Cardiac Remodeling and Dysfunction in Obesity‐Associated Cardiomyopathy

doi: 10.1161/JAHA.124.035234

Figure Lengend Snippet: A through L , WT mice received NS or MitoTEMPO were subjected to ND or HFD feeding for 24 wks. A , Experimental schematic diagram showing the process of HFD‐induced obesity cardiomyopathy. After 12 wks of HFD feeding, MitoTEMPO was injected into obese mice at a dose of 10 mg/kg per day for 12 wks. B , C , LV fractional shortening ( B ) and ratio between mitral E wave and A wave ( C ) were measured by echocardiography in indicated mice (n=12 per group). D , Histogram of HW/TL in indicated mice. HW indicated wet weight (n=12 per group). E , Representative images of cardiac WGA staining, Oil Red O staining, and Masson trichrome staining in perivascular and interstitial area in indicated heart. F , Quantification of cardiomyocyte cross‐sectional area by WGA (n=6 per group). G , Quantification of LV collagen volume in interstitial area by Masson trichrome staining (n=6 per group). H , Quantification of myocardial lipid accumulation by Oil Red O staining (n=6 per group). I , Tissue lysate in indicated heart were immunoprecipitated with TXNIP antibody, and immunoblot assays were performed using NLRP3, TXNIP, and thioredoxin antibodies. J , K , Protein of IL‐1β ( J ) and IL‐18 ( K ) in the heart of WT mice fed an HFD or ND for 24 wks with or without MitoTEMPO treatment. IL‐1β and IL‐18 protein were detected by ELISA. Values were normalized to total protein level (n=6 per group). L , Representative immunoblots of ASC, procapase‐1, cleaved caspase‐1 p20, IL‐1β, and IL‐18 in the indicated heart. ASC indicates apoptosis‐associated speck like protein; HFD, high‐fat diet; HW/TL, heart weight/tibia length ratio; IL, interleukin; IP:TXNIP, immunoprecipitation:thioredoxin‐interacting protein; LV, left ventricular; ND, normal diet; ns, not significant; NLRP3, nucleotide‐binding oligomerization domain‐like receptor 3; NRVM, neonatal rat ventricular myocyte; ORO, Oil Red O; NS, normal saline; WGA, wheat germ agglutinin; and WT, wild type.

Article Snippet: Selective NLRP3 inhibitor MCC950 sodium (HY‐12815A) and mitochondria ROS scavenger MitoTEMPO (HY‐112879) were obtained from from Medchem Express (Shanghai, China).

Techniques: Injection, Staining, Immunoprecipitation, Western Blot, Enzyme-linked Immunosorbent Assay, Binding Assay, Saline

Journal: Biomedicines

Article Title: A Mitochondrial Perspective on Noncommunicable Diseases

doi: 10.3390/biomedicines11030647

Figure Lengend Snippet: Antioxidants targeting mitochondrial oxidative damage.

Article Snippet: ROS scavenger/ROS generation inhibitor , MitoQ , Blocks the generation of ROS and mitochondrial protein thiol oxidation , Nervous system diseases; Endocrinology and metabolic disease; Tumors; Cardiovascular diseases , Clinical Phase II , [ , , ] .

Techniques:

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: HDAC class I inhibitor domatinostat sensitizes pancreatic cancer to chemotherapy by targeting cancer stem cell compartment via FOXM1 modulation

