Journal: Scientific Reports

Article Title: p66Shc activation promotes increased oxidative phosphorylation and renders CNS cells more vulnerable to amyloid beta toxicity

doi: 10.1038/s41598-018-35114-y

Figure Lengend Snippet: Ectopic expression and activation of p66Shc in HT22 cells promotes a reduction in aerobic glycolysis enzyme levels. ( A ) Immunoblot analysis of extracts from HT22 cells transiently transfected with either pcDNA control plasmid or a p66Shc-HA expression vector. DOPPA treatment (100 nM) promoted both increased p66Shc phosphorylation and repressed PDH phosphorylation in p66Shc-HA transfected cells. DOPPA exposure also led to a reduction in levels of the aerobic glycolysis enzymes PDK1, LDHA and PKM2 in p66Shc-HA expressing cells compared to control cells. ( B ) Densitometric analysis of blots revealed a significant increase in S36 phosphorylation of p66Shc and a concomitant decrease in PDH phosphorylation and protein levels of PDK1, LDHA and PKM2 in p66Shc-HA expressing cells following DOPPA exposure. Data presented are the mean ± SEM of 3 independent experiments (*P < 0.05, **P < 0.01).

Article Snippet: The following primary antibodies were used: p66SHC (AM00143PU-N; Acris Antibodies), pSer p66SHC (566807; EMD Millipore), SHC (610878; BD Biosciences), HA-tag (MMS-101P; Covance), PDH (ab110334; abcam), Actin (sc-47778; Santa Cruz), pser 232 PDH (AP1063; EMD Millipore), LDHA (#2012; Cell Signalling), PDK1 (ADI-KAP-PK112-F; Enzo Life Sciences), and PKM2 (#3198; Cell Signalling).

Techniques: Expressing, Activation Assay, Western Blot, Transfection, Plasmid Preparation

Journal: Scientific Reports

Article Title: p66Shc activation promotes increased oxidative phosphorylation and renders CNS cells more vulnerable to amyloid beta toxicity

doi: 10.1038/s41598-018-35114-y

Figure Lengend Snippet: Activation of endogenous p66Shc in B12 cells promotes a reduction in the levels of aerobic glycolysis enzymes. ( A ) Immunoblot analysis of extracts from B12 cells revealed increased phosphorylation of p66Shc following 24-hour DOPPA (100 nM) exposure compared to untreated control cells. DOPPA exposure also promoted decreased phosphorylation of pyruvate dehydrogenase (PDH) and led to a reduction in levels of the aerobic glycolysis enzymes pyruvate dehydrogenase kinase 1 (PDK1), lactate dehydrogenase A (LDHA) and pyruvate kinase 2 (PKM2) compared to control cells. ( B ) Densitometric analysis of blots revealed a significant increase in S36 phosphorylation of p66Shc and a concomitant decrease in PDH phosphorylation and protein levels of PDK1, LDHA and PKM2 following DOPPA exposure. Data presented are the mean ± SEM of 3 independent experiments (*P < 0.05, **P < 0.01).

Article Snippet: The following primary antibodies were used: p66SHC (AM00143PU-N; Acris Antibodies), pSer p66SHC (566807; EMD Millipore), SHC (610878; BD Biosciences), HA-tag (MMS-101P; Covance), PDH (ab110334; abcam), Actin (sc-47778; Santa Cruz), pser 232 PDH (AP1063; EMD Millipore), LDHA (#2012; Cell Signalling), PDK1 (ADI-KAP-PK112-F; Enzo Life Sciences), and PKM2 (#3198; Cell Signalling).

Techniques: Activation Assay, Western Blot

Journal: Scientific Reports

Article Title: p66Shc activation promotes increased oxidative phosphorylation and renders CNS cells more vulnerable to amyloid beta toxicity

doi: 10.1038/s41598-018-35114-y

Figure Lengend Snippet: Phosphorylation and activation of endogenous p66Shc in B12 cells leads to an increase in mitochondrial oxidative metabolism. ( A ) Oxygen consumption rate of B12 cells, with and without DOPPA (100 nM) treatment for 24 hours, was measured in real-time using a Seahorse XFe24 Flux Analyzer. After normalization to protein content, B12 cells treated with DOPPA displayed significant increases in ( B ) basal respiration, ( C ) maximal respiration, ( D ) spare respiratory capacity, ( E ) ATP production, and ( F ) proton leak when compared to untreated cells. Data presented are the mean ± SEM of 3 independent experiments (*P < 0.05).

Article Snippet: The following primary antibodies were used: p66SHC (AM00143PU-N; Acris Antibodies), pSer p66SHC (566807; EMD Millipore), SHC (610878; BD Biosciences), HA-tag (MMS-101P; Covance), PDH (ab110334; abcam), Actin (sc-47778; Santa Cruz), pser 232 PDH (AP1063; EMD Millipore), LDHA (#2012; Cell Signalling), PDK1 (ADI-KAP-PK112-F; Enzo Life Sciences), and PKM2 (#3198; Cell Signalling).

