Journal: Neural Regeneration Research

Article Title: A novel mechanism of PHB2-mediated mitophagy participating in the development of Parkinson's disease

doi: 10.4103/1673-5374.389356

Figure Lengend Snippet: Effect of MPP + on PHB2 protein expression and mitochondrial function in SH-SY5Y cells. SH-SY5Y cells were treated with PHB2-shRNA and PHB2-Over Exp, and then induced with 1 mM MPP + for 24 hours. (A, B) PHB2 protein concentration in SH-SY5Y cells after 1 mM MPP + treatment for 6, 12, 24, and 48 hours. (A) Western blots showing PHB2 protein expression in MPP + -treated cells at different times. (B) Quantitative analysis of PHB2 protein expression. (C–G) Protein expression changes of Nrf2, HO-1, NQO-1, and PHB2. (C) Western blots showing Nrf2, HO-1, NQO-1, and PHB2 protein expression in MPP + -treated and PHB2-shRNA- or PHB2-Over Exp-treated cells. (D–G) Quantitative analysis of Nrf2, HO-1, NQO-1, and PHB2 protein expression. (H, I) The effect of PHB2 expression on mitochondrial membrane potential induced by MPP + . (H) JC-1 staining of SH-SY5Y cells treated with PHB2-shRNA and PHB2-Over Exp. Original magnification 10×, scale bar: 50 μm. (I) Quantitative analysis of JC-1 staining: the green/red ratio reflects changes in the mitochondrial membrane potential. (J, K) The effect of changes in PHB2 expression on ROS production under MPP + induction. (J) Intracellular ROS fluorescence. SH-SY5Y cells were treated with PHB2-shRNA and PHB2-Over Exp and labeled with DCFH-DA (green fluorescence). Original magnification 4×, scale bar: 100 μm. (K) Quantitative analysis of ROS (DCFH-DA) in SH-SY5Y cells. (L) Electron microscopy images of morphological changes in mitochondria in PHB2-shRNA-treated and MPP + -induced cells. Red boxes show magnified mitochondria. Scale bars: 1 μm. Data are expressed as mean ± SEM ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, vs . control group; # P < 0.05,## P < 0.01, vs . MPP + group (one-way analysis of variance with Tukey's multiple comparison test). DCFH-DA: 2′,7′-Dichlorodihydrofluorescein diacetate; HO-1: heme oxygenase-1; JC-1: 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide; MPP + : 1-methyl-4-phenylpyridinium; NQO-1: NAD(P)H quinone dehydrogenase 1; Nrf2: nuclear factor erythroid 2-related factor 2; Over Exp: overexpression; PHB2: prohibitin 2; ROS: reactive oxygen species; SH-SY5Y: human neuroblastoma cell line.

Article Snippet: Membranes were incubated overnight at 4°C in solutions with primary antibodies against PHB2 (rabbit, 1:2000, Proteintech, Chicago, IL, USA, Cat# 12295-1-AP, RRID: AB_2164779), Parkin (mouse, 1:2000, Abcam, Cambridge, UK, Cat# ab77924, RRID: AB_1566559), LC3B (rabbit, 1:2000, Abcam, Cat# ab192890, RRID: AB_2827794), p-PERK Thr980 (rabbit, 1:2000, Cell Signaling Technology, Danvers, MA, USA, Cat# 3179, RRID: AB_2095853), nuclear factor erythroid 2-related factor (Nrf2; rabbit, 1:2000, Cell Signaling Technology, Cat# 12721, RRID: AB_2715528), heme oxygenase 1 (HO-1; rabbit, 1:2000, Cell Signaling Technology, Cat# 43966, RRID: AB_2799254), quinone oxidoreductase 1 (NQO-1; mouse, 1:2000, Cell Signaling Technology, Cat #3187, RRID: AB_2154354), and β-actin (mouse, 1:5000, CWBio, Beijing, China, Cat# CW0096, RRID: AB_2665433).