doi: 10.1186/s13046-022-02295-4

Figure Lengend Snippet: Domatinostat affects PDAC stem cells by modulating oxidative stress. A. The effect of domatinostat (0,5 μM) on PANC1 and ASPC1 spheroid cultures. Cells (1000/mL) seeded in a matrigel drop and sphere medium, were treated with and without domatinostat and collected 7 days after treatment. Images of one spheroid for each condition in a representative experiment is shown (white scale bar: 50 μm, magnification 20X). On the right, bar graphs show the numbers of spheroids for well (mean ± SD of 2 or more separate experiments each one with technical triplicate). B. PANC1 and ASPC1 spheroids viability treated with and without domatinostat (0.5 μM and 1 μM) was assessed by cell titer luminescence assay (see ) (mean ± SD of 2 or more separate experiments each one with technical triplicate). C. Flow cytometry assay shows CD133 protein expression decrease after domatinostat (0.5 and 1 μM) treatment for 16 h in PANC1 and ASPC1 cells. D. qRT-PCR analysis shows Oct-4 levels drop when PANC1, and ASPC1 spheroids are treated with domatinostat (0.5 μM) for 16 h. E. Cellular ROS production is visualized by Hydroethidine (HE) staining. PANC1 and ASPC1 spheroids were treated with domatinostat (0.5 μM) alone and in combination with N-acetylcysteine (NAC, 5 mM), as ROS scavenger, at the indicated timing. Cells were stained for HE as described in Material and Methods section and visualized by flow cytometry. F. Mitochondrial ROS amount is analyzed by mitosox staining. PANC1 and ASPC1 spheroids treated with or without domatinostat (0.5 μM) alone at the indicated timing were fixed, stained for mitosox (red) and measured by Opera Phenix confocal microscopy. The mitosox positive cells are counted by Harmony software as described in Material and Methods section. Representative images (20X magnification) show stained cells (red) and mitosox counted positive cells (green). G.-H. The observed increase in ROS amount is related to an increase of apoptotic cancer stem cells upon domatinostat treatment. PANC1 and ASPC1 spheroids, treated as previously, were stained for AnnexinV-FITC and CD133-APC as described in Material and Methods section and visualized by flow cytometry. In G. PANC1 and ASPC1 spheroids were treated with domatinostat (0.5 μM) alone and in combination with NAC, 5 mM, as ROS scavenger, at the indicated timing. In H. PANC1 and ASPC1 spheroids were treated with domatinostat (0.5 μM) alone and in combination with Mitoquinone mesylate (MitoQ) 100 nM, as mitochondrial ROS scavenger, at the indicated timing. (Statistically significant results by ANOVA test are reported *** indicates P < 0.0001, ** indicates P < 0.005 and * indicates P < 0.05)

Article Snippet: Intriguingly, apoptotic effect induced by domatinostat was almost completely reverted by concomitant treatment with the mitochondria ROS scavenger mitoquinone mesylate (MitoQ) (Fig. H).

Techniques: Luminescence Assay, Flow Cytometry, Expressing, Quantitative RT-PCR, Staining, Confocal Microscopy, Software

Journal: Nature Communications

Article Title: Ether phospholipids are required for mitochondrial reactive oxygen species homeostasis

doi: 10.1038/s41467-023-37924-9

Figure Lengend Snippet: a Mitochondrial ROS were measured by MitoSOX staining and flow cytometry in the indicated cells grown for 60 hr in DMSO or 10 nM IACS-010759. The experiment was repeated independently three times and representative results were shown. b Quantification of mitochondrial ROS (MFI comparison) in sensitive (PATC66/108) and resistant (PATC124/148) groups after 60 hr treatment with 10 nM IACS-010759. Three biologically independent replicates per cell line. Data represent mean ± S.D between the sensitive group (2 cell lines) and resistant group (2 cell lines). c – f Cells were transfected with the hydrogen peroxide indicator pCS2+MLS-Hyper7 (mitochondrial matrix-targeted) ( c ) and pCS2 + HyPer7-NES (cytoplasm-targeted) ( e ), and treated with 10 nM IACS-010759 at the indicated times. Mitochondria were defined by staining with an antibody against the mitochondrial outer membrane protein TOMM20. Nuclei were stained with Hoechst 33578. Scale bar, 10μm. d Quantification of fluorescence intensity in ( c ). f Quantification of fluorescence intensity in ( e ). For ( d ) and ( f ), at least 50 cells with positive green fluorescence from three biologically independent replicates were calculated for each group. g Cell death was detected by propidium iodide staining and flow cytometry in PATC124 cells treated with DMSO or 10 nM IACS-010759 for 2 days, in the presence or absence of the mitochondrial-ROS inducer MitoPQ (10 μM). Data represent mean ± S.D of three biologically independent replicates. h Cell death in PATC66 cells treated with DMSO or 10 nM IACS-010759 for 3 days, in the presence or absence of the 1 μM mitochondrial ROS scavenger, MitoQ. Data represent mean ± S.D of three biologically independent replicates. i , j Mitochondrial ROS ( i ) and cell viability ( j ) were detected by flow cytometry in PATC124 cells infected with control sgRNA (sgCTRL) or sgRNA targeting SOD2 (sgSOD2) following treatment with10 nM IACS-010759 for 3 days. Data represent mean ± S.D of three biologically independent replicates. Statistical analysis by two-tailed Students’ unpaired t test with significance indicated ( b , d , f , g – j ). Source data are provided as a Source Data file.

