Journal: CNS Neuroscience & Therapeutics

Article Title: Cleaving PINK1 or PGAM5 ? Involvement of PARL in Methamphetamine‐Induced Excessive Mitophagy and Neuronal Necroptosis

doi: 10.1111/cns.70293

Figure Lengend Snippet: PARL overexpression rescued excessive mitophagy and defective autophagic flux in primary neurons. (A) Detection of Parkin and LC3 protein expression by Western blot. (B) Statistical results of the relative expression levels of Parkin and LC3II/I proteins. (C) Tom20 (green) and Parkin (red) costaining. Scale bar = 5 μm. (D) Tom20 (green) and LC3 (red) costaining. Scale bar = 5 μm. (E, I) Fluorescence analysis of neurons transfected with mCherry‐GFP‐LC3 adenovirus. The yellow puncta indicate autophagosomes, and the free red puncta indicate autolysosomes. Scale bar = 5 μm. (F) LC3 (green) and LAMP1 (red) costaining. Scale bar = 5 μm. (G) Quantitative analysis of colocalization of Parkin and Tom20. (H) Quantitative analysis of colocalization of LC3 and Tom20. (J) Quantitative analysis of colocalization of LC3 and LAMP1. ** p < 0.01 and **** p < 0.0001 versus Ad group; # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 for the PARL + Meth group versus Ad + Meth group.

Article Snippet: The primary neurons were fixed with 4% paraformaldehyde for 30 min and then rinsed with 0.3% Triton X‐100 for 15 min. After 5% bovine serum albumin blockage, the cells were incubated at 4°C overnight with the following primary antibodies: LC3 (12741, 1:200), RIP1 (3493, 1:200), and Tom20 (42406S, 1:200) antibodies were obtained from Cell Signaling Technology; PARL (NBP1‐80878) antibody was obtained from Novus Biologicals, RIP3 (sc‐374,639, 1:200), LC3 (sc‐398,822, 1:200), Tom20 (sc‐17,764, 1:200), and PINK1 (sc‐514,353, 1:200) antibodies were obtained from Santa Cruz Technology; p‐MLKL (ab196436, 1:200) antibody was obtained from Abcam; PGAM5 (68116‐1‐Ig, 1:1000), MLKL (66675‐1‐Ig, 1:200), RIP3 (17563‐1‐AP, 1:200), LAMP1 (67300‐1‐Ig, 1:200), and Parkin (14060‐1‐AP, 1:200) were obtained from Proteintech.

Techniques: Over Expression, Expressing, Western Blot, Fluorescence, Transfection

Journal: CNS Neuroscience & Therapeutics

Article Title: Cleaving PINK1 or PGAM5 ? Involvement of PARL in Methamphetamine‐Induced Excessive Mitophagy and Neuronal Necroptosis

doi: 10.1111/cns.70293

Figure Lengend Snippet: PARL overexpression reversed the Meth‐induced neuronal necroptosis. (A) The Calcein‐AM/PI double staining in control and PARL overexpression neurons after Meth treatment. Scale bar = 50 μm. (B) Quantitative analysis of the proportions of dead cells in neurons. (C) LDH levels in different groups. (D) The level of TNF‐α in the cell culture supernatants detected by ELISA. (E) MLKL, RIP3, RIP1, Drp1, and Fis1 protein expression. (F) Statistical results of the relative levels of MLKL, RIP3, RIP1, Drp1, and Fis1 proteins. (G) Tom20 (green) and p‐MLKL (red) co‐staining. Scale bar = 5 μm. (H) Quantitative analysis of colocalization of p‐MLKL and Tom20. Data are shown as the mean ± SEM. One‐way ANOVA was used to determine the statistical significance between multiple groups. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 versus Ad group; # p < 0.05, ## p < 0.01, ### p < 0.001, and #### p < 0.0001 for the PARL + Meth group versus Ad + Meth group.