Techniques: Expressing, shRNA, Protein Concentration, Western Blot, Membrane, Staining, Fluorescence, Labeling, Electron Microscopy, Control, Comparison, Over Expression

Journal: Neural Regeneration Research

Article Title: A novel mechanism of PHB2-mediated mitophagy participating in the development of Parkinson's disease

doi: 10.4103/1673-5374.389356

Figure Lengend Snippet: Effect of PHB2 on mitophagy in a PD cell model. (A–F) PHB2 and LC3II/LC3I protein expression in SH-SY5Y cells after treatment with PHB2-shRNA or PHB2-Over Exp and MPP + (1 mM, 24 hours). (A) Protein expression levels of PHB2 and LC3 in SH-SY5Y cells treated with PHB2-shRNA under MPP + induction. (B, C) Quantitative analysis of PHB2 and LC3II/LC3I protein expression. (D) Protein expression levels of PHB2 and LC3 in SH-SY5Y cells treated with PHB2-Over Exp under MPP + induction. (E, F) Quantitative analysis of PHB2 and LC3II/LC3I protein expression. (G) Cellular localization of LC3 and TOM20 or TIM23 in SH-SY5Y cells treated with PHB2-shRNA under MPP + induction. Immunofluorescence changes of LC3 and mitochondrial proteins (TOM20 and TIM23). Red fluorescence represents TOM20 and TIM23, green fluorescence represents LC3, and blue fluorescence represents DAPI. Original magnification 20×, Scale bars: 20 μm. Data are expressed as mean ± SEM ( n = 3). ** P < 0.01, *** P < 0.001, vs . control group (one-way analysis of variance with Tukey's multiple comparison test). DAPI: 4′,6-Diamidino-2-phenylindole; LC3: microtubule-associated protein 1 light chain 3; MPP + : 1-methyl-4-phenylpyridinium; Over Exp: overexpression; PHB2: prohibitin 2; SH-SY5Y: human neuroblastoma cell line; TIM23: translocase of inner mitochondrial membrane 23; TOM20: translocase of outer mitochondrial membrane 20.

Article Snippet: Membranes were incubated overnight at 4°C in solutions with primary antibodies against PHB2 (rabbit, 1:2000, Proteintech, Chicago, IL, USA, Cat# 12295-1-AP, RRID: AB_2164779), Parkin (mouse, 1:2000, Abcam, Cambridge, UK, Cat# ab77924, RRID: AB_1566559), LC3B (rabbit, 1:2000, Abcam, Cat# ab192890, RRID: AB_2827794), p-PERK Thr980 (rabbit, 1:2000, Cell Signaling Technology, Danvers, MA, USA, Cat# 3179, RRID: AB_2095853), nuclear factor erythroid 2-related factor (Nrf2; rabbit, 1:2000, Cell Signaling Technology, Cat# 12721, RRID: AB_2715528), heme oxygenase 1 (HO-1; rabbit, 1:2000, Cell Signaling Technology, Cat# 43966, RRID: AB_2799254), quinone oxidoreductase 1 (NQO-1; mouse, 1:2000, Cell Signaling Technology, Cat #3187, RRID: AB_2154354), and β-actin (mouse, 1:5000, CWBio, Beijing, China, Cat# CW0096, RRID: AB_2665433).

Techniques: Expressing, shRNA, Immunofluorescence, Fluorescence, Control, Comparison, Over Expression, Membrane

Journal: Neural Regeneration Research

Article Title: A novel mechanism of PHB2-mediated mitophagy participating in the development of Parkinson's disease

doi: 10.4103/1673-5374.389356

Figure Lengend Snippet: Parkin increases PHB2/LC3-mediated mitophagy in SH-SY5Y cells. (A–D) Protein levels of Parkin, PHB2, and autophagy marker (LC3) in SH-SY5Y cells after treatment with Parkin-shRNA, Parkin-Over Exp, and MPP + (1 mM, 24 hours). (A) Protein expression levels of Parkin, LC3, and PHB2 in SH-SY5Y cells treated with Parkin-shRNA or Parkin-Over Exp under MPP + induction. (B–D) Quantitative analysis of Parkin, and LC3II/LC3I, PHB2 protein expression. (E) Immunofluorescence analysis of PHB2 and LC3. Cellular localization of LC3 and PHB2 in SH-SY5Y cells treated with Parkin-shRNA or Parkin-Over Exp under MPP + induction. Red fluorescence represents PHB2, green fluorescence represents LC3, and blue fluorescence represents DAPI. Original magnification 40×, Scale bar: 20 μm. Data are expressed as mean ± SEM ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, vs. control group; # P < 0.05,### P < 0.001, vs . MPP + group (one-way analysis of variance with Tukey's multiple comparison test). DAPI: 4′,6-Diamidino-2-phenylindole; LC3: microtubule-associated protein 1 light chain 3; MPP + : 1-methyl-4-phenylpyridinium; Over Exp: overexpression; Parkin: parkin RBR E3 ubiquitin-protein ligase; PHB2: prohibitin 2; SH-SY5Y: human neuroblastoma cell line.