Article Snippet: Data represent mean ± S.D of three biologically independent replicates. h Cell death in PATC66 cells treated with DMSO or 10 nM IACS-010759 for 3 days, in the presence or absence of the 1 μM mitochondrial ROS scavenger, MitoQ.

Techniques: Staining, Flow Cytometry, Comparison, Transfection, Membrane, Fluorescence, Infection, Control, Two Tailed Test

Journal: Nature Communications

Article Title: Ether phospholipids are required for mitochondrial reactive oxygen species homeostasis

doi: 10.1038/s41467-023-37924-9

Figure Lengend Snippet: a Heatmap of the lipid metabolites detected at significantly different levels in the treatment-sensitive cells (PATC66/108) compared to resistant ones (PATC124/148). Metabolites with P < 0.05 between the two groups were listed. N = 5 for each cell line. b – d Mitochondrial-ROS ( b ), lipid-ROS ( c ) and cell death ( d ) as measured with MitoSOX, BODIPY 581/591 C11, and propidium iodide, respectively in PATC148 cells upon 3 days of treatment with DMSO or 10 nM IACS-010759, in the presence or absence of the SCD1 inhibitor A939572 (10 μM). Data represent mean ± S.D of three biologically independent replicates. e PATC148 xenograft tumor growth in mice treated with vehicle or 5 mg/kg IACS-010759, once every other day, alone or in combination with the SCD1 inhibitor A939572 (7.8 mg/kg, once every other day). Mice were treated with fasting/feeding cycle protocol. Tumor measurements were made on the 33 rd day of experiment, 9 hr after the last treatment. N = 4 for vehicle and combination groups; N = 5 for IACS-010759 and A939572 groups. Data represent mean ± SEM. Statistical analysis by ordinary one-way ANOVA followed by Tukey’s multiple comparisons test. f Heatmap of lipid species detected in purified mitochondria from sensitive group (PATC66/108) and resistant group (PATC124/148). Metabolites with P < 0.05 (by two-tailed Students’ unpaired t test) between the two groups were listed. N = 3 for each cell line. g – i Mitochondrial-ROS ( g ), lipid-ROS ( h ), and cell death ( i ) as measured with MitoSOX, BODIPY 581/591 C11, and propidium iodide, respectively in PATC124 cells infected with control sgRNA (sgCTRL) or sgRNA targeting GNPAT, AGPS, and FAR1, and treated for 3 days with DMSO or 10 nM IACS-010759. Data represent mean ± S.D of three biologically independent replicates. Statistical analysis by two-tailed Students’ unpaired t test with significance indicated ( b – d , g – i ). Source data are provided as a Source Data file.

Article Snippet: Data represent mean ± S.D of three biologically independent replicates. h Cell death in PATC66 cells treated with DMSO or 10 nM IACS-010759 for 3 days, in the presence or absence of the 1 μM mitochondrial ROS scavenger, MitoQ.

Techniques: Purification, Two Tailed Test, Infection, Control

Journal: Nature Communications

Article Title: Ether phospholipids are required for mitochondrial reactive oxygen species homeostasis

doi: 10.1038/s41467-023-37924-9

Figure Lengend Snippet: a – c Mitochondrial ROS ( a ), lipid peroxidation ( b ), and cell death ( c ) as measured with MitoSOX, BODIPY 581/591 C11, and propidium iodide, respectively in PATC124 cells infected with control sgRNA (sgCTRL) or sgRNA targeting GNPAT (sgGNPAT) in the presence or absence of 100 μM O-C16-18:1 PC. Data represent mean ± S.D of 3 biologically independent replicates. d – e Mitochondrial ROS level ( d ) and cell death ( e ) in sgCTRL- and sgGNPAT-PATC124 cells, in the presence of 100 μM oleic acid or 100 μM O-C16-18:1 PC. Data represent mean ± S.D of three biologically independent replicates. f , g Growth curves of sub-cutaneous xenograft tumors derived from PATC148 cells infected with control sgRNA (sgCTRL) or sgRNA targeting GNPAT (sgGNPAT) and treated with vehicle or 5 mg/kg IACS-010759. Tumor volume was measured every 3–5 days for 24 days. N = 5–8 per group. h , i Mitochondrial ROS were detected with MitoSOX upon treatment with DMSO or 10 nM IACS-010759 in PATC 53 ( h ) and PATC108 ( i ) cells grown in the presence or absence of 100 μM ether-MUFA (O-C16-18:1 PC). Data represent mean ± S.D of three biologically independent replicates. j , k Cell viability was detected by propidium iodide staining after treatment with DMSO or 10 nM IACS-010759 in PATC53 ( j ) and PATC108 ( k ) cells grown in the presence or absence of 100 μM O-C16-18:1 PC. Data represent mean ± S.D of 3 biologically independent replicates. Statistical analysis by two-tailed Students’ unpaired t test with significance indicated ( b – d , g – i ). Source data are provided as a Source Data file.