Techniques: Activation Assay

Journal: Scientific Reports

Article Title: p66Shc activation promotes increased oxidative phosphorylation and renders CNS cells more vulnerable to amyloid beta toxicity

doi: 10.1038/s41598-018-35114-y

Figure Lengend Snippet: p66Shc activation promotes an increase in mitochondrial membrane potential (∆𝜓m) and ROS production in B12 cells. ( A ) B12 cells were stained with the ∆𝜓m sensitive fluorochrome TMRM (red), while nuclei were stained with Hoechst stain (blue) and visualized by fluorescence microscopy. Quantification of TMRM fluorescence (right panel) revealed a significant elevation of ∆𝜓m in DOPPA (100 nM) treated B12 cells when compared to untreated control cells. (B) B12 cells were stained with Mitotracker CMX-ROS (Red) and visualized by fluorescence microscopy. Quantification of Mitotracker CMX-ROS (right panel) revealed a significant increase in mitochondrial ROS production following DOPPA treatment (100 nM) compared to control cells. Data presented are the mean ± SEM of 3 independent experiments (**P < 0.01; ****P < 0.001).

Article Snippet: The following primary antibodies were used: p66SHC (AM00143PU-N; Acris Antibodies), pSer p66SHC (566807; EMD Millipore), SHC (610878; BD Biosciences), HA-tag (MMS-101P; Covance), PDH (ab110334; abcam), Actin (sc-47778; Santa Cruz), pser 232 PDH (AP1063; EMD Millipore), LDHA (#2012; Cell Signalling), PDK1 (ADI-KAP-PK112-F; Enzo Life Sciences), and PKM2 (#3198; Cell Signalling).

Techniques: Activation Assay, Staining, Fluorescence, Microscopy

Journal: Scientific Reports

Article Title: p66Shc activation promotes increased oxidative phosphorylation and renders CNS cells more vulnerable to amyloid beta toxicity

doi: 10.1038/s41598-018-35114-y

Figure Lengend Snippet: Ectopic expression of p66Shc in HT22 cells promotes increased mitochondrial membrane potential and ROS production following DOPPA exposure. ( A ) HT22 cells were transfected with either pcDNA or a p66Shc-HA expression plasmid, treated with DOPPA (100 nM) and stained with TMRM. Stained cells were visualized by fluorescence microscopy and fluorescence intensity was quantified (right panel). ( B ) HT22 cells transfected as indicated and treated with DOPPA (100 nM) were stained with Mitotracker CMX-ROS and visualized by fluorescence microscopy. Fluorescence intensity of stained cells was quantified (right panel). HT22 cells transfected with p66Shc and treated with DOPPA exhibited significantly higher TMRM and Mitotracker CMX-ROS staining compared to pcDNA control transfected cells. Nuclei were stained with Hoechst stain (blue). Data presented are the mean ± SEM of 3 independent experiments (**P < 0.01; ****P < 0.001).

Article Snippet: The following primary antibodies were used: p66SHC (AM00143PU-N; Acris Antibodies), pSer p66SHC (566807; EMD Millipore), SHC (610878; BD Biosciences), HA-tag (MMS-101P; Covance), PDH (ab110334; abcam), Actin (sc-47778; Santa Cruz), pser 232 PDH (AP1063; EMD Millipore), LDHA (#2012; Cell Signalling), PDK1 (ADI-KAP-PK112-F; Enzo Life Sciences), and PKM2 (#3198; Cell Signalling).

Techniques: Expressing, Transfection, Plasmid Preparation, Staining, Fluorescence, Microscopy

Journal: Scientific Reports

Article Title: p66Shc activation promotes increased oxidative phosphorylation and renders CNS cells more vulnerable to amyloid beta toxicity

doi: 10.1038/s41598-018-35114-y

Figure Lengend Snippet: Silencing p66Shc expression promotes aerobic glycolysis while reducing mitochondrial ROS production. ( A ) Immunoblot analysis of extracts from B12 cells transfected with p66Shc specific siRNA. Knockdown of p66Shc expression resulted in elevated levels of PDK1, LDHA and PKM2 in addition to increased phosphorylation of PDH. This effect was also observed in B12 cells with silenced p66Shc expression treated with DOPPA. ( B ) Densitometric analysis of immunoblots. ( C ) Mitotracker CMX-ROS (red) staining was significantly decreased in B12 cells with silenced p66Shc expression when compared to control cells. Nuclei were stained with Hoechst stain (blue). Data presented are the mean ± SEM of 3 independent experiments (*P < 0.05, **P < 0.01; ***P < 0.001).

Article Snippet: The following primary antibodies were used: p66SHC (AM00143PU-N; Acris Antibodies), pSer p66SHC (566807; EMD Millipore), SHC (610878; BD Biosciences), HA-tag (MMS-101P; Covance), PDH (ab110334; abcam), Actin (sc-47778; Santa Cruz), pser 232 PDH (AP1063; EMD Millipore), LDHA (#2012; Cell Signalling), PDK1 (ADI-KAP-PK112-F; Enzo Life Sciences), and PKM2 (#3198; Cell Signalling).

Techniques: Expressing, Western Blot, Transfection, Staining

Journal: Scientific Reports

Article Title: p66Shc activation promotes increased oxidative phosphorylation and renders CNS cells more vulnerable to amyloid beta toxicity

doi: 10.1038/s41598-018-35114-y

Figure Lengend Snippet: Aβ exposure promotes p66Shc activation and a reduction in aerobic glycolysis enzyme levels in B12 cells. ( A ) Immunoblot analysis of B12 cells treated with Aβ 1–42 (20 µM) for 24 hours. ( B ) Densitometric analysis of immunoblots revealed a significant increase in p66Shc phosphorylation and a concomitant decrease in PDH phosphorylation and levels of PDK1, LDHA, and PKM2 following Aβ exposure. Data presented are the mean ± SEM of 3