Article Snippet: Membranes were incubated overnight at 4°C in solutions with primary antibodies against PHB2 (rabbit, 1:2000, Proteintech, Chicago, IL, USA, Cat# 12295-1-AP, RRID: AB_2164779), Parkin (mouse, 1:2000, Abcam, Cambridge, UK, Cat# ab77924, RRID: AB_1566559), LC3B (rabbit, 1:2000, Abcam, Cat# ab192890, RRID: AB_2827794), p-PERK Thr980 (rabbit, 1:2000, Cell Signaling Technology, Danvers, MA, USA, Cat# 3179, RRID: AB_2095853), nuclear factor erythroid 2-related factor (Nrf2; rabbit, 1:2000, Cell Signaling Technology, Cat# 12721, RRID: AB_2715528), heme oxygenase 1 (HO-1; rabbit, 1:2000, Cell Signaling Technology, Cat# 43966, RRID: AB_2799254), quinone oxidoreductase 1 (NQO-1; mouse, 1:2000, Cell Signaling Technology, Cat #3187, RRID: AB_2154354), and β-actin (mouse, 1:5000, CWBio, Beijing, China, Cat# CW0096, RRID: AB_2665433).

Techniques: Marker, shRNA, Expressing, Immunofluorescence, Fluorescence, Control, Comparison, Over Expression, Ubiquitin Proteomics

Journal: Neural Regeneration Research

Article Title: A novel mechanism of PHB2-mediated mitophagy participating in the development of Parkinson's disease

doi: 10.4103/1673-5374.389356

Figure Lengend Snippet: Parkin regulates anti-oxidative stress protein expression via PHB2. (A–D) SH-SY5Y cells were treated with PHB2-shRNA, Parkin-Over Exp, and MPP + (1 mM, 24 hours). Changes in Nrf2, HO-1, and NQO-1 protein levels and quantitative analysis. (A) Protein expression levels of Nrf2, HO-1, and NQO-1 in SH-SY5Y cells treated with PHB2-shRNA or Parkin-Over Exp under MPP + induction. (B–D) Quantitative analysis of Nrf2, HO-1, and NQO-1 protein expression. Data are expressed as mean ± SEM ( n = 3). *** P < 0.001, vs. control group; # P < 0.05,### P < 0.001, vs. MPP + group (one-way analysis of variance with Tukey's multiple comparison test). HO-1: Heme oxygenase-1; MPP + : 1-methyl-4-phenylpyridinium; NQO-1: NAD(P)H quinone dehydrogenase 1; Nrf2: nuclear factor erythroid 2-related factor 2; Over Exp: overexpression; Parkin: parkin RBR E3 ubiquitin-protein ligase; PHB2: prohibitin 2; SH-SY5Y: human neuroblastoma cell line.

Article Snippet: Membranes were incubated overnight at 4°C in solutions with primary antibodies against PHB2 (rabbit, 1:2000, Proteintech, Chicago, IL, USA, Cat# 12295-1-AP, RRID: AB_2164779), Parkin (mouse, 1:2000, Abcam, Cambridge, UK, Cat# ab77924, RRID: AB_1566559), LC3B (rabbit, 1:2000, Abcam, Cat# ab192890, RRID: AB_2827794), p-PERK Thr980 (rabbit, 1:2000, Cell Signaling Technology, Danvers, MA, USA, Cat# 3179, RRID: AB_2095853), nuclear factor erythroid 2-related factor (Nrf2; rabbit, 1:2000, Cell Signaling Technology, Cat# 12721, RRID: AB_2715528), heme oxygenase 1 (HO-1; rabbit, 1:2000, Cell Signaling Technology, Cat# 43966, RRID: AB_2799254), quinone oxidoreductase 1 (NQO-1; mouse, 1:2000, Cell Signaling Technology, Cat #3187, RRID: AB_2154354), and β-actin (mouse, 1:5000, CWBio, Beijing, China, Cat# CW0096, RRID: AB_2665433).

Techniques: Expressing, shRNA, Control, Comparison, Over Expression, Ubiquitin Proteomics