Article Snippet: Data represent mean ± S.D of three biologically independent replicates. h Cell death in PATC66 cells treated with DMSO or 10 nM IACS-010759 for 3 days, in the presence or absence of the 1 μM mitochondrial ROS scavenger, MitoQ.

Techniques: Infection, Control, Derivative Assay, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Ether phospholipids are required for mitochondrial reactive oxygen species homeostasis

doi: 10.1038/s41467-023-37924-9

Figure Lengend Snippet: a , b BN-PAGE showed mitochondrial supercomplexes (SC) in sensitive (PATC66/108) and resistant (PATC124/148) cells ( a ). Quantifications of the high molecular weight supercomplex (hwmSC) are shown in ( b ). Data represent mean ± S.D of three biologically independent replicates. c , d Mitochondrial supercomplexes as shown with BN-PAGE ( c ) and quantifications of hwmSCs ( d ) in sensitive (PATC66/108) cells grown in the presence or absence of 100 μM ether-MUFA (O-C16-18:1 phosphatidylcholine (PC)) for 24 hr. Data represent mean ± S.D of 4 biologically independent replicates. e , f Mitochondrial supercomplexes as shown with BN-PAGE ( e ) and quantifications of hwmSCs ( f ) in sensitive (PATC66/108) cells grown in the presence or absence of 100 μM ether-PUFA (O-C16-20:3 PC) for 24 hr. Data represent mean ± S.D of three biologically independent replicates. g , h Mitochondrial supercomplexes as shown with BN-PAGE ( g ) and quantifications of hwmSCs ( h ) in PATC124 cells infected with control sgRNA (sgCTRL) or sgRNA targeting GNPAT(sgGNPAT) in the presence or absence of 100 μM O-C16-18:1 PC or 100 μM ether-PUFA (O-C16-20:3 PC) for 24 hr. Complex V subunit protein APT5A was detected by SDS-PAGE western blotting as control protein. Data represent mean ± S.D of 5 biologically independent replicates. i Schematic representation of the major conclusions. Peroxisome-derived ether phospholipids, especially those linked with MUFAs, enhance mitochondrial ETC supercomplexes assembly to maintain redox balance and promote resistance to mitochondrial complex I inhibition. Impairment of MUFAs-linked ether phospholipids synthesis by targeting SCD1, GNPAT, or APGS synergizes with mitochondrial complex I inhibition to induce mitochondrial ROS and cell death. Statistical analysis by two-tailed Students’ unpaired t test with significance indicated ( b , d , f , h ). Source data are provided as a Source Data file.

Article Snippet: Data represent mean ± S.D of three biologically independent replicates. h Cell death in PATC66 cells treated with DMSO or 10 nM IACS-010759 for 3 days, in the presence or absence of the 1 μM mitochondrial ROS scavenger, MitoQ.

Techniques: High Molecular Weight, Infection, Control, SDS Page, Western Blot, Derivative Assay, Inhibition, Two Tailed Test

Journal: Archives of Toxicology

Article Title: Ferroptosis regulation through Nrf2 and implications for neurodegenerative diseases

doi: 10.1007/s00204-023-03660-8

Figure Lengend Snippet: Characterization of ferroptosis in neurodegenerative diseases (NDs) pathological conditions. There exists a significant correlation between ferroptosis and NDs. Ferroptosis contributes to the advancement of NDs, whereas the expression of Nrf2 and its transcriptionally regulated peptides (GSH, GPX4) decreases with aging. In contrast to the impairment of the antioxidant system, heightened levels of reactive oxygen species (ROS) and increased lipid peroxidation, combined with brain iron deposition, induce ferroptosis. Ferroptosis results in alterations in mitochondrial morphology, neuronal damage and eventual cell death. Drawing by Inkscape

Article Snippet: MitoQ , Radical Trapping Antioxidant (RTA, Targets mitochondrial ROS scavenging → rescues mitochondrial integrity and function) , Oxidative cell death , RSL3-treated neuronal HT22 cells , Mitochondrial ROS, lipid peroxidation↓ , Jelinek et al. ( ) .

