Journal: Animals : an Open Access Journal from MDPI

Article Title: Effect of Zearalenone-Induced Ferroptosis on Mice Spermatogenesis

doi: 10.3390/ani12213026

Figure Lengend Snippet: List of primers employed for q-RT-PCR.

Article Snippet: The proteins were then transferred onto PVDF membranes (GE Bioscience, Newark, NJ, USA) and then blocked with 5% BSA (dissolved in TBST) for 1 h and the membrane was incubated with anti-SYCP3 (ab97672; Abcam, Cambridge, UK), anti-DDX4 (bs-22896R; Bioss Biotech, Beijing, China), anti-SOX9 (ab185966; Abcam, Cambridge, UK), anti-SLC7A11 (bs-6883R; Bioss Biotech, Beijing, China), anti-GPX4 (bs-3884R; Bioss Biotech, Beijing, China), anti-4-HNE (ab48506; Abcam), and anti-β-actin (bs-0061R; Bioss Biotech, Beijing, China) overnight at 4 °C.

Techniques: Sequencing

Journal: Animals : an Open Access Journal from MDPI

Article Title: Effect of Zearalenone-Induced Ferroptosis on Mice Spermatogenesis

doi: 10.3390/ani12213026

Figure Lengend Snippet: Expressions of genes, proteins involved in ferroptosis in testis of mice in each group. ( A ) Nrf2 mRNA expression. ( B ) SLC7A11 mRNA expression. ( C ) GPX4 mRNA expression. ( D ) Western blot detection of SLC7A11, GPX4, and 4-HNE protein. ( E ) Image J analysis showing the grey value of SLC7A11. ( F ) Image J analysis showing the grey value of GPX4. ( G ) Image J analysis showing the grey value of 4-HNE. The data are expressed as means ± SEM, with n ≥ 3; Different lowercase letters (a, b, c, d) indicate significant differences ( p < 0.05).

Article Snippet: The proteins were then transferred onto PVDF membranes (GE Bioscience, Newark, NJ, USA) and then blocked with 5% BSA (dissolved in TBST) for 1 h and the membrane was incubated with anti-SYCP3 (ab97672; Abcam, Cambridge, UK), anti-DDX4 (bs-22896R; Bioss Biotech, Beijing, China), anti-SOX9 (ab185966; Abcam, Cambridge, UK), anti-SLC7A11 (bs-6883R; Bioss Biotech, Beijing, China), anti-GPX4 (bs-3884R; Bioss Biotech, Beijing, China), anti-4-HNE (ab48506; Abcam), and anti-β-actin (bs-0061R; Bioss Biotech, Beijing, China) overnight at 4 °C.

Techniques: Expressing, Western Blot

Journal:

Article Title: NEW STRATEGIES FOR THE ISOLATION AND ACTIVITY DETERMINATION OF NATURALLY OCCURRING TYPE-4 GLUTATHIONE PEROXIDASE

doi: 10.1016/j.pep.2008.07.014

Figure Lengend Snippet: SDS-PAGE-assessed total protein and immunodetectable protein at various stages of GPx4 purification. Proteins were dissolved in NuPAGE LDS sample buffer under reducing conditions and heated to 70 °C before loading onto a NuPAGE 4-12% acrylamide/bis-acrylamide gel. Protein bands (lanes 1-6) were visualized by Coomassie Blue staining or by immunoblotting (lane 7). Sample lanes with amounts of applied protein are as follows: (1) crude tissue lysate (20 μg); (2) supernatant from 50% saturated (NH4)2SO4 (20 μg); (3) dialyzed precipitate from 70% saturated (NH4)2SO4 (20 μg); (4) active fraction pool from DEAE-Sepharose column (5 μg); (5) active pool from Sephadex G-50 column (0.4 μg); (6) active pool from HiTrap SP column (0.2 μg); (7) anti-GPx4 immunoblot of active pool from HiTrap SP column; (8) protein molecular mass standards.

Article Snippet: The rabbit anti-human GPx4 polyclonal antibody was from Gene Tex (San Antonio, TX).

Techniques: SDS Page, Purification, Acrylamide Gel Assay, Staining, Western Blot

Journal:

Article Title: NEW STRATEGIES FOR THE ISOLATION AND ACTIVITY DETERMINATION OF NATURALLY OCCURRING TYPE-4 GLUTATHIONE PEROXIDASE

doi: 10.1016/j.pep.2008.07.014

Figure Lengend Snippet: Enzymatic activity at different stages of GPx4 purification a

Article Snippet: The rabbit anti-human GPx4 polyclonal antibody was from Gene Tex (San Antonio, TX).

Techniques: Activity Assay, Purification

Journal:

Article Title: NEW STRATEGIES FOR THE ISOLATION AND ACTIVITY DETERMINATION OF NATURALLY OCCURRING TYPE-4 GLUTATHIONE PEROXIDASE

doi: 10.1016/j.pep.2008.07.014

Figure Lengend Snippet: HPTLC-assessed GPx4 activity based on 7α-OOH reduction. Each 0.41 mL reaction mixture contained 3 mM GSH, 1 mM EDTA, 0.1 mM DFO, 0.1% Triton X-100, 25 μM 7α-OOH, and GPx4 (2.1 μg/mL) in PBS at 37 °C. At the indicated time points, samples were extracted and recovered lipid fractions were analyzed by HPTLC, using a TMPD spray followed by H2SO4 for analyte detection. (A) HPTLC profile showing changes in 7α-OOH and 7α-OH levels as a function of incubation time. Profile for a control mixture containing everything except GPx4 is also shown. (B) Plot of time-dependent changes in 7α-OOH (●) and 7α-OH (○) level, as determined by densitometry. The inset shows first-order plots for 7α-OOH loss (●) and 7α-OH accumulation (○), the specific enzymatic activity determined from the former being 0.84 ± 0.01 U/mg, and from the latter, 0.80 ± 0.05 U/mg. Values are means ± deviations from duplicate determinations.

Article Snippet: The rabbit anti-human GPx4 polyclonal antibody was from Gene Tex (San Antonio, TX).

Techniques: High Performance Thin Layer Chromatography, Activity Assay, Incubation, Control

Journal:

Article Title: NEW STRATEGIES FOR THE ISOLATION AND ACTIVITY DETERMINATION OF NATURALLY OCCURRING TYPE-4 GLUTATHIONE PEROXIDASE

doi: 10.1016/j.pep.2008.07.014

Figure Lengend Snippet: Kinetics of GPx4-catalyzed 7α-OOH reduction. Each 0.5 mL reaction mixture contained 3 mM GSH, 1 mM EDTA, 0.1 mM DFO, 0.1% Triton X-100, 25 μM 7α-OOH, and GPx4 in PBS at 37 °C. GPx4 concentration was either 0.9 μg/mL (△) or 1.8 μg/mL (▲). First-order plots of 7α-OOH decay as a function of incubation time are shown. Data from a control lacking GPx4 are plotted alongside (○). Values are means from duplicate experiments.

Article Snippet: The rabbit anti-human GPx4 polyclonal antibody was from Gene Tex (San Antonio, TX).

Techniques: Concentration Assay, Incubation, Control

Journal: Frontiers in Pharmacology

Article Title: Combination decoction of Astragalus mongholicus and Salvia miltiorrhiza mitigates pressure-overload cardiac dysfunction by inhibiting multiple ferroptosis pathways

doi: 10.3389/fphar.2024.1447546

Figure Lengend Snippet: HDC metabolite identification and molecular docking. (A) Chromatograms of HDCH, Control_S, HDCH_S, and HDCL_S under NEG and POS ion modes; (B) Venn diagram of metabolites in different samples. (C) Molecular Docking Results of Astragaloside IV and Salvianolic Acid B with DHODH, FSP1, and GPX4. HDCH, High Dosage Astragalus mongholicus and Salvia miltiorrhiza Decoction; Control_S, Normal Rat Serum; HDCH_S, High Dosage Astragalus mongholicus and Salvia miltiorrhiza Medication-Containing Serum; HDCL_S, Low Dosage Astragalus mongholicus and Salvia miltiorrhiza Medication-Containing Serum; NEG, Negative Ion Mode; POS, Positive Ion Mode.