Journal: Neural Regeneration Research

Article Title: A novel mechanism of PHB2-mediated mitophagy participating in the development of Parkinson's disease

doi: 10.4103/1673-5374.389356

Figure Lengend Snippet: Silencing PHB2 inhibits mitophagy and aggravates dopaminergic neuronal loss in PD mice. (A) Schematic of PHB2-shRNA and MPTP treatment in mice. (B, C) Quantitative analysis of PHB2 protein levels in the midbrain of C57BL/6J mice after PHB2-shRNA injection. (B) Protein expression levels of PHB2 in C57BL/6J mice treated with PHB2-shRNA. (C) Quantitative analysis of PHB2 protein expression. (D–F) An acute PD model was established by intraperitoneal injection of MPTP in C57BL/6J mice after PHB2-shRNA injection. Changes and quantitative analysis of LC3II/LC3I and PHB2 protein levels in the midbrain. (D) Protein expression levels of PHB2 and LC3 after MPTP injection in PD model mice. (E, F) Quantitative analysis of PHB2 and LC3II/LC3I protein expression. (G, H) TH immunofluorescence (Alexa Fluor 488, green fluorescence) shows dopaminergic neurons in the substantia nigra in PD mice with silenced PHB2. (G) Immunofluorescence of TH-positive neurons in the substantia nigra. Mice were treated with PHB2-shRNA and MPTP. Original magnification 10×, Scale bar: 200 μm. (H) Quantitative analysis of TH-positive neurons in the substantia nigra. (I) Fluorescence co-localization of LC3 and PHB2, TIM23, or TOM20 in the substantia nigra. Mice were treated with PHB2-shRNA and MPTP. Red fluorescence (Alexa Fluor 555): PHB2, TOM20, and TIM23, green fluorescence (Alexa Fluor 488): LC3, and blue fluorescence: DAPI. Original magnification 20×, Scale bars: 20 μm. Data are expressed as mean ± SEM ( n = 3). * P < 0.05, *** P < 0.001, vs. control group; $ P < 0.05, $$ P < 0.01, $$$ P < 0.001, vs. MPTP group (one-way analysis of variance with Tukey's multiple comparison test). LC3: Microtubule-associated protein 1 light chain 3; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; PD: Parkinson's disease; PHB2: Prohibitin 2; TH: tyrosine hydroxylase; TIM23: translocase of inner mitochondrial membrane 23; TOM20: translocase of outer mitochondrial membrane 20.

Article Snippet: Membranes were incubated overnight at 4°C in solutions with primary antibodies against PHB2 (rabbit, 1:2000, Proteintech, Chicago, IL, USA, Cat# 12295-1-AP, RRID: AB_2164779), Parkin (mouse, 1:2000, Abcam, Cambridge, UK, Cat# ab77924, RRID: AB_1566559), LC3B (rabbit, 1:2000, Abcam, Cat# ab192890, RRID: AB_2827794), p-PERK Thr980 (rabbit, 1:2000, Cell Signaling Technology, Danvers, MA, USA, Cat# 3179, RRID: AB_2095853), nuclear factor erythroid 2-related factor (Nrf2; rabbit, 1:2000, Cell Signaling Technology, Cat# 12721, RRID: AB_2715528), heme oxygenase 1 (HO-1; rabbit, 1:2000, Cell Signaling Technology, Cat# 43966, RRID: AB_2799254), quinone oxidoreductase 1 (NQO-1; mouse, 1:2000, Cell Signaling Technology, Cat #3187, RRID: AB_2154354), and β-actin (mouse, 1:5000, CWBio, Beijing, China, Cat# CW0096, RRID: AB_2665433).

Techniques: shRNA, Injection, Expressing, Immunofluorescence, Fluorescence, Control, Comparison, Membrane

Journal: Neural Regeneration Research

Article Title: A novel mechanism of PHB2-mediated mitophagy participating in the development of Parkinson's disease

doi: 10.4103/1673-5374.389356

Figure Lengend Snippet: Silencing PHB2 reduces antioxidative stress protein expression and aggravates motor defecits in PD mice. (A–D) Changes and quantitative analysis of Nrf2, HO-1, and NQO-1 protein levels in the midbrain of PHB2-shRNA and MPTP-treated PD mice. (E) Quantitative analysis of mice in the tail suspension and rotarod tests. Data are expressed as mean ± SEM ( n = 5). * P < 0.05, ** P < 0.01, vs. control group; $ P < 0.05, $$$ P < 0.001, vs . MPTP group (one-way analysis of variance with Tukey's multiple comparison test). HO-1: Heme oxygenase-1; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; NQO-1: NAD(P)H quinone dehydrogenase 1; Nrf2: nuclear factor erythroid 2-related factor 2; PD: Parkinson's disease; PHB2: prohibitin 2.