Article Snippet: The primary antibodies used were p62 (ab109012, 1:10,000) antibody was obtained from Abcam; LC3 (12,741, 1:1000), RIP1 (3493, 1:1000), and MLKL (37,705, 1:1000) antibodies were obtained from Cell Signaling Technology; PARL (NBP1‐80878, 1:500) and PINK1 (BC100‐494, 1:1000) antibodies were obtained from Novus Biologicals; RIP3 (sc‐374,639, 1:500) and β‐actin (sc‐47,778, 1:500) antibodies were obtained from Santa Cruz Technology; PGAM5 (28445‐1‐AP, 1:2000), Fis1 (10956‐1‐AP, 1:4000), Drp1 (12957‐1‐AP, 1:2000), and Parkin (14060‐1‐AP, 1:2000) antibodies were obtained from Proteintech.

Techniques: Over Expression, Double Staining, Control, Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Staining

Journal: CNS Neuroscience & Therapeutics

Article Title: Cleaving PINK1 or PGAM5 ? Involvement of PARL in Methamphetamine‐Induced Excessive Mitophagy and Neuronal Necroptosis

doi: 10.1111/cns.70293

Figure Lengend Snippet: Knockdown of PGAM5 alleviated neuronal necroptosis in primary neurons. (A) The Calcein‐AM/PI double staining in control and PGAM5 downregulation neurons after Meth treatment. Scale bar = 50 μm. (B) Quantitative analysis of the proportions of dead cells in neurons. (C) The LDH release level was measured in different groups. (D) The expression level of TNF‐α in the cell culture supernatants was analyzed by ELISA. (E) Detection of MLKL, RIP3, RIP1, Drp1, and Fis1 protein expression by Western blot. (F) Statistical results of the relative expression levels of MLKL, RIP3, RIP1, Drp1, and Fis1 proteins. (G) Tom20 (green) and p‐MLKL (red) costaining. Scale bar = 5 μm. (H) Quantitative analysis of colocalization of p‐MLKL and Tom20. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 versus ShNC group; # p < 0.05, ## p < 0.01, and #### p < 0.0001 for the ShPGAM5 + Meth group versus ShNC + Meth group.

Article Snippet: The primary antibodies used were p62 (ab109012, 1:10,000) antibody was obtained from Abcam; LC3 (12,741, 1:1000), RIP1 (3493, 1:1000), and MLKL (37,705, 1:1000) antibodies were obtained from Cell Signaling Technology; PARL (NBP1‐80878, 1:500) and PINK1 (BC100‐494, 1:1000) antibodies were obtained from Novus Biologicals; RIP3 (sc‐374,639, 1:500) and β‐actin (sc‐47,778, 1:500) antibodies were obtained from Santa Cruz Technology; PGAM5 (28445‐1‐AP, 1:2000), Fis1 (10956‐1‐AP, 1:4000), Drp1 (12957‐1‐AP, 1:2000), and Parkin (14060‐1‐AP, 1:2000) antibodies were obtained from Proteintech.

Techniques: Knockdown, Double Staining, Control, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: CNS Neuroscience & Therapeutics

Article Title: Cleaving PINK1 or PGAM5 ? Involvement of PARL in Methamphetamine‐Induced Excessive Mitophagy and Neuronal Necroptosis

doi: 10.1111/cns.70293

Figure Lengend Snippet: PARL overexpression ameliorated neuronal loss after Meth exposure. (A) PARL overexpression significantly increased the number of Nissl‐positive cells in the hippocampus after Meth exposure. (B) TEM images showing mitochondrial morphology and autophagosome containing unhealthy mitochondria in the hippocampus. (C) Detection of Drp1, Fis1, MLKL, RIP3, RIP1, PINK1, PGAM5, Parkin, and LC3 protein expression by Western blot. (D) Statistical results of the relative expression levels of Drp1, Fis1, MLKL, RIP3, RIP1, PINK1, PGAM5, Parkin, and LC3 proteins. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 versus AAV group; # p < 0.05, ## p < 0.01, and ### p < 0.001 for the PARL + Meth group versus AAV + Meth group.