Article Snippet: Sodium Penicillin for injection (42220302, Shandong Lukang, China), anhydrous ethanol (100092683, Sinopharm Chemical Reagent Co., Ltd., China), xylene (1002341922, Sinopharm Chemical Reagent Co., Ltd., China), 0.1 M phosphate buffer (PB) (T16865, saint-Bio, China), 1% osmium tetroxide (18,456, Ted Pella Inc), ×5 loading buffer (P0015L, P0285, Beyotime, China), Quick Color Pre-Stained Gel kit 12.5% (S6172, Uelandy, China), Tris-glycine SDS-PAGE running buffer (G2027-1L, Solarbio, China), NC membrane (66,485, Bio Trace, United States), non-fat milk powder (P0216, Beyotime, China), TBST (T1085, Solarbio, China), Anti-GPX4 mAb (67763-1-Ig, Proteintech, China), Anti-FSP1 mAb (68049-1-Ig, Proteintech, China), Anti-DHODH mAb (67977-1-Ig, Proteintech, China), Anti-GAPDH Rabbit pAb (GB11002, Servicebio, China), Horseradish Peroxidase-conjugated secondary antibody (A0208, A0216, Beyotime, China), SuperSignal ECL chemiluminescence kit (P0018M, Beyotime, China), Antibody Stripping Solution (WB6200, NCM biotech, China).

Techniques: Control

Journal: Frontiers in Pharmacology

Article Title: Combination decoction of Astragalus mongholicus and Salvia miltiorrhiza mitigates pressure-overload cardiac dysfunction by inhibiting multiple ferroptosis pathways

doi: 10.3389/fphar.2024.1447546

Figure Lengend Snippet: HDC ameliorates H9c2 cell dysfunction by inhibiting ferroptosis through the elevation of GPX4 levels. (A) Western blotting analysis of GPX4 levels in H9c2 cells from various groups to evaluate the effect of AngII and various doses of HDC on GPX4 content. (B–F) All panels represent the conditions of H9c2 cells under HDC intervention, assessing the effects of GPX4 inhibition and non-inhibition in varying FSP1 and DHODH states. (B–E) Measurements in H9c2 cells include GSH levels, MDA content, Fe 2+ levels, cell viability, and NT-proBNP levels. (F) Flow cytometry images of H9c2 cells post JC-1 staining and quantitative analysis of the proportion of green fluorescence signal in flow cytometry. p * < 0.05, p ** < 0.01, p **** < 0.0001. represents an individual sample data point. HDC, Astragalus mongholicus and Salvia miltiorrhiza Combination; GPX4, Glutathione Peroxidase 4; GAPDH, Glyceraldehyde 3-Phosphate Dehydrogenase; AngII, Angiotensin II; L-S, Low Dosage Astragalus mongholicus and Salvia miltiorrhiza Medication-Containing Serum; H-S, High Dosage Astragalus mongholicus and Salvia miltiorrhiza Medication-Containing Serum; GSH, Reduced Glutathione; FSP1, Ferroptosis Suppressor Protein 1; DHODH, Dihydroorotate Dehydrogenase; E, Erastin; B, Brequinar; i, Inhibitor of FSP1; MDA, Malondialdehyde; NT-proBNP, N-terminal pro B-type Natriuretic Peptide; PE-A, Phycoerythrin Area; FITC-A, Fluorescein Isothiocyanate Area.

Article Snippet: Sodium Penicillin for injection (42220302, Shandong Lukang, China), anhydrous ethanol (100092683, Sinopharm Chemical Reagent Co., Ltd., China), xylene (1002341922, Sinopharm Chemical Reagent Co., Ltd., China), 0.1 M phosphate buffer (PB) (T16865, saint-Bio, China), 1% osmium tetroxide (18,456, Ted Pella Inc), ×5 loading buffer (P0015L, P0285, Beyotime, China), Quick Color Pre-Stained Gel kit 12.5% (S6172, Uelandy, China), Tris-glycine SDS-PAGE running buffer (G2027-1L, Solarbio, China), NC membrane (66,485, Bio Trace, United States), non-fat milk powder (P0216, Beyotime, China), TBST (T1085, Solarbio, China), Anti-GPX4 mAb (67763-1-Ig, Proteintech, China), Anti-FSP1 mAb (68049-1-Ig, Proteintech, China), Anti-DHODH mAb (67977-1-Ig, Proteintech, China), Anti-GAPDH Rabbit pAb (GB11002, Servicebio, China), Horseradish Peroxidase-conjugated secondary antibody (A0208, A0216, Beyotime, China), SuperSignal ECL chemiluminescence kit (P0018M, Beyotime, China), Antibody Stripping Solution (WB6200, NCM biotech, China).

Techniques: Western Blot, Inhibition, Flow Cytometry, Staining, Fluorescence

Journal: Frontiers in Pharmacology

Article Title: Combination decoction of Astragalus mongholicus and Salvia miltiorrhiza mitigates pressure-overload cardiac dysfunction by inhibiting multiple ferroptosis pathways

doi: 10.3389/fphar.2024.1447546

Figure Lengend Snippet: HDC improves HF by inhibiting ferroptosis via an increase in GPX4 levels. (A) Western blotting analysis of GPX4 levels in the myocardium from SD rats of various groups to evaluate the impact of HDC on GPX4 content. (B–L) All panels depict the effects of GPX4 inhibition and non-inhibition under HDC treatment in rats with various FSP1 and DHODH states. (B–E, J) In HF rats, measurements include myocardial GSH levels, mitochondrial morphology (TEM images, indicates one of the mitochondria), MDA levels, Fe 2+ content, and serum NT-proBNP levels. (F, G) The heart mass to tibia length ratio and gross cardiac images in HF rats. (H, I) M-mode echocardiography images of the left ventricle adjacent to the sternum and quantitative analysis of LVEF in HF rats. (L) Masson’s trichrome staining of myocardial tissue in HF rats (×50, ×100, ×200 magnifications), with blue staining indicating collagen deposition, suggestive of myocardial fibrosis. (K) Quantitative analysis of fibrotic area based on Masson’s trichrome staining. p*** < 0.001, p **** < 0.0001. represents an individual sample data point. HDC, Astragalus mongholicus and Salvia miltiorrhiza Combination; HF, Heart failure; GPX4, Glutathione Peroxidase 4; GAPDH, Glyceraldehyde 3-Phosphate Dehydrogenase; TAC, Transverse Aortic Constriction; L, Low Dosage Astragalus mongholicus and Salvia miltiorrhiza Decoction; H, High Dosage Astragalus mongholicus and Salvia miltiorrhiza Decoction; FSP1, Ferroptosis Suppressor Protein 1; DHODH, Dihydroorotate Dehydrogenase; GSH, Reduced Glutathione; E, Erastin; B, Brequinar; i, Inhibitor of FSP1; TEM, Transmission Electron Microscopy; MDA, Malondialdehyde; NT-proBNP, N-terminal pro B-type Natriuretic Peptide; HM, Heart Mass; TL, Tibia Length; LVEF, Left Ventricular Ejection Fraction.

Article Snippet: Sodium Penicillin for injection (42220302, Shandong Lukang, China), anhydrous ethanol (100092683, Sinopharm Chemical Reagent Co., Ltd., China), xylene (1002341922, Sinopharm Chemical Reagent Co., Ltd., China), 0.1 M phosphate buffer (PB) (T16865, saint-Bio, China), 1% osmium tetroxide (18,456, Ted Pella Inc), ×5 loading buffer (P0015L, P0285, Beyotime, China), Quick Color Pre-Stained Gel kit 12.5% (S6172, Uelandy, China), Tris-glycine SDS-PAGE running buffer (G2027-1L, Solarbio, China), NC membrane (66,485, Bio Trace, United States), non-fat milk powder (P0216, Beyotime, China), TBST (T1085, Solarbio, China), Anti-GPX4 mAb (67763-1-Ig, Proteintech, China), Anti-FSP1 mAb (68049-1-Ig, Proteintech, China), Anti-DHODH mAb (67977-1-Ig, Proteintech, China), Anti-GAPDH Rabbit pAb (GB11002, Servicebio, China), Horseradish Peroxidase-conjugated secondary antibody (A0208, A0216, Beyotime, China), SuperSignal ECL chemiluminescence kit (P0018M, Beyotime, China), Antibody Stripping Solution (WB6200, NCM biotech, China).