Article Snippet: Membranes were incubated overnight at 4°C in solutions with primary antibodies against PHB2 (rabbit, 1:2000, Proteintech, Chicago, IL, USA, Cat# 12295-1-AP, RRID: AB_2164779), Parkin (mouse, 1:2000, Abcam, Cambridge, UK, Cat# ab77924, RRID: AB_1566559), LC3B (rabbit, 1:2000, Abcam, Cat# ab192890, RRID: AB_2827794), p-PERK Thr980 (rabbit, 1:2000, Cell Signaling Technology, Danvers, MA, USA, Cat# 3179, RRID: AB_2095853), nuclear factor erythroid 2-related factor (Nrf2; rabbit, 1:2000, Cell Signaling Technology, Cat# 12721, RRID: AB_2715528), heme oxygenase 1 (HO-1; rabbit, 1:2000, Cell Signaling Technology, Cat# 43966, RRID: AB_2799254), quinone oxidoreductase 1 (NQO-1; mouse, 1:2000, Cell Signaling Technology, Cat #3187, RRID: AB_2154354), and β-actin (mouse, 1:5000, CWBio, Beijing, China, Cat# CW0096, RRID: AB_2665433).

Techniques: Expressing, shRNA, Suspension, Control, Comparison

Journal: BioMed Research International

Article Title: Alternative Splicing Generates Different Parkin Protein Isoforms: Evidences in Human, Rat, and Mouse Brain

doi: 10.1155/2014/690796

Figure Lengend Snippet: List of antibodies targeting PARK2 isoforms.

Article Snippet: , GTX25667 Parkin antibody CR20121213_GTX25667 , GeneTex International Corporation.

Techniques:

Journal: Cell Death & Disease

Article Title: Inhibition of PINK1/Parkin-dependent mitophagy sensitizes multidrug-resistant cancer cells to B5G1, a new betulinic acid analog

doi: 10.1038/s41419-019-1470-z

Figure Lengend Snippet: a After treatment with B5G1 (6 µM) for the indicated times, PINK1 and p-Parkin (Ser65) expression levels were detected by western blotting. β-actin was used as a loading control. b HepG2/ADM cells treated with B5G1 (6 µM) for the indicated times were lysed and then separated into mitochondrial fractions and cytosolic fractions. Parkin translocation was measured by Western blotting. β-actin and VDAC were used as loading controls for the cytosolic and mitochondrial proteins, respectively. c After treatment with B5G1 (6 µM) for 12 h, HepG2/ADM cells were lysed with CO-IP lysis buffer, and the interaction between PINK1 and Parkin was measured by Co-IP assay. The asterisk indicated the band of Parkin. d HepG2/ADM cells were pretreated with NC or PINK1 siRNA, followed by treatment with B5G1 (6 µM) for 12 h. PINK1 expression levels were detected by western blotting. GAPDH was used as a loading control. e HepG2/ADM cells were transfected with NC or PINK1 siRNA for 24 h and then treated with B5G1 (6 µM) for another 24 h. Mitochondrial colocalization with LC3 was observed by a fluorescence microscope. Magnification: ×630; scale bar: 10 μm. f HepG2/ADM cells were transfected with NC or Parkin siRNA for 24 h and then treated with B5G1 (6 µM) for 12 h. Parkin and LC3 expression levels were detected by western blotting. β-actin was used as a loading control. g HepG2/ADM cells were transfected with NC or Parkin siRNA for 24 h and then treated with B5G1 (6 µM) for 24 h. Mitochondrial colocalization with LC3 was detected by immunofluorescence. Magnification: ×630; scale bar: 10 μm

Article Snippet: Antibodies against p-Parkin (Ser65) were obtained from Biorbyt-Biotechnology Company (Cambridge, Cambridgeshire, UK).

Techniques: Expressing, Western Blot, Control, Translocation Assay, Co-Immunoprecipitation Assay, Lysis, Transfection, Fluorescence, Microscopy, Immunofluorescence

Journal: Cell Death & Disease

Article Title: Inhibition of PINK1/Parkin-dependent mitophagy sensitizes multidrug-resistant cancer cells to B5G1, a new betulinic acid analog

doi: 10.1038/s41419-019-1470-z

Figure Lengend Snippet: a Tumor volumes were measured every other day. At the end of the treatment period, the tumors were taken out and photographed ( n = 6). * P < 0.05, ** P < 0.01, *** P < 0.001 vs vehicle. b The finall tumors weight were measured. *** P < 0.001. c The mice body weight before and after B5G1 treatment. d The tumor tissues were subjected to H&E staining and immunohistochemistry staining for ki67 and cleaved caspase-3. Scale bar: 50 μm. e The tumor tissues were subjected to immunohistochemistry staining for PINK1, p-Parkin (Ser65), and COX IV. Scale bar: 50 μm.

Article Snippet: Antibodies against p-Parkin (Ser65) were obtained from Biorbyt-Biotechnology Company (Cambridge, Cambridgeshire, UK).

Techniques: Staining, Immunohistochemistry