Article Snippet: The primary antibodies used were p62 (ab109012, 1:10,000) antibody was obtained from Abcam; LC3 (12,741, 1:1000), RIP1 (3493, 1:1000), and MLKL (37,705, 1:1000) antibodies were obtained from Cell Signaling Technology; PARL (NBP1‐80878, 1:500) and PINK1 (BC100‐494, 1:1000) antibodies were obtained from Novus Biologicals; RIP3 (sc‐374,639, 1:500) and β‐actin (sc‐47,778, 1:500) antibodies were obtained from Santa Cruz Technology; PGAM5 (28445‐1‐AP, 1:2000), Fis1 (10956‐1‐AP, 1:4000), Drp1 (12957‐1‐AP, 1:2000), and Parkin (14060‐1‐AP, 1:2000) antibodies were obtained from Proteintech.

Techniques: Over Expression, Expressing, Western Blot

Journal: CNS Neuroscience & Therapeutics

Article Title: Cleaving PINK1 or PGAM5 ? Involvement of PARL in Methamphetamine‐Induced Excessive Mitophagy and Neuronal Necroptosis

doi: 10.1111/cns.70293

Figure Lengend Snippet: PGAM5 knockdown ameliorated neuronal loss after Meth exposure. (A) PGAM5 downregulation significantly increased the number of Nissl‐positive cells in the hippocampus after Meth exposure. (B) TEM images showing mitochondrial morphology and autophagosome containing unhealthy mitochondria in the hippocampus. (C) Detection of MLKL, RIP3, RIP1, Drp1, and Fis1 protein expression by Western blot. (D) Statistical results of the relative expression levels of MLKL, RIP3, RIP1, Drp1, and Fis1 proteins. * p < 0.05 and ** p < 0.01, versus AAV group; # p < 0.05 and ## p < 0.01 for the ShPGAM5 + Meth group versus AAV + Meth group.

Article Snippet: The primary antibodies used were p62 (ab109012, 1:10,000) antibody was obtained from Abcam; LC3 (12,741, 1:1000), RIP1 (3493, 1:1000), and MLKL (37,705, 1:1000) antibodies were obtained from Cell Signaling Technology; PARL (NBP1‐80878, 1:500) and PINK1 (BC100‐494, 1:1000) antibodies were obtained from Novus Biologicals; RIP3 (sc‐374,639, 1:500) and β‐actin (sc‐47,778, 1:500) antibodies were obtained from Santa Cruz Technology; PGAM5 (28445‐1‐AP, 1:2000), Fis1 (10956‐1‐AP, 1:4000), Drp1 (12957‐1‐AP, 1:2000), and Parkin (14060‐1‐AP, 1:2000) antibodies were obtained from Proteintech.

Techniques: Knockdown, Expressing, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Ailanthone blocks mitophagy to promote mtDNA leakage through BAX-BAK1 pores and suppress hepatocellular carcinoma cell proliferation

doi: 10.3389/fphar.2024.1509482

Figure Lengend Snippet: Ailanthone reduces PRKN-mediated BAX degradation and promotes BAX-BAK1 mitochondrial pores formation. (A) WB assays were performed to assess the BAX and BAK1 proteins expression in Huh7 cells treated with ailanthone for 48 h. (B) Huh7 cells were treated with 1.2 μM ailanthone for 48 h with or without CCCP (10 μM for 24 h), BAX and PRKN proteins expression in mitochondria and cytoplasm were measured by WB. (C, D) Huh7 cells were treated with 1.2 μM ailanthone for 48 h. The lysates were immunoprecipitated against BAX, and immunoblotting assays for BAX and BAK1 were performed (C) . The colocalization of BAX and BAK1 was observed (1000×, scale bar: 50 μm). The colocalization area of BAX with BAK1 per cell was quantified (D) . (E) Huh7 cells were treated with MSN-50 (1 μM for 3 h), MSN-50 was then removed, and the cells were treated with 0.6 μM ailanthone for 48 h. CCK8 assay measured cell viability. (F, G) Huh7 cells were treated with 1.2 μM ailanthone for 48 h with or without CCCP (10 μM for 24 h), BAX and PRKN proteins expression were measured by WB (F) . The colocalization of BAX and PRKN proteins were observed (1000×, scale bar: 50 μm). The colocalization area of BAX with PRKN per cell was quantified (G) . (H, I) IP analysis of BAX and BAK1 in Huh7 cells treated with 1.2 μM ailanthone for 48 h was conducted. The ubiquitinated modified BAX and BAK1 signals were visualized using a pan-ubiquitin antibody. The quantitative analysis of ubiquitinated BAX and BAK1 proteins expression was normalized to their respective levels in the IP group. Bar, SD. * p < 0.05 or ** p < 0.01.

