park2  (Addgene inc)

Journal: Molecular Neurodegeneration

Article Title: Mitochondrial CISD1/Cisd accumulation blocks mitophagy and genetic or pharmacological inhibition rescues neurodegenerative phenotypes in Pink1/parkin models

doi: 10.1186/s13024-024-00701-3

Figure Lengend Snippet: Drosophila Cisd is functionally more similar to CISD1. A Immunoblots of protein lysates from RPE1 cells ± YFP-Parkin overexpression (OE) treated with antimycin A (4 µM) and oligomycin (10 µM) for the indicated time to induce mitophagy, probed for mitophagy marker pUb, and degradation of TOM20 and CISD1/2 (CISD1, Proteintech, 16006-1-AP; and CISD2, Proteintech, 13318-1-AP), alongside respective loading control total Ub and Tubulin. Blot is representative of 3 replicate experiments. B Confocal micrographs of U2OS cells transfected with human CISD1-FLAG or CISD2-FLAG, counter-stained with antibodies against TOM20 (mitochondria) or Calnexin (ER). C Confocal micrographs of Drosophila larval neurons expressing transgenic mito-GFP and WT control, human CISD1-HA or CISD2-HA driven by nSyb -GAL4. D Immunoblots of protein lysates of 2- and 20-day-old whole flies expressing the indicated transgenes via da -GAL4 versus WT control, probed for pUb and CISD1/2 with (CISD2, Proteintech, 13318-1-AP). Scale bars = 10 μm

Article Snippet: A Immunoblots of protein lysates from RPE1 cells ± YFP-Parkin overexpression (OE) treated with antimycin A (4 μM) and oligomycin (10 μM) for the indicated time to induce mitophagy, probed for mitophagy marker pUb, and degradation of TOM20 and CISD1/2 (CISD1, Proteintech, 16006-1-AP; and CISD2, Proteintech, 13318-1-AP), alongside respective loading control total Ub and Tubulin.

Techniques: Western Blot, Over Expression, Marker, Control, Transfection, Staining, Expressing, Transgenic Assay

Journal: Molecular Neurodegeneration

Article Title: Mitochondrial CISD1/Cisd accumulation blocks mitophagy and genetic or pharmacological inhibition rescues neurodegenerative phenotypes in Pink1/parkin models

doi: 10.1186/s13024-024-00701-3

Figure Lengend Snippet: Cisd overexpression blocks mitophagy flux. A Immunoblot analysis of whole fly lysates from 2-day-old flies of the indicated genotypes, analysed for pUb, and Tubulin or total protein levels as loading controls. Blot is representative of 3 replicate experiments. Cisd overexpression was driven by da -GAL4. B Confocal microscopy analysis of adult Drosophila flight muscle from 2-day-old flies of WT control, parkin mutant and Cisd overexpression driven by da -GAL4 immunostained for mitochondria (ATP5A) and pUb. C – F Confocal analysis of mitophagy reporter mito -QC (OMM-localised tandem RFP-GFP) of WT or Cisd overexpression driven by nSyb -GAL4, in larval ( C , D ) or adult ( E , F ) neurons with ‘red-only’ mitolysosomes shown. D , F Number of mitolysosomes quantified shown in C and E. Data points indicate individual animals analysed. Statistical analysis: unpaired t-test; ** P < 0.01; **** P < 0.0001. Scale bars = 10 μm

Article Snippet: A Immunoblots of protein lysates from RPE1 cells ± YFP-Parkin overexpression (OE) treated with antimycin A (4 μM) and oligomycin (10 μM) for the indicated time to induce mitophagy, probed for mitophagy marker pUb, and degradation of TOM20 and CISD1/2 (CISD1, Proteintech, 16006-1-AP; and CISD2, Proteintech, 13318-1-AP), alongside respective loading control total Ub and Tubulin.

Techniques: Over Expression, Western Blot, Confocal Microscopy, Control, Mutagenesis

Journal: Molecular Neurodegeneration

Article Title: Mitochondrial CISD1/Cisd accumulation blocks mitophagy and genetic or pharmacological inhibition rescues neurodegenerative phenotypes in Pink1/parkin models