Techniques: Expressing

Journal: Archives of Toxicology

Article Title: Ferroptosis regulation through Nrf2 and implications for neurodegenerative diseases

doi: 10.1007/s00204-023-03660-8

Figure Lengend Snippet: Nrf2-regulated iron death mechanisms in neurodegenerative diseases. a Blood–brain barrier (BBB), astrocytes, and neurons. Brain capillary endothelial cells (BCECs) safeguard the function of the BBB. Astrocytes on the abluminal surface of BCECs promote brain iron uptake. Astrocytes are primarily responsible for releasing iron into neurons while attenuating iron toxicity. b the transcytosis model. After Fe 3+ entering the blood circulation forms a complex (holo-Tf) with transferrin (Tf), it binds to TfR1 on the surface of brain microvascular endothelial cells (BMECs), and then the Tf–TfR1 complex enters BMECs through clathrin-mediated endocytosis. One of the two models of iron import across BCEC—the transcytosis model. Holo-Tf is transported directly by vesicles to extraluminal sites for release into the brain. c Nrf2 in astrocytes activates non-cell-autonomous protection of nearby neurons and alleviates oxidative stress (OS) by mediating antioxidant responses. GSH produced in the brain via activated Nrf2 pathway is predominantly derived from astrocytes, whose neuroprotective function depends on the transport of GSH precursors from astrocytes to motor neurons. d Regulation of iron in neurons and mechanisms of Nrf2-regulated ferroptosis in neurodegenerative diseases. The key components of ferroptosis are the lipid and antioxidant systems, as well as iron metabolism. metabolism of iron: The Tf–TfR1 complex carrying Fe 3+ is endocytosed into neurons, Fe 3+ is separated from Tf, and then reduced to Fe 2+ by six transmembrane epithelial antigen-prostate 3 (STEAP3), and Fe 2+ is pumped into the cytoplasm through divalent metal transporter 1 (DMT1), which is also a classic model of iron import across BCEC. Alternatively, Fe 3+ reduced by cellular prion protein (PrPC) is transported into neurons by DMT1. In addition, solute carrier family 39 member 14 (SLC39A14) can also transport Fe 2+ into the cell. Intracellular iron can be stored in two forms: as Fe 3+ stored in ferritin or as active unbound iron called the labile iron pool (LIP). Poly-(rC)-binding protein1/2 (PCBP1/2) are in charge of transferring the iron to ferritin. In some cases, nuclear receptor coactivator 4 (NCOA4) mediates ferritin autophagy, releasing iron. Heme oxygenase 1 (HO1) catalyzes the degradation of heme and can also release Fe 2+ . With the aid of amyloid precursor protein (APP), which is transmitted by soluble tau protein to stabilize FPN1, elevated Fe 2+ can be expelled through Ferroportin1 (FPN1)/copper cyanine (Cp) or FPN1/hephaestin (Heph). Through the IRP–IRE interaction, iron can, when overloaded, increase the expression of ferritin, FPN1, and APP, while blocking the ordinary function of furin, upregulating secretase, and speeding up the deposition of Aβ. By the Fenton reaction, excessive Fe 2+ produces ROS and encourages the oxidation of PUFA on the membranes of cells (PE-PUFA), and finally triggers ferroptosis. Lipid metabolism: Lysophospholipid acyltransferase 3 (LPCAT3), long-chain fatty acid CoA ligase 4 (ACSL4), and other enzyme-sensitize membrane lipids to lipid peroxidation, which, catalyzed by lipoxygenases (LOX), accumulates PUFA-OOH, triggering ferroptosis. Antioxidant system: GSH–GPX4 axis in cytoplasm and mitochondria, ferroptosis suppressor protein 1 (FSP1)–CoQ10 axis in plasma membrane, dihydroorotate dehydrogenase (DHODH)–CoQ 10 H 2 , and GTP cyclohydrolase1–tetrahydrobiopterin (GCH1–BH 4 ) axis in mitochondria. Mitochondrial PTEN-inducible putative kinase 1 (PINK1) expression is regulated by Nrf2 under OS conditions. Keap1–Nrf2–ARE axis: Under OS conditions, Keap1 releases Nrf2, and the increase of Nrf2 levels in the cytoplasm increases its nuclear translocation. Upon nuclear import, Nrf2 forms a heterodimer with the small muscular aponeurotic fibrosarcoma (Maf) protein, enabling Nrf2 to bind to AREs in the upstream promoter regions of a variety of target genes, leading to their transcriptional activation. Drawing by Inkscape