Article Snippet: The primary antibodies as follows: PINK1 (Proteintech, 23274-1-AP, 1:500), PRKN (Proteintech, 66674-1-Ig, 1:100; Proteintech, 14060-1-AP, 1:200), BAK1 (Proteintech, 29552-1-AP, 1:200), BAX (Proteintech, 60267-1-Ig, 1:200).

Techniques: Expressing, Immunoprecipitation, Western Blot, CCK-8 Assay, Modification, Ubiquitin Proteomics

Journal: Frontiers in Pharmacology

Article Title: Ailanthone blocks mitophagy to promote mtDNA leakage through BAX-BAK1 pores and suppress hepatocellular carcinoma cell proliferation

doi: 10.3389/fphar.2024.1509482

Figure Lengend Snippet: Ailanthone blocks PINK1-PRKN-mediated mitophagy while promoting BAX-BAK1 mitochondrial pores formation and inducing inflammation in vivo . (A, B) IHC detection of LC3, PINK1, PRKN, BAX, BAK1, TNF-α, IL-1β, and IL-6 proteins expression in tumour tissues (100×, scale bar: 500 μm). (C, D) The colocalization of PINK1 with PRKN and BAX with BAK1 in tumour tissues was observed by confocal microscopy imaging (400×, scale bar: 100 μm). The colocalization areas were quantified. Bar, SD. ** p < 0.01.

Article Snippet: The primary antibodies as follows: PINK1 (Proteintech, 23274-1-AP, 1:500), PRKN (Proteintech, 66674-1-Ig, 1:100; Proteintech, 14060-1-AP, 1:200), BAK1 (Proteintech, 29552-1-AP, 1:200), BAX (Proteintech, 60267-1-Ig, 1:200).

Techniques: In Vivo, Expressing, Confocal Microscopy, Imaging

Journal: Frontiers in Pharmacology

Article Title: Ailanthone blocks mitophagy to promote mtDNA leakage through BAX-BAK1 pores and suppress hepatocellular carcinoma cell proliferation

doi: 10.3389/fphar.2024.1509482

Figure Lengend Snippet: Schematic representation of ailanthone blocks mitophagy to promote mtDNA leakage through BAX-BAK1 pores and suppress hepatocellular carcinoma cell proliferation. Ailanthone induces mitochondrial damage while blocking PINK1-PRKN-mediated mitophagy, leading to the accumulation of dysfunctional mitochondria in cells, thereby inhibiting hepatocellular carcinoma (HCC) cell proliferation. Furthermore, the inhibition of PRKN protein by ailanthone reduced the ubiquitination and degradation of BAX, promoting the localization of BAX on the outer mitochondrial membrane, forming BAX-BAK1 mitochondrial pores, aggravating the release of mtDNA, and inducing inflammatory responses. Ultimately, the accumulation of damaged mitochondria and the release of inflammatory factors both contribute to the suppressive effect of ailanthone on HCC cell proliferation.

Article Snippet: The primary antibodies as follows: PINK1 (Proteintech, 23274-1-AP, 1:500), PRKN (Proteintech, 66674-1-Ig, 1:100; Proteintech, 14060-1-AP, 1:200), BAK1 (Proteintech, 29552-1-AP, 1:200), BAX (Proteintech, 60267-1-Ig, 1:200).