doi: 10.1186/s13024-024-00701-3

Figure Lengend Snippet: Cisd overexpression causes autophagosome accumulation and prevents autophagy. A Confocal micrographs of WT control versus Cisd overexpressing adult flight muscle via Mef2 -GAL4, immunostained for p62 alongside imaging mCherry-Atg8a autophagosome reporter. B Electron micrographs of flight muscle as in A, showing multiple autophagic vesicles (inset) in proximity to disrupted mitochondria (arrowheads). C Larval neurons of WT control versus Cisd overexpressing or Atg5 knockdown (via nSyb -GAL4 driver) animals immunostained for p62 alongside ATP5a (mitochondria) and DAPI. D Immunoblot analysis of protein lysates from whole flies overexpressing Cisd (via da -GAL4) or WT controls. Blots were probed with antibodies against p62, Atg8a (LC3), Cisd (CISD2, Proteintech, 13318-1-AP) and Tubulin. Quantification of replicate blots is shown in Fig. S A, B. E Quantification of the number of autolysosomes shown in F. Data points indicate individual animals analysed. Statistical analysis: unpaired t-test; *** P < 0.001. F Confocal microscopy analysis of adult flight muscle WT control versus Cisd overexpressing animals co-expressing the autophagy flux reporter GFP-mCherry-Atg8a driven by Mef2 -GAL4. G , H Locomotor climbing assay of 2-day-old adult flies expressing the indicated transgenes (via da -GAL4). I Immunoblot analysis of equivalent samples analysed in G and H, probed for autophagy markers (p62 and Atg8a), Cisd and Tubulin. Quantification of replicate blots is shown in Fig. S C. Statistical analyses: Kruskal-Wallis non-parametric test with Dunn’s post-hoc correction. *** P < 0.001; **** P < 0.0001. Scale bars = 10 μm for light microscopy, or indicated on image for EM

Article Snippet: A Immunoblots of protein lysates from RPE1 cells ± YFP-Parkin overexpression (OE) treated with antimycin A (4 μM) and oligomycin (10 μM) for the indicated time to induce mitophagy, probed for mitophagy marker pUb, and degradation of TOM20 and CISD1/2 (CISD1, Proteintech, 16006-1-AP; and CISD2, Proteintech, 13318-1-AP), alongside respective loading control total Ub and Tubulin.

Techniques: Over Expression, Control, Imaging, Knockdown, Western Blot, Confocal Microscopy, Expressing, Climbing Assay, Light Microscopy

Journal: Molecules and Cells

Article Title: PARK2 Induces Osteoclastogenesis through Activation of the NF-κB Pathway

doi: 10.14348/molcells.2022.0058

Figure Lengend Snippet: (A) The mRNA levels of PARK2 and osteoclast marker genes were analyzed by real-time PCR in BMMs cultured with RANKL and M-CSF for the indicated days. *** P < 0.001 versus day 0. (B) PARK2 protein expression during osteoclast differentiation were analyzed by western blotting. (C) The relative level of PARK2 protein was determined by Image J software. *** P < 0.001 versus BMM. Data were presented as mean ± SD.

Article Snippet: PARK2 plasmid (#59416) was purchased from Addgene (USA).

Techniques: Marker, Real-time Polymerase Chain Reaction, Cell Culture, Expressing, Western Blot, Software

Journal: Molecules and Cells

Article Title: PARK2 Induces Osteoclastogenesis through Activation of the NF-κB Pathway

doi: 10.14348/molcells.2022.0058

Figure Lengend Snippet: (A) pOCs transfected with control (Con) or PARK2 siRNA were subjected to western blotting and real-time PCR analyses. * P < 0.05 versus the control siRNA group. (B) pOCs transfected with control or PARK2 siRNA were further cultured with RANKL and M-CSF for 3 days and then stained for TRAP. TRAP + multinucleated cells more than 3, 10 nuclei were counted. Scale bars = 200 μm. * P < 0.05, *** P < 0.001. (C) pOCs transfected with control or PARK2 siRNA were cultured with RANKL and M-CSF until day 6. The mRNA expression levels of ACP5, DC-STMAP, MMP9 and v-ATPase were analyzed by real-time PCR. * P < 0.05; ** P < 0.01; *** P < 0.001. Data were presented as mean ± SD. (D) pOCs transfected with control or PARK2 siRNA were cultured on dentine discs until day 12. The dark areas in gray images indicate the resorbed surface of dentin slices. The percentage of resorbed area and depth of resorption pits were presented as mean ± SD. * P < 0.05; ** P < 0.01.

Article Snippet: PARK2 plasmid (#59416) was purchased from Addgene (USA).

Techniques: Transfection, Control, Western Blot, Real-time Polymerase Chain Reaction, Cell Culture, Staining, Expressing

Journal: Molecules and Cells

Article Title: PARK2 Induces Osteoclastogenesis through Activation of the NF-κB Pathway

doi: 10.14348/molcells.2022.0058

Figure Lengend Snippet: (A) BMMs transfected with control (Con) vector or PARK2 plasmid were subjected to western blotting (left panel) and real-time PCR (right panel) analyses. *** P < 0.001. (B) BMMs transfected with either control or PARK2 plasmid were cultured with RANKL and M-CSF for 5 days and stained for TRAP. Representative images (left panel) and quantification of TRAP + multinucleated cells are shown (right panel). Scale bars = 200 μm. *** P < 0.001. (C) mRNA levels of ACP5, DC-STAMP, MMP9 and OSCAR were measured by real-time PCR. * P < 0.05; *** P < 0.001.