Techniques: Western Blot, Inhibition, Staining, Transmission Assay, Electron Microscopy

Journal: Frontiers in Pharmacology

Article Title: Combination decoction of Astragalus mongholicus and Salvia miltiorrhiza mitigates pressure-overload cardiac dysfunction by inhibiting multiple ferroptosis pathways

doi: 10.3389/fphar.2024.1447546

Figure Lengend Snippet: HDC ameliorates H9c2 cell dysfunction by increasing FSP1 levels to inhibit ferroptosis. (A) Western blotting analysis of FSP1 levels in H9c2 cells from various groups to assess the effects of AngII and different doses of HDC on FSP1 content. (B–F) These panels represent the HDC-treated cells, examining the effects of FSP1 inhibition and non-inhibition under varying GPX4 and DHODH states. (B–E) Measurements in H9c2 cells include CoQ10H2 levels, MDA levels, Fe 2+ content, cell viability, and NT-proBNP levels. (F) Flow cytometry images of H9c2 cells post JC-1 staining and quantitative analysis of the green fluorescence signal proportion in flow cytometry charts. p ** < 0.01, p **** < 0.0001. represents an individual sample data point. HDC, Astragalus mongholicus and Salvia miltiorrhiza Combination; FSP1, Ferroptosis Suppressor Protein 1; GAPDH, Glyceraldehyde 3-Phosphate Dehydrogenase; AngII, Angiotensin II; L-S, Low Dosage Astragalus mongholicus and Salvia miltiorrhiza Medication-Containing Serum; H-S, High Dosage Astragalus mongholicus and Salvia miltiorrhiza Medication-Containing Serum; CoQ10H2, Reduced Coenzyme Q10; GPX4, Glutathione Peroxidase 4; DHODH, Dihydroorotate Dehydrogenase; i, Inhibitor of FSP1; B, Brequinar; E, Erastin; MDA, Malondialdehyde; NT-proBNP, N-terminal pro B-type Natriuretic Peptide; PE-A, Phycoerythrin Area; FITC-A, Fluorescein Isothiocyanate Area.

Article Snippet: Sodium Penicillin for injection (42220302, Shandong Lukang, China), anhydrous ethanol (100092683, Sinopharm Chemical Reagent Co., Ltd., China), xylene (1002341922, Sinopharm Chemical Reagent Co., Ltd., China), 0.1 M phosphate buffer (PB) (T16865, saint-Bio, China), 1% osmium tetroxide (18,456, Ted Pella Inc), ×5 loading buffer (P0015L, P0285, Beyotime, China), Quick Color Pre-Stained Gel kit 12.5% (S6172, Uelandy, China), Tris-glycine SDS-PAGE running buffer (G2027-1L, Solarbio, China), NC membrane (66,485, Bio Trace, United States), non-fat milk powder (P0216, Beyotime, China), TBST (T1085, Solarbio, China), Anti-GPX4 mAb (67763-1-Ig, Proteintech, China), Anti-FSP1 mAb (68049-1-Ig, Proteintech, China), Anti-DHODH mAb (67977-1-Ig, Proteintech, China), Anti-GAPDH Rabbit pAb (GB11002, Servicebio, China), Horseradish Peroxidase-conjugated secondary antibody (A0208, A0216, Beyotime, China), SuperSignal ECL chemiluminescence kit (P0018M, Beyotime, China), Antibody Stripping Solution (WB6200, NCM biotech, China).

Techniques: Western Blot, Inhibition, Flow Cytometry, Staining, Fluorescence

Journal: Frontiers in Pharmacology

Article Title: Combination decoction of Astragalus mongholicus and Salvia miltiorrhiza mitigates pressure-overload cardiac dysfunction by inhibiting multiple ferroptosis pathways

doi: 10.3389/fphar.2024.1447546

Figure Lengend Snippet: HDC improves HF by inhibiting ferroptosis through the elevation of FSP1 levels. (A) Western blotting analysis of FSP1 levels in the myocardium from SD rats of various groups to evaluate the impact of HDC on FSP1 content. (B–K) Analysis in HDC-treated rats under various GPX4 and DHODH states, assessing the effects of both FSP1 inhibition and non-inhibition. (B–E, J) Measurements in HF rats include myocardial CoQ10H2 levels, mitochondrial morphology (TEM images, indicates one of the mitochondria), MDA levels, Fe 2+ content, and serum NT-proBNP levels. (F, G) Heart mass to tibia length ratio and gross cardiac images in HF rats. (H, I) Parasternal long-axis M-mode echocardiography images of the left ventricle and quantitative analysis of LVEF in rats. (L) Masson’s trichrome staining of myocardial tissue in HF rats (×50, ×100, ×200 magnifications), where blue staining indicates collagen deposition, indicative of myocardial fibrosis. (K) Quantitative analysis of the fibrotic area based on Masson’s trichrome staining. p * < 0.05, p ** < 0.01, p *** < 0.001, p **** < 0.0001. represents an individual sample data point. HDC, Astragalus mongholicus and Salvia miltiorrhiza Combination; HF, Heart failure; FSP1, Ferroptosis Suppressor Protein 1; GAPDH, Glyceraldehyde 3-Phosphate Dehydrogenase; TAC, Transverse Aortic Constriction; L, Low Dosage Astragalus mongholicus and Salvia miltiorrhiza Decoction; H, High Dosage Astragalus mongholicus and Salvia miltiorrhiza Decoction; CoQ10H2, Reduced Coenzyme Q10; GPX4, Glutathione Peroxidase 4; DHODH, Dihydroorotate Dehydrogenase; i, Inhibitor of FSP1; B, Brequinar; E, Erastin; TEM, Transmission Electron Microscopy; MDA, Malondialdehyde; NT-proBNP, N-terminal pro B-type Natriuretic Peptide; HM, Heart Mass; TL, Tibia Length; LVEF, Left Ventricular Ejection Fraction.

Article Snippet: Sodium Penicillin for injection (42220302, Shandong Lukang, China), anhydrous ethanol (100092683, Sinopharm Chemical Reagent Co., Ltd., China), xylene (1002341922, Sinopharm Chemical Reagent Co., Ltd., China), 0.1 M phosphate buffer (PB) (T16865, saint-Bio, China), 1% osmium tetroxide (18,456, Ted Pella Inc), ×5 loading buffer (P0015L, P0285, Beyotime, China), Quick Color Pre-Stained Gel kit 12.5% (S6172, Uelandy, China), Tris-glycine SDS-PAGE running buffer (G2027-1L, Solarbio, China), NC membrane (66,485, Bio Trace, United States), non-fat milk powder (P0216, Beyotime, China), TBST (T1085, Solarbio, China), Anti-GPX4 mAb (67763-1-Ig, Proteintech, China), Anti-FSP1 mAb (68049-1-Ig, Proteintech, China), Anti-DHODH mAb (67977-1-Ig, Proteintech, China), Anti-GAPDH Rabbit pAb (GB11002, Servicebio, China), Horseradish Peroxidase-conjugated secondary antibody (A0208, A0216, Beyotime, China), SuperSignal ECL chemiluminescence kit (P0018M, Beyotime, China), Antibody Stripping Solution (WB6200, NCM biotech, China).