Techniques: Blocking Assay, Inhibition, Ubiquitin Proteomics, Membrane

Journal: Bone Research

Article Title: Enhanced SIRT3 expression restores mitochondrial quality control mechanism to reverse osteogenic impairment in type 2 diabetes mellitus

doi: 10.1038/s41413-024-00399-5

Figure Lengend Snippet: The diabetic microenvironment induces mitophagy impairment in osteoblasts. a , b Enrichment Analysis for differentially expressed genes between the CTRL and HFD&STZ groups using GO and KEGG databases. c Detection of osteoblast mitochondrial ultrastructure by transmission electron microscopy (TEM) in femurs from CTRL and HFD&STZ mice. d Visualization of mitochondria in MC3T3-E1 cells after HGPA treatment using Mitotracker Green dye. e Representative images of JC-1 staining in CTRL and HGPA-treated MC3T3-E1 cells. Red fluorescence represented intact mitochondria with normal membrane potential and green fluorescence represented impaired mitochondria with reduced membrane potential. Scale bar, 200 μm. f Representative images of MitoTracker-labeled mitochondria (deep red), Mtphagy-labeled mitophagy process (red), and Lyso Dye-labeled lysosomes (green), of which the co-localization indicates the occurrence of mitophagy. Scale bar, 50 μm. g , h Western blot images and quantification of LC3 in MC3T3-E1 cells treated with gradient concentrations of HGPA, with or without Baf-A1 (10 nmol/L). i Representative IF staining of TOM20 (red), PRKN (green), and MitoTracker (deep red) in MC3T3-E1 cells from CTRL and HGPA groups stimulated by CCCP for 6 h. Scale bar, 50 μm. j Western blot results of mitophagy-related proteins including PINK1, PRKN, SQSTM1, and LC3 in MC3T3-E1 cells stimulated by HGPA for various duration. k Western blot analysis of PINK1 and PRKN in bone from CTRL and HFD&STZ mice. l qPCR analysis of Pink1 and Prkn genes. n = 6 per group. Data presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The primary antibodies used for IF staining were as follows: OCN (bs0470R, Bioss USA), RUNX2 (R25634, Zen-bio), TOM20 (A19403, ABclonal, China), PRKN (66674-1, Proteintech), PINK1 (A7131, ABclonal, China), SIRT3 (HA722251, HUABIO), CTSK (bs-1611R, Bioss), FOXO3 (66428-1, Proteintech).

Techniques: Transmission Assay, Electron Microscopy, Staining, Fluorescence, Membrane, Labeling, Western Blot

Journal: Bone Research

Article Title: Enhanced SIRT3 expression restores mitochondrial quality control mechanism to reverse osteogenic impairment in type 2 diabetes mellitus