Article Snippet: PARK2 plasmid (#59416) was purchased from Addgene (USA).

Techniques: Transfection, Control, Plasmid Preparation, Western Blot, Real-time Polymerase Chain Reaction, Cell Culture, Staining

Journal: Molecules and Cells

Article Title: PARK2 Induces Osteoclastogenesis through Activation of the NF-κB Pathway

doi: 10.14348/molcells.2022.0058

Figure Lengend Snippet: (A and B) pOCs transfected with control (Con) siRNA or PARK2 siRNA were starved with serum-free medium and then stimulated with RANKL (500 ng/ml) for the indicated time. Both phospho-forms and total forms of the NF-κB signaling components (A) and MAPKs (B) were determined by western blotting. (C) pOCs transfected with control siRNA or PARK2 siRNA were serum-starved and then stimulated with RANKL (500 ng/ml) for 15 min. Representative confocal images are presented. Scale bars = 20 μm. *** P < 0.001. (D) BMMs transfected with control or PARK2 plasmid were cultured with or without BAY 11-7085 (5 μM) for 2 days. Protein levels of phospho-forms and total forms of p65 were detected by western blotting.

Article Snippet: PARK2 plasmid (#59416) was purchased from Addgene (USA).

Techniques: Transfection, Control, Western Blot, Plasmid Preparation, Cell Culture

Journal: Molecules and Cells

Article Title: PARK2 Induces Osteoclastogenesis through Activation of the NF-κB Pathway

doi: 10.14348/molcells.2022.0058

Figure Lengend Snippet: (A) BMMs were cultured in the presence of LPS (100 ng/ml), IL-1β (10 ng/ml), and TNFα (40 ng/ml) for 24 h and mRNA levels of PARK2 were determined by real-time PCR. ** P < 0.01; *** P < 0.001. Con, control. (B) pOCs transfected with control siRNA or PARK2 siRNA were cultured in the presence of LPS (100 ng/ml), IL-1β (10 ng/ml), or TNFα (40 ng/ml) with RANKL and M-CSF until day 4 and stained for TRAP. Scale bars = 200 μm. (C) mRNA levels of ACP5, DC-STAMP, OC-STAMP, and v-ATPase in PARK2 or control siRNA transfected pOC cultured with RANKL and LPS (100 ng/ml) were measured by real-time PCR. * P < 0.05; ** P < 0.01; *** P < 0.001. (D) pOCs transfected with control siRNA or PARK2 siRNA were serum-starved and stimulated with LPS (200 ng/ml) for 15 min. Protein levels of the phospho-forms and total forms of IKK, IκB, and p65 were determined by western blotting.

Article Snippet: PARK2 plasmid (#59416) was purchased from Addgene (USA).

Techniques: Cell Culture, Real-time Polymerase Chain Reaction, Control, Transfection, Staining, Western Blot

Journal: Molecules and Cells

Article Title: PARK2 Induces Osteoclastogenesis through Activation of the NF-κB Pathway

doi: 10.14348/molcells.2022.0058

Figure Lengend Snippet: (A) LPS (2.5 mg/kg) or vehicle (PBS) was injected at the midline of calvariae. Either control (Con) or PARK2 siRNA was locally injected near the LPS-injected site. The knock-down efficiency was determined by real-time PCR. * P < 0.05; *** P < 0.001. (B) Representative μCT images (left panel) and measurements of bone volume per tissue volume (BV/TV) (right panel) are shown. * P < 0.05; *** P < 0.001. (C) Total calvariae were stained for TRAP activity (left panel). Quantitative values of TRAP + area (%) are shown at the right panel. * P < 0.05; *** P < 0.001.

Article Snippet: PARK2 plasmid (#59416) was purchased from Addgene (USA).

Techniques: Injection, Control, Knockdown, Real-time Polymerase Chain Reaction, Staining, Activity Assay

Journal: Molecules and Cells

Article Title: PARK2 Induces Osteoclastogenesis through Activation of the NF-κB Pathway

doi: 10.14348/molcells.2022.0058

Figure Lengend Snippet: Upon activation of RANK by RANKL, TRAF6 is recruited and activated. Downstream of TRAF6, PARK2 may interact with NEMO and enhance its linear ubiquitination, resulting in the activation of the IKK complex. The activated IKK complex facilitates proteasomal degradation of IκB and triggers p65 translocation to the nucleus, leading to gene transcription of osteoclast marker genes like ACP5, MMP9, and Ctsk.

Article Snippet: PARK2 plasmid (#59416) was purchased from Addgene (USA).

Techniques: Activation Assay, Ubiquitin Proteomics, Translocation Assay, Marker