Techniques: Western Blot, Inhibition, Staining, Transmission Assay, Electron Microscopy

Journal: Frontiers in Pharmacology

Article Title: Combination decoction of Astragalus mongholicus and Salvia miltiorrhiza mitigates pressure-overload cardiac dysfunction by inhibiting multiple ferroptosis pathways

doi: 10.3389/fphar.2024.1447546

Figure Lengend Snippet: HDC ameliorates H9c2 cell dysfunction by inhibiting ferroptosis through the elevation of DHODH levels. (A) "Western blotting analysis of DHODH levels in H9c2 cells from various groups, assessing the effects of AngII and different doses of HDC on DHODH content. (B–F) Experiments conducted on cells treated with HDC, evaluating the impact of inhibiting or not inhibiting DHODH under different GPX4 and iFSP1 states. (B–E) Measurements in H9c2 cells include CoQ10H2 levels, MDA levels, Fe 2+ content, cell viability, and NT-proBNP levels. Flow cytometry images of H9c2 cells post JC-1 staining and quantitative analysis of the green fluorescence signal proportion in flow cytometry charts. p ns ≥ 0.05, p * < 0.05, p ** < 0.01, p *** < 0.001, p **** < 0.0001. represents an individual sample data point. HDC, Astragalus mongholicus and Salvia miltiorrhiza Combination; DHODH, Dihydroorotate Dehydrogenase; WB, Western blotting; GAPDH, Glyceraldehyde 3-Phosphate Dehydrogenase; AngII, Angiotensin II; L-S, Low Dosage Astragalus mongholicus and Salvia miltiorrhiza Medication-Containing Serum; H-S, High Dosage Astragalus mongholicus and Salvia miltiorrhiza Medication-Containing Serum; CoQ10H2, Reduced Coenzyme Q10; GPX4, Glutathione Peroxidase 4; FSP1, Ferroptosis Suppressor Protein 1; B, Brequinar; i, Inhibitor of FSP1; E, Erastin; MDA, Malondialdehyde; NT-proBNP, N-terminal pro B-type Natriuretic Peptide; PE-A, Phycoerythrin Area; FITC-A, Fluorescein Isothiocyanate Area.

Article Snippet: Sodium Penicillin for injection (42220302, Shandong Lukang, China), anhydrous ethanol (100092683, Sinopharm Chemical Reagent Co., Ltd., China), xylene (1002341922, Sinopharm Chemical Reagent Co., Ltd., China), 0.1 M phosphate buffer (PB) (T16865, saint-Bio, China), 1% osmium tetroxide (18,456, Ted Pella Inc), ×5 loading buffer (P0015L, P0285, Beyotime, China), Quick Color Pre-Stained Gel kit 12.5% (S6172, Uelandy, China), Tris-glycine SDS-PAGE running buffer (G2027-1L, Solarbio, China), NC membrane (66,485, Bio Trace, United States), non-fat milk powder (P0216, Beyotime, China), TBST (T1085, Solarbio, China), Anti-GPX4 mAb (67763-1-Ig, Proteintech, China), Anti-FSP1 mAb (68049-1-Ig, Proteintech, China), Anti-DHODH mAb (67977-1-Ig, Proteintech, China), Anti-GAPDH Rabbit pAb (GB11002, Servicebio, China), Horseradish Peroxidase-conjugated secondary antibody (A0208, A0216, Beyotime, China), SuperSignal ECL chemiluminescence kit (P0018M, Beyotime, China), Antibody Stripping Solution (WB6200, NCM biotech, China).

Techniques: Western Blot, Flow Cytometry, Staining, Fluorescence

Journal: Frontiers in Pharmacology

Article Title: Combination decoction of Astragalus mongholicus and Salvia miltiorrhiza mitigates pressure-overload cardiac dysfunction by inhibiting multiple ferroptosis pathways

doi: 10.3389/fphar.2024.1447546

Figure Lengend Snippet: HDC mitigates HF by inhibiting ferroptosis through the elevation of DHODH levels. (A) Western blotting analysis of DHODH levels in the myocardium from SD rats of various groups to assess the impact of HDC on DHODH content. (B–L) Analysis in HDC-treated rats under different GPX4 and FSP1 states, evaluating the effects of DHODH inhibition and non-inhibition. (B–E, J) Measurements in HF rats include myocardial CoQ10H2 levels, mitochondrial morphology (TEM images, indicates one of the mitochondria), MDA levels, Fe 2+ content, and serum NT-proBNP levels. (F, G) Heart mass to tibia length ratio and gross cardiac images in HF rats. (H, I) Parasternal long-axis M-mode echocardiography images of the left ventricle and quantitative analysis of LVEF in rats. (L) Masson’s trichrome staining of myocardial tissue in HF rats (×50, ×100, ×200 magnifications), where blue staining indicates collagen deposition, suggestive of myocardial fibrosis. (K) Quantitative analysis of the fibrotic area based on Masson’s trichrome staining. p ns ≥0.05, p *<0.05, p **<0.01, p ***<0.001, p ****<0.0001. represents an individual sample data point. HDC, Astragalus mongholicus and Salvia miltiorrhiza Combination; HF, Heart failure; DHODH, Dihydroorotate Dehydrogenase; GAPDH, Glyceraldehyde 3-Phosphate Dehydrogenase; TAC, Transverse Aortic Constriction; L, Low Dosage Astragalus mongholicus and Salvia miltiorrhiza Decoction; H, High Dosage Astragalus mongholicus and Salvia miltiorrhiza Decoction; CoQ10H2, Reduced Coenzyme Q10; GPX4, Glutathione Peroxidase 4; FSP1, Ferroptosis Suppressor Protein 1; B, Brequinar; i, Inhibitor of FSP1; E, Erastin; TEM, Transmission Electron Microscopy; MDA, Malondialdehyde; NT-proBNP, N-terminal pro B-type Natriuretic Peptide; HM, Heart Mass; TL, Tibia Length; LVEF, Left Ventricular Ejection Fraction.

Article Snippet: Sodium Penicillin for injection (42220302, Shandong Lukang, China), anhydrous ethanol (100092683, Sinopharm Chemical Reagent Co., Ltd., China), xylene (1002341922, Sinopharm Chemical Reagent Co., Ltd., China), 0.1 M phosphate buffer (PB) (T16865, saint-Bio, China), 1% osmium tetroxide (18,456, Ted Pella Inc), ×5 loading buffer (P0015L, P0285, Beyotime, China), Quick Color Pre-Stained Gel kit 12.5% (S6172, Uelandy, China), Tris-glycine SDS-PAGE running buffer (G2027-1L, Solarbio, China), NC membrane (66,485, Bio Trace, United States), non-fat milk powder (P0216, Beyotime, China), TBST (T1085, Solarbio, China), Anti-GPX4 mAb (67763-1-Ig, Proteintech, China), Anti-FSP1 mAb (68049-1-Ig, Proteintech, China), Anti-DHODH mAb (67977-1-Ig, Proteintech, China), Anti-GAPDH Rabbit pAb (GB11002, Servicebio, China), Horseradish Peroxidase-conjugated secondary antibody (A0208, A0216, Beyotime, China), SuperSignal ECL chemiluminescence kit (P0018M, Beyotime, China), Antibody Stripping Solution (WB6200, NCM biotech, China).

Techniques: Western Blot, Inhibition, Staining, Transmission Assay, Electron Microscopy

Journal: Frontiers in Pharmacology

Article Title: Combination decoction of Astragalus mongholicus and Salvia miltiorrhiza mitigates pressure-overload cardiac dysfunction by inhibiting multiple ferroptosis pathways

doi: 10.3389/fphar.2024.1447546

Figure Lengend Snippet: Comparative analysis of the effectiveness of GPX4, FSP1, and DHODH in mediating HDC’s improvement of H9c2 cell dysfunction. In this figure, H9c2 cells are divided into three groups: the AngII + H-S + i + E group, primarily mediated by DHODH for HDC’s protective effect; the AngII + H-S + B + E group, primarily mediated by FSP1 for HDC’s protective effect; and the AngII + H-S + B + i group, primarily mediated by GPX4 for HDC’s protective effect. (A–F) Measurements in these three groups of H9c2 cells include GSH levels, CoQ10H2 levels, MDA levels, Fe 2+ content, cell viability, and NT-proBNP levels. (G) Flow cytometry images post JC-1 staining of the H9c2 cells and the quantitative analysis of the proportion of green fluorescence signal. p ns ≥0.05, p *<0.05, p **<0.01, p ****<0.0001. represents an individual sample data point. GPX4, Glutathione Peroxidase 4; FSP1, Ferroptosis Suppressor Protein 1; DHODH, Dihydroorotate Dehydrogenase; HDC, Astragalus mongholicus and Salvia miltiorrhiza Combination; AngII, Angiotensin II; H-S, High Dosage Astragalus mongholicus and Salvia miltiorrhiza Medication-Containing Serum; i (iFSP1), Inhibitor of FSP1; E, Erastin; B, Brequinar; GSH, Reduced Glutathione; CoQ10H2, Reduced Coenzyme Q10; MDA, Malondialdehyde; NT-proBNP, N-terminal pro B-type Natriuretic Peptide; PE-A, Phycoerythrin Area; FITC-A, Fluorescein Isothiocyanate Area.