doi: 10.1038/s41413-024-00399-5

Figure Lengend Snippet: Activation of SIRT3 promotes mitophagy and ameliorates osteoblast dysfunction in MC3T3-E1 cells. a Relative protein and mRNA expression of SIRT3 in MC3T3-E1 cells transfected with Lv- Ctrl and Lv- Sirt3 . b , c Representative images and quantification of mitochondria in Lv- Ctrl , Lv- Sirt3 , HGPA+Lv- Ctrl , HGPA+Lv- Sirt3 groups. Scale bar, 20 μm. d Representative images of MitoTracker-labeled mitochondria (deep red), Mtphagy Dye-labeled mitophagy (red), and Lyso Dye-labeled lysosomes (green), of which the co-localization indicates the occurrence of mitophagy. Scale bar, 100 μm. e Representative IF staining of TOM20 (red), PRKN (green), and MitoTracker (deep red) in MC3T3-E1 cells from HGPA+Lv- Ctrl and HGPA+Lv- Sirt3 groups stimulated by CCCP for 6 h. Scale bar, 100 μm. f , g Western blot images and quantification of mitophagy-related proteins, including PINK1, PRKN, LC3, and SQSTM1 in the above groups. h qPCR analysis of Alpl , Runx2 , Col1a1 and Ibsp genes in 3 independent replicative experiments. i Representative images and quantification of ALP (7 days) and ARS (21 days) staining of MC3T3-E1 cells from above four group. j , k Western blot results and quantitative analysis of RUNX2, ALP, COL1A1 in MC3T3-E1 cells. l , n Representative images and quantification of ALP staining of MC3T3-E1 cells treated with HGPA, HGPA + HKL and HGPA+mdivi-1. Scale bar, 1 000 μm. o , m Western blot analysis of RUNX2 and ALP in MC3T3-E1 cells with different intervention. Data presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The primary antibodies used for IF staining were as follows: OCN (bs0470R, Bioss USA), RUNX2 (R25634, Zen-bio), TOM20 (A19403, ABclonal, China), PRKN (66674-1, Proteintech), PINK1 (A7131, ABclonal, China), SIRT3 (HA722251, HUABIO), CTSK (bs-1611R, Bioss), FOXO3 (66428-1, Proteintech).

Techniques: Activation Assay, Expressing, Transfection, Labeling, Staining, Western Blot

Journal: Journal of Asthma and Allergy

Article Title: NLRP3 Inflammasome Deficiency Alleviates Inflammation and Oxidative Stress by Promoting PINK1/Parkin-Mediated Mitophagy in Allergic Rhinitis Mice and Nasal Epithelial Cells

doi: 10.2147/JAA.S467774

Figure Lengend Snippet: Effects of MCC950 on NLRP3 inflammasome and mitophagy in IL-13-stimulated HNEpCs. HNEpCs were pretreated with 10 μM MCC950 for 1 h and then incubated with IL-13 (10 ng/mL) for 24 h. ( A ) Representative images of NLRP3 and IL-1β in each group (Magnification: 400×). ( B and C ) The quantification of NLRP3 and IL-1β immunofluorescent staining in HNEpCs. ( D–H ) Western blot analysis and quantification of PINK1, Parkin, LC3, and TOM20 in HNEpCs. Data are presented as mean ± SEM (n=3). ** P <0.01.

Article Snippet: The fixed HNEpCs were immunostained with the corresponding primary antibodies against NLRP3 (68102-1-Ig, PROTEINTECH), IL-1β (16806-1-AP, PROTEINTECH), LC3 (SAB1305552, Sigma-Aldrich), Parkin (66674-1-Ig, PROTEINTECH), and COX IV (11242-1-AP, 1:100, PROTEINTECH).

Techniques: Incubation, Staining, Western Blot

Journal: Journal of Asthma and Allergy

Article Title: NLRP3 Inflammasome Deficiency Alleviates Inflammation and Oxidative Stress by Promoting PINK1/Parkin-Mediated Mitophagy in Allergic Rhinitis Mice and Nasal Epithelial Cells

doi: 10.2147/JAA.S467774

Figure Lengend Snippet: NLRP3 inhibition enhances PINK1/Parkin-mediated mitophagy in IL-13 treated HNEpCs. HNEpCs were transfected with NC siRNA or PINK1 siRNA, and then added the remaining reagents in order. ( A–E ) Western blot analysis and quantification of PINK1, Parkin, LC3, and TOM20 in HNEpCs. ( F ) The co-localization of Parkin (green) with COX IV (red) were determined by IF analysis. Magnification: 400×. Data are presented as mean ± SEM (n=3). ** P <0.01.

Article Snippet: The fixed HNEpCs were immunostained with the corresponding primary antibodies against NLRP3 (68102-1-Ig, PROTEINTECH), IL-1β (16806-1-AP, PROTEINTECH), LC3 (SAB1305552, Sigma-Aldrich), Parkin (66674-1-Ig, PROTEINTECH), and COX IV (11242-1-AP, 1:100, PROTEINTECH).

Techniques: Inhibition, Transfection, Western Blot