Article Snippet: MitoQ , Radical Trapping Antioxidant (RTA, Targets mitochondrial ROS scavenging → rescues mitochondrial integrity and function) , Oxidative cell death , RSL3-treated neuronal HT22 cells , Mitochondrial ROS, lipid peroxidation↓ , Jelinek et al. ( ) .

Techniques: Produced, Derivative Assay, Binding Assay, Transferring, Expressing, Blocking Assay, Membrane, Clinical Proteomics, Translocation Assay, Activation Assay

Journal: Archives of Toxicology

Article Title: Ferroptosis regulation through Nrf2 and implications for neurodegenerative diseases

doi: 10.1007/s00204-023-03660-8

Figure Lengend Snippet: Signaling molecules associated with ferroptosis and regulated by Nrf2

Article Snippet: MitoQ , Radical Trapping Antioxidant (RTA, Targets mitochondrial ROS scavenging → rescues mitochondrial integrity and function) , Oxidative cell death , RSL3-treated neuronal HT22 cells , Mitochondrial ROS, lipid peroxidation↓ , Jelinek et al. ( ) .

Techniques: Binding Assay, Functional Assay, Clinical Proteomics, Membrane

Journal: Ecotoxicology and environmental safety

Article Title: Arsenic exposure provoked prostatic PANoptosis by inducing mitochondrial dysfunction in mice and WPMY-1 cells.

doi: 10.1016/j.ecoenv.2025.118139

Figure Lengend Snippet: Fig. 4. NaAsO2 exposure triggered mitochondrial damage-activated PANoptosis in WPMY-1 cells. (A) MMP was measured using JC-1 probes (scale bar, 20 μm). (B−C) DCFDA (B) and MitoSOX (C) assays reveal intracellular ROS and mtROS levels in WPMY-1 cells after the indicated treatment. (D−F) Effects of NAC on NaAsO2- triggered apoptosis (D−E) and LDH activity (F). (G) Effects of NaAsO2 and NAC on protein levels of Bcl-xL, Bax, ZBP1, RIPK1, RIPK3, MLKL, p-MLKL, GSDME, caspase-3, GSDME-N, cleaved caspase-3, IL-18, and IL-1β in WPMY-1 cells. (H) Effects of NaAsO2 and Mito-TEMPO on protein levels of ZBP1, RIPK1, RIPK3, MLKL, p- MLKL, GSDME, caspase-3, GSDME-N, cleaved caspase-3, IL-18, and IL-1β in WPMY-1 cells. The corresponding quantification data for G and H are provided in Figs. S3D and S4D, respectively. Results are displayed as the mean ± SE based on three repeated trials. *: P < 0.05, **: P < 0.01.

Article Snippet: WPMY-1 cells underwent treatment with 0, 1, 2, and 4 μM NaAsO2 (S7400; SigmaAldrich, MO, USA) for 48 h. The inhibitors Necrostatin-1 (Nec-1; HY15760; MedChemExpress (MCE), NJ, USA) for necroptosis, 2-Bromopalmitic acid (2-BP, HY-111770, MCE) for GSDME broad-spectrum palmitoylation, Z-DEVD-FMK (HY-12466, MCE) for caspase-3, and the mtROS scavenger Mito-TEMPO (HY-125944, MCE) were used in the following treatments: cells were initially exposed to 100 μM Nec-1, 50 μM 2-BP, 50 μM Z-DEVD-FMK, and 60 μM Mito-TEMPO for 3, 3.5, 3, and 6 h, respectively, and subsequently underwent 48 h of exposure to 4 μM NaAsO2.

Techniques: Activity Assay