Article Snippet: Sodium Penicillin for injection (42220302, Shandong Lukang, China), anhydrous ethanol (100092683, Sinopharm Chemical Reagent Co., Ltd., China), xylene (1002341922, Sinopharm Chemical Reagent Co., Ltd., China), 0.1 M phosphate buffer (PB) (T16865, saint-Bio, China), 1% osmium tetroxide (18,456, Ted Pella Inc), ×5 loading buffer (P0015L, P0285, Beyotime, China), Quick Color Pre-Stained Gel kit 12.5% (S6172, Uelandy, China), Tris-glycine SDS-PAGE running buffer (G2027-1L, Solarbio, China), NC membrane (66,485, Bio Trace, United States), non-fat milk powder (P0216, Beyotime, China), TBST (T1085, Solarbio, China), Anti-GPX4 mAb (67763-1-Ig, Proteintech, China), Anti-FSP1 mAb (68049-1-Ig, Proteintech, China), Anti-DHODH mAb (67977-1-Ig, Proteintech, China), Anti-GAPDH Rabbit pAb (GB11002, Servicebio, China), Horseradish Peroxidase-conjugated secondary antibody (A0208, A0216, Beyotime, China), SuperSignal ECL chemiluminescence kit (P0018M, Beyotime, China), Antibody Stripping Solution (WB6200, NCM biotech, China).

Techniques: Flow Cytometry, Staining, Fluorescence

Journal: Frontiers in Pharmacology

Article Title: Combination decoction of Astragalus mongholicus and Salvia miltiorrhiza mitigates pressure-overload cardiac dysfunction by inhibiting multiple ferroptosis pathways

doi: 10.3389/fphar.2024.1447546

Figure Lengend Snippet: Comparative analysis of the effectiveness of GPX4, FSP1, and DHODH in mediating HDC’s amelioration of HF in SD rats. In this study, SD rats with HF were divided into three groups: TAC + H + i + E group, primarily mediated by DHODH for HDC’s protective effect; TAC + H + B + E group, primarily mediated by FSP1 for HDC’s protective effect; and TAC + H + B + I group, primarily mediated by GPX4 for HDC’s protective effect. (A–D) Measurements in these groups include myocardial GSH levels, MDA levels, Fe 2+ content, and serum CoQ10H2 levels in SD rats. (E) Parasternal long-axis echocardiography images of the left ventricle in these groups of SD rats. (H) Quantitative calculation of LVEF based on echocardiography images. (F) Gross cardiac images of the rats in these groups. (I) Heart mass to tibia length ratio in these groups of SD rats. (G) TEM images of myocardial tissue, indicates one of the mitochondria. (J) Serum NT-proBNP levels in these groups of SD rats. (L) Masson’s trichrome staining of myocardial tissue (×50, ×100, ×200 magnifications), where blue staining indicates collagen deposition, suggestive of myocardial fibrosis. (K) Quantitative analysis of the fibrotic area ratio based on Masson’s trichrome staining images. p ns ≥0.05, p *<0.05, p **<0.01, p ****<0.0001. represents an individual sample data point. GPX4, Glutathione Peroxidase 4; FSP1, Ferroptosis Suppressor Protein 1; DHODH, Dihydroorotate Dehydrogenase; HDC, Astragalus mongholicus and Salvia miltiorrhiza Combination; HF, Heart failure; TAC, Transverse Aortic Constriction; H, High Dosage Astragalus mongholicus and Salvia miltiorrhiza Decoction; i, Inhibitor of FSP1; E, Erastin; B, Brequinar; GSH, Reduced Glutathione; CoQ10H2, Reduced Coenzyme Q10; MDA, Malondialdehyde; LVEF, Left Ventricular Ejection Fraction; NT-proBNP, N-terminal pro B-type Natriuretic Peptide; PE-A, Phycoerythrin Area; FITC-A, Fluorescein Isothiocyanate Area.

Article Snippet: Sodium Penicillin for injection (42220302, Shandong Lukang, China), anhydrous ethanol (100092683, Sinopharm Chemical Reagent Co., Ltd., China), xylene (1002341922, Sinopharm Chemical Reagent Co., Ltd., China), 0.1 M phosphate buffer (PB) (T16865, saint-Bio, China), 1% osmium tetroxide (18,456, Ted Pella Inc), ×5 loading buffer (P0015L, P0285, Beyotime, China), Quick Color Pre-Stained Gel kit 12.5% (S6172, Uelandy, China), Tris-glycine SDS-PAGE running buffer (G2027-1L, Solarbio, China), NC membrane (66,485, Bio Trace, United States), non-fat milk powder (P0216, Beyotime, China), TBST (T1085, Solarbio, China), Anti-GPX4 mAb (67763-1-Ig, Proteintech, China), Anti-FSP1 mAb (68049-1-Ig, Proteintech, China), Anti-DHODH mAb (67977-1-Ig, Proteintech, China), Anti-GAPDH Rabbit pAb (GB11002, Servicebio, China), Horseradish Peroxidase-conjugated secondary antibody (A0208, A0216, Beyotime, China), SuperSignal ECL chemiluminescence kit (P0018M, Beyotime, China), Antibody Stripping Solution (WB6200, NCM biotech, China).

Techniques: Staining

Journal: Redox Biology

Article Title: Phospholipid peroxidation inhibits autophagy via stimulating the delipidation of oxidized LC3-PE

doi: 10.1016/j.redox.2022.102421

Figure Lengend Snippet: Phospholipid peroxidation induced by RSL3 inhibits autophagy. a , Immunoblots of LC3 and GPX4 expression in Gpx4 fl/fl MEF incubated with RSL3 (100 nM, 6 h), rapamycin (50 nM, 12 h) or sequentially incubated with RSL3 and rapamycin (100 nM RSL3, 6 h followed by 50 nM rapamycin, 12 h). b , Relative densitometric quantitation of LC3-II/I and GPX4 in (a). n = 3 samples. c , Immunoblots of LC3 and GPX4 expression in NSC34 incubated with RSL3 (100 nM, 6 h), rapamycin (50 nM, 12 h) or sequentially incubated with RSL3 and rapamycin (100 nM RSL3 for 6 h, followed by 50 nM rapamycin for 12 h). d , Relative densitometric quantitation of LC3-II/I and GPX4 in (c). n = 3 samples. e , Representative fluorescence micrographs of GFP-LC3 expression (green) in Gpx4 fl/fl MEFs transiently transfected with GFP-LC3, followed by the indicated treatments as described for (a). Scale bar, 10 μm. f , GFP-LC3 puncta observed in (e) were counted. g , TEM images of Gpx4 fl/fl MEFs treated as described for (a). Top panel showed low magnification. Bottom panel showed magnified images of the areas marked in red in the top panel. Arrows indicated autophagosomes. Scale bars, Top panel 2 μm, Bottom panel 500 nm. h , Live cell fluorescence imaging of lipid hydroperoxide formation in Gpx4 fl/fl MEFs using LiperFluo, green. Cells were transiently transfected with RFP-LC3, followed by the indicated treatments as described for (a). Scale bar, 10 μm. i , RFP-LC3 puncta observed in (h) were counted. j, Immunoblots of LC3, GPX4 and P62 in Gpx4 −/− cells after treatment with 50 nM rapamycin for 12 h. k , Immunoblots of LC3 and EGFP in GPX4 overexpressing Gpx4 fl/fl MEFs after treatment with 50 nM rapamycin for 12 h. l , Relative densitometric quantitation of LC3-II/I in (k). n = 4 samples. Data are mean ± s.d.. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way ANOVA followed by Tukey's multiple comparisons test). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Anti-15Lipoxygenase 1 (Abcam, ab244205), anti-Glutathione Peroxidase 4 (GPX4) (Abcam, ab125066), anti-Calnexin (Proteintech, 10427-2-AP), anti-ATG4B (CST, 13507S), anti-ATG4B (Proteintech, 15131-1-AP), anti-LC3B (CST, 2775), anti-GABARAP (Abgent, AP1821a), anti-SQSTM1/p62 (Abcam, ab56416), anti-GFP (Abcam, ab290), anti-FLAG (Sigma, F1804), anti-Tom20 (Proteintech, 11802-1-AP), anti-GAPDH (Fudebio-tech, FD0063), anti-β-actin (Fudebio-tech, FD0060), normal rabbit IgG (Santa Cruz, sc-2027), goat anti-mouse IgG-HRP (Fudebio-tech, FDM007), goat anti-rabbit IgG-HRP (Fudebio-tech, FDR007), 1 S , 3 R -RSL3 (TargetMol, T3646), Fer-1 (Targetmol, T6500), PD146176 (Sigma, P4620), ML355 (Selleck, S6557), Zileuton (Selleck, S1443), Rapamycin (TargetMol, T1537), Bafilomycin A1 (BaF1, Sigma, B1793) Lipofectamine™ LTX Reagent with PLUS™ Reagent (Thermo Fisher Scientific, 15338100), Protein A/G Plus-Agarose Immunoprecipitation Reagent (Santa Cruz, sc-2003), DAPI (Beyotime, C1002), LiperFluo (DOJINDO, L248), Lipoxygenase Inhibitor Screening Assay Kit (Cayman, 760700).

Techniques: Western Blot, Expressing, Incubation, Quantitation Assay, Fluorescence, Transfection, Imaging

Journal: Redox Biology

Article Title: Phospholipid peroxidation inhibits autophagy via stimulating the delipidation of oxidized LC3-PE

doi: 10.1016/j.redox.2022.102421

Figure Lengend Snippet: Peroxized PE diminishes the formation of LC3-II. a , Quantitative assessment of PE molecular species (sn 1/ sn 2 ) in control and AA-supplemented Gpx4 fl/fl MEFs. n = 4 samples. b , Quantitative assessment of oxPEs in Gpx4 fl/fl MEFs treated as indicated. n = 4 samples. c , Immunoblots showing autophagy inhibition in Gpx4 fl/fl MEFs treated with RSL3 in the presence of AA. d , Relative densitometric quantitation of LC3-II, P62 and GPX4 in (c). n = 4 samples. e , Immunoblot showing autophagy inhibition in NSC34 cells treated with RSL3 in the presence of AA. f , Relative densitometric quantitation of LC3-II, P62 and GPX4 in (e). n = 3 samples. g , Immunoblots of cell lysates from Gpx4 fl/fl MEFs treated as indicated and described in the methods section. h , AA-initiated metabolic and GPX4-dependent regulation of oxygenated PE ferroptotic signals. i , Effects of PE-OOH on autophagy in Gpx4 fl/fl MEFs. Cell lysates were isolated and immunoblotted for the indicated proteins. j , Relative densitometric quantitation of LC3-II/I, P62 and GPX4 in (i). n = 3 samples. k , Effects of PE-OOH on autophagy in NSC34 cells. Cell lysates were isolated and immunoblotted for the indicated proteins. l , Relative densitometric quantitation of LC3-II/I, P62 and GPX4 in (k). n = 3 samples. Student's t -test was used for the statistical analysis of (a). Two-way ANOVA with Holm-Sidak's multiple comparisons test was used for the statistical analysis of (b). One-way ANOVA followed by Tukey's multiple comparisons test was used for the statistical analysis of (d, f, j, l). Data are mean ± s.d.. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Anti-15Lipoxygenase 1 (Abcam, ab244205), anti-Glutathione Peroxidase 4 (GPX4) (Abcam, ab125066), anti-Calnexin (Proteintech, 10427-2-AP), anti-ATG4B (CST, 13507S), anti-ATG4B (Proteintech, 15131-1-AP), anti-LC3B (CST, 2775), anti-GABARAP (Abgent, AP1821a), anti-SQSTM1/p62 (Abcam, ab56416), anti-GFP (Abcam, ab290), anti-FLAG (Sigma, F1804), anti-Tom20 (Proteintech, 11802-1-AP), anti-GAPDH (Fudebio-tech, FD0063), anti-β-actin (Fudebio-tech, FD0060), normal rabbit IgG (Santa Cruz, sc-2027), goat anti-mouse IgG-HRP (Fudebio-tech, FDM007), goat anti-rabbit IgG-HRP (Fudebio-tech, FDR007), 1 S , 3 R -RSL3 (TargetMol, T3646), Fer-1 (Targetmol, T6500), PD146176 (Sigma, P4620), ML355 (Selleck, S6557), Zileuton (Selleck, S1443), Rapamycin (TargetMol, T1537), Bafilomycin A1 (BaF1, Sigma, B1793) Lipofectamine™ LTX Reagent with PLUS™ Reagent (Thermo Fisher Scientific, 15338100), Protein A/G Plus-Agarose Immunoprecipitation Reagent (Santa Cruz, sc-2003), DAPI (Beyotime, C1002), LiperFluo (DOJINDO, L248), Lipoxygenase Inhibitor Screening Assay Kit (Cayman, 760700).

Techniques: Western Blot, Inhibition, Quantitation Assay, Isolation

Journal: Redox Biology

Article Title: Phospholipid peroxidation inhibits autophagy via stimulating the delipidation of oxidized LC3-PE

doi: 10.1016/j.redox.2022.102421

Figure Lengend Snippet: ALOX15-induced phospholipid peroxidation inhibits the formation of LC3-PE. a , Effects of LOX inhibitors on autophagy. Gpx4 fl/fl MEFs were treated with RSL3 and rapamycin as indicated in the absence or presence of the indicated LOX inhibitors followed by immunoblotting of cell lysates. b , Densitometric quantitation of the ratio of LC3-II to GAPDH in (a). n = 3 samples. c , Gpx4 fl/fl MEFs transiently expressing GFP-LC3 (green) were treated with RSL3 and/or rapamycin as indicated before fixation. Scale bar, 10 μm. Top panel showed low magnification. Bottom panel showed magnified images of the marked areas in the top panel. d , Quantitative analysis of the number of GFP-LC3 puncta observed in (c), counted in randomly selected fields by using ImageJ software. e , Immunoblots of ALOX15-EGFP overexpressing Gpx4 fl/fl MEFs after treatment with 50 nM rapamycin for 12 h. f , Immunoprecipitation of GFP-LC3 and ALOX15-FLAG in Gpx4 fl/fl MEFs. g , Immunoprecipitation of LC3 and ALOX15 in Gpx4 fl/fl MEFs. Data are mean ± s.d.. *** P < 0.001 (one-way ANOVA followed by Tukey's multiple comparisons test). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Anti-15Lipoxygenase 1 (Abcam, ab244205), anti-Glutathione Peroxidase 4 (GPX4) (Abcam, ab125066), anti-Calnexin (Proteintech, 10427-2-AP), anti-ATG4B (CST, 13507S), anti-ATG4B (Proteintech, 15131-1-AP), anti-LC3B (CST, 2775), anti-GABARAP (Abgent, AP1821a), anti-SQSTM1/p62 (Abcam, ab56416), anti-GFP (Abcam, ab290), anti-FLAG (Sigma, F1804), anti-Tom20 (Proteintech, 11802-1-AP), anti-GAPDH (Fudebio-tech, FD0063), anti-β-actin (Fudebio-tech, FD0060), normal rabbit IgG (Santa Cruz, sc-2027), goat anti-mouse IgG-HRP (Fudebio-tech, FDM007), goat anti-rabbit IgG-HRP (Fudebio-tech, FDR007), 1 S , 3 R -RSL3 (TargetMol, T3646), Fer-1 (Targetmol, T6500), PD146176 (Sigma, P4620), ML355 (Selleck, S6557), Zileuton (Selleck, S1443), Rapamycin (TargetMol, T1537), Bafilomycin A1 (BaF1, Sigma, B1793) Lipofectamine™ LTX Reagent with PLUS™ Reagent (Thermo Fisher Scientific, 15338100), Protein A/G Plus-Agarose Immunoprecipitation Reagent (Santa Cruz, sc-2003), DAPI (Beyotime, C1002), LiperFluo (DOJINDO, L248), Lipoxygenase Inhibitor Screening Assay Kit (Cayman, 760700).

Techniques: Western Blot, Quantitation Assay, Expressing, Software, Immunoprecipitation

Journal: Redox Biology

Article Title: Phospholipid peroxidation inhibits autophagy via stimulating the delipidation of oxidized LC3-PE

doi: 10.1016/j.redox.2022.102421

Figure Lengend Snippet: Peroxidized LC3-PE is a preferential substrate for ATG4B. a , Immunoblots of Gpx4 fl/fl MEFs treated as described in the methods section. b , Immunoblots of ALOX15-EGFP overexpressing Gpx4 fl/fl MEFs after treatment with FMK-9a (1 μM, 6 h). c , Immunoblots of Hela and ATG4B KO Hela (ATG4B −/- ) with the indicated treatments. d , ATG4B activity detection by immunoblotting. Cells were treated as indicated, and GFP-LC3 was detected. e , Relative densitometric quantitation of GFP-LC3-II in (d). n = 3 samples. f , Immunoblots of ER-ALOX15-FLAG-cherry overexpressing Gpx4 fl/fl MEFs after treatment with 50 nM rapamycin for 12 h. g , Relative densitometric quantitation of LC3-II and GPX4 in (f) are shown. n = 3 samples. h , Detection of ATG4B activity in ATG4B KO HeLa cells by SDS-PAGE. i , Fold changes of GFP-LC3-II and GPX4 in (h). n = 4 samples. j , Immunoprecipitation of GFP-LC3 and pcDNA3.1(+)-ATG4B in Gpx4 fl/fl MEFs. k , Work flow for quantification of PE and oxidation products in LC3-II by LC-MS. Cells were disrupted by sonication and the lysates immunoprecipitated using anti-LC3 antibodies. The pellets were then incubated with ALOX15 to get oxidized PE, followed by incubation with ATG4B to release the lipids for LC-MS analysis. l , Immunoprecipitation of LC3 in Gpx4 fl/fl MEFs incubated with 500 nM rapamycin for 14 h. m, In vitro oxidation of LC3-PE by ALOX15 and its enzymatic reaction with ATG4B. Samples were subjected to SDS-PAGE. n , Band intensity of LC3-II in (m). n = 3 samples. o , Relative abundance of each class of phospholipids in LC3-II. CL, cardiolipin; PC, phosphatidylcholine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine. p , Representative normal-phase LC-MS chromatogram of phospholipids in LC3-II. q , The amounts of oxidized PE in LC3-II. n = 7 samples. One-way ANOVA followed by Tukey's multiple comparisons test was used for the statistical analysis of (e, g, i, n). Student's t -test was used for the statistical analysis of (q). Data are mean ± s.d.. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Anti-15Lipoxygenase 1 (Abcam, ab244205), anti-Glutathione Peroxidase 4 (GPX4) (Abcam, ab125066), anti-Calnexin (Proteintech, 10427-2-AP), anti-ATG4B (CST, 13507S), anti-ATG4B (Proteintech, 15131-1-AP), anti-LC3B (CST, 2775), anti-GABARAP (Abgent, AP1821a), anti-SQSTM1/p62 (Abcam, ab56416), anti-GFP (Abcam, ab290), anti-FLAG (Sigma, F1804), anti-Tom20 (Proteintech, 11802-1-AP), anti-GAPDH (Fudebio-tech, FD0063), anti-β-actin (Fudebio-tech, FD0060), normal rabbit IgG (Santa Cruz, sc-2027), goat anti-mouse IgG-HRP (Fudebio-tech, FDM007), goat anti-rabbit IgG-HRP (Fudebio-tech, FDR007), 1 S , 3 R -RSL3 (TargetMol, T3646), Fer-1 (Targetmol, T6500), PD146176 (Sigma, P4620), ML355 (Selleck, S6557), Zileuton (Selleck, S1443), Rapamycin (TargetMol, T1537), Bafilomycin A1 (BaF1, Sigma, B1793) Lipofectamine™ LTX Reagent with PLUS™ Reagent (Thermo Fisher Scientific, 15338100), Protein A/G Plus-Agarose Immunoprecipitation Reagent (Santa Cruz, sc-2003), DAPI (Beyotime, C1002), LiperFluo (DOJINDO, L248), Lipoxygenase Inhibitor Screening Assay Kit (Cayman, 760700).

Techniques: Western Blot, Activity Assay, Quantitation Assay, SDS Page, Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, Sonication, Incubation, In Vitro

Journal: Redox Biology

Article Title: Phospholipid peroxidation inhibits autophagy via stimulating the delipidation of oxidized LC3-PE

doi: 10.1016/j.redox.2022.102421

Figure Lengend Snippet: ATG4B shows a higher affinity for PE-OOH than PE. a , Ribbon models of human ATG4B (h ATG4B) (ATG4B PDB ID: 2CY7 ) interactions with PE (i) and PE-OOH (ii). b , Ribbon models of mouse ATG4B (m ATG4B) interactions with PE (i) and PE-OOH (ii). c , Binding energies of PE and PE-OOH with h ATG4B and m ATG4B. d , Molecular dynamics simulation of h ATG4B interacting with PE and PE-OOH. e , Molecular dynamics simulation of m ATG4B interacting with PE and PE-OOH. f , Liposome binding assay of ATG4B towards PE and PE-OOH. Recombinant ATG4B (500 ng) was incubated with liposomes (400 nmol) composed of PC, PC/PE (1:1) or PC/PE-OOH (1:1). g , Dot blot binding analysis of recombinant ATG4B to immobilized PE and PE-OOH. h , The protease activity of ATG4B is regulated by phospholipid peroxidation. i , Relative band intensity of GFP-LC3-II for the data shown in (h). n = 3 samples. Differences between the groups were analyzed by two-way ANOVA with Sidak's multiple comparisons test. Data are mean ± s.d.. *** P < 0.001. j , Thermal stabilization of ATG4B in Gpx4 fl/fl MEFs incubated with PE (20 μM, 1 h) and PE-OOH (20 μM, 1 h) was determined using a standard cellular thermal shift protocol with heat treatment from 37 °C to 77 °C. k , Relative band intensity of ATG4B for the data shown in (j). n = 3 samples. Data are mean ± s.d.. l , A schematic summarizing the role of PE and PE-OOH in the regulation of autophagosome formation. Lipidation of cytoplasmic LC3-I to LC3-II by covalent conjugation of the C-terminal glycine is an essential step for phagophore formation. PE peroxidation induced by ALOX15 results in the formation of oxidized PE, which is also a substrate for LC3-I lipidation. ATG4B shows a higher affinity for PE-OOH than PE.

Article Snippet: Anti-15Lipoxygenase 1 (Abcam, ab244205), anti-Glutathione Peroxidase 4 (GPX4) (Abcam, ab125066), anti-Calnexin (Proteintech, 10427-2-AP), anti-ATG4B (CST, 13507S), anti-ATG4B (Proteintech, 15131-1-AP), anti-LC3B (CST, 2775), anti-GABARAP (Abgent, AP1821a), anti-SQSTM1/p62 (Abcam, ab56416), anti-GFP (Abcam, ab290), anti-FLAG (Sigma, F1804), anti-Tom20 (Proteintech, 11802-1-AP), anti-GAPDH (Fudebio-tech, FD0063), anti-β-actin (Fudebio-tech, FD0060), normal rabbit IgG (Santa Cruz, sc-2027), goat anti-mouse IgG-HRP (Fudebio-tech, FDM007), goat anti-rabbit IgG-HRP (Fudebio-tech, FDR007), 1 S , 3 R -RSL3 (TargetMol, T3646), Fer-1 (Targetmol, T6500), PD146176 (Sigma, P4620), ML355 (Selleck, S6557), Zileuton (Selleck, S1443), Rapamycin (TargetMol, T1537), Bafilomycin A1 (BaF1, Sigma, B1793) Lipofectamine™ LTX Reagent with PLUS™ Reagent (Thermo Fisher Scientific, 15338100), Protein A/G Plus-Agarose Immunoprecipitation Reagent (Santa Cruz, sc-2003), DAPI (Beyotime, C1002), LiperFluo (DOJINDO, L248), Lipoxygenase Inhibitor Screening Assay Kit (Cayman, 760700).

Techniques: Binding Assay, Recombinant, Incubation, Dot Blot, Activity Assay, Conjugation Assay

Journal: Redox Biology

Article Title: Phospholipid peroxidation inhibits autophagy via stimulating the delipidation of oxidized LC3-PE

doi: 10.1016/j.redox.2022.102421

Figure Lengend Snippet: Phospholipid peroxidation inhibits autophagy in vivo . a , Immunoblot of midbrain tissues of female C57BL/6NCr mice received intraperitoneal injection of RSL3 and Fer-1. b , Relative densitometric quantitation of LC3-II/I in (a). n = 6–7 samples. c , Immunoblots of midbrain tissues of male C57BL/6 mice with ICV injection of RSL3 and rapamycin. d , Relative densitometric quantitation of LC3-II/I, P62 and GPX4 in (c). n = 3 mice. e , Immunoblots of subcellular fractions from the midbrain tissues of mice in (c). Cyto, cytosol; ER, endoplasmic reticulum; Mito, mitochondria. f , Score plot of the OPLS-DA. Groups of mice: control, RSL3, rapamycin, RSL3 + rapamycin. n = 7 mice. g , Volcano plot of the pairwise comparison between rapamycin and RSL3 + rapamycin groups represented by the log 2 (fold change) plotted against the -log 10 ( P value). h , Immunoblotting of midbrain tissues of Gpx4 fl/fl mice with ICV injection of PBS, AAV-EGFP, AAV- Cre -EGFP and rapamycin. i , Relative densitometric quantitation of LC3-II/I, P62 and GPX4 in (h). n = 4 mice. j , Immunoblots of subcellular fractions from the midbrain tissues of mice in (h). k , Score plot of the OPLS-DA. Groups of mice: control, AAV-EGFP, AAV- Cre -EGFP, rapamycin, AAV- Cre -EGFP + rapamycin. n = 6 mice. l , Volcano plot is represented by the log 2 (fold change) of rapamycin vs AAV- Cre -EGFP + rapamycin groups plotted against the -log 10 ( P value). m , Immunoblotting of midbrain tissues of C57BL/6 mice with ICV injection of PBS, AAV-EGFP, AAV-ALOX15-EGFP and rapamycin. n , Relative densitometric quantitation of LC3-II/I and P62 in (m). n = 3 mice. o , Immunoblots of midbrain tissue in WT and Alox15 KO mice with ICV injection of rapamycin. p , Relative densitometric quantitation of LC3-II/I and P62 in (o). n = 4 mice. One-way ANOVA followed by Tukey's multiple comparisons test was used for the statistical analysis of (b, d, i, n, p). Data are mean ± s.d.. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Anti-15Lipoxygenase 1 (Abcam, ab244205), anti-Glutathione Peroxidase 4 (GPX4) (Abcam, ab125066), anti-Calnexin (Proteintech, 10427-2-AP), anti-ATG4B (CST, 13507S), anti-ATG4B (Proteintech, 15131-1-AP), anti-LC3B (CST, 2775), anti-GABARAP (Abgent, AP1821a), anti-SQSTM1/p62 (Abcam, ab56416), anti-GFP (Abcam, ab290), anti-FLAG (Sigma, F1804), anti-Tom20 (Proteintech, 11802-1-AP), anti-GAPDH (Fudebio-tech, FD0063), anti-β-actin (Fudebio-tech, FD0060), normal rabbit IgG (Santa Cruz, sc-2027), goat anti-mouse IgG-HRP (Fudebio-tech, FDM007), goat anti-rabbit IgG-HRP (Fudebio-tech, FDR007), 1 S , 3 R -RSL3 (TargetMol, T3646), Fer-1 (Targetmol, T6500), PD146176 (Sigma, P4620), ML355 (Selleck, S6557), Zileuton (Selleck, S1443), Rapamycin (TargetMol, T1537), Bafilomycin A1 (BaF1, Sigma, B1793) Lipofectamine™ LTX Reagent with PLUS™ Reagent (Thermo Fisher Scientific, 15338100), Protein A/G Plus-Agarose Immunoprecipitation Reagent (Santa Cruz, sc-2003), DAPI (Beyotime, C1002), LiperFluo (DOJINDO, L248), Lipoxygenase Inhibitor Screening Assay Kit (Cayman, 760700).

Techniques: In Vivo, Western Blot, Injection, Quantitation Assay

Journal: BMC Ophthalmology

Article Title: Lens epithelium cell ferroptosis mediated by m 6 A-lncRNA and GPX4 expression in lens tissue of age-related cataract

doi: 10.1186/s12886-023-03205-8

Figure Lengend Snippet: Differentially m 6 A-lncRNAs and acted on target genes associated with ferroptosis

Article Snippet: After being washed, binding of anti GPX4 IgG was detected with alkaline phosphatase-conjugated goat anti-rabbit IgG antibody (1:10,000; Santa Cruz, Dallas, TX, USA).

Techniques: Methylation

Journal: BMC Ophthalmology

Article Title: Lens epithelium cell ferroptosis mediated by m 6 A-lncRNA and GPX4 expression in lens tissue of age-related cataract

doi: 10.1186/s12886-023-03205-8

Figure Lengend Snippet: Differentially m 6 A-lncRNAs and acted on target genes associated with ARC

Article Snippet: After being washed, binding of anti GPX4 IgG was detected with alkaline phosphatase-conjugated goat anti-rabbit IgG antibody (1:10,000; Santa Cruz, Dallas, TX, USA).

Techniques: Methylation

Journal: BMC Ophthalmology

Article Title: Lens epithelium cell ferroptosis mediated by m 6 A-lncRNA and GPX4 expression in lens tissue of age-related cataract

doi: 10.1186/s12886-023-03205-8

Figure Lengend Snippet: GPX4 may a target of lncRNA ENST00000586817 by cis mechanism and inhibit LECs ferroptosis in ARC. ( A ) MeRIP-Seq analysis on the distribution of m 6 A peak within lncRNA ENST00000586817. ( B ) Diagram showing ENST00000586817 and GPX4 locus on chromosome 19. ( C ) qRT-PCR analysis for validation the expression of DE-lncRNA ENST00000586817 in human LECs (n = 10 each). qRT-PCR data were normalized against the GAPDH level. ( D ) TEM analysis indicated that mitochondria were increased in ARC (n = 3 each). ( E ) qRT-PCR analysis was performed on the GPX4 in human LECs (n = 10 each). qRT-PCR data were normalized against the GAPDH level. ( F ) Western blot analysis for the expression of GPX4 protein (n = 3 each). Significance value: ns, no significance, * p < 0.05, *** * p < 0.0001

Article Snippet: After being washed, binding of anti GPX4 IgG was detected with alkaline phosphatase-conjugated goat anti-rabbit IgG antibody (1:10,000; Santa Cruz, Dallas, TX, USA).

Techniques: Quantitative RT-PCR, Biomarker Discovery, Expressing, Western Blot

Journal: iScience

Article Title: The role of HIF-1α/HO-1 pathway in hippocampal neuronal ferroptosis in epilepsy

doi: 10.1016/j.isci.2023.108098

Figure Lengend Snippet: Validation of excessive oxidative stress of hippocampus in PTZ kindling epilepsy mice models (A) Relative activity of antioxidant enzymes in hippocampus, including SOD, CAT, and GSH-Px. (B and C) western blot and RT-qPCR results for GPX4 in hippocampus. (D) ROS staining of CA1, CA3, and DG regions in hippocampus. (E) Relative MDA content hippocampus. Unpaired two-tailed Student’s t-test was used to compare two groups. The data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Article Snippet: Rabbit anti-GPX4 monoclonal antibody , Abmart , Cat# T56959F.

Techniques: Biomarker Discovery, Activity Assay, Western Blot, Quantitative RT-PCR, Staining, Two Tailed Test

Journal: iScience

Article Title: The role of HIF-1α/HO-1 pathway in hippocampal neuronal ferroptosis in epilepsy

doi: 10.1016/j.isci.2023.108098

Figure Lengend Snippet:

Article Snippet: Rabbit anti-GPX4 monoclonal antibody , Abmart , Cat# T56959F.

Techniques: Recombinant, cDNA Synthesis, Iron Assay, Enzyme-linked Immunosorbent Assay, Multiple Displacement Amplification, Software

Journal: iScience

Article Title: The role of HIF-1α/HO-1 pathway in hippocampal neuronal ferroptosis in epilepsy

doi: 10.1016/j.isci.2023.108098

Figure Lengend Snippet: Primer sequences information

Article Snippet: Rabbit anti-GPX4 monoclonal antibody , Abmart , Cat# T56959F.

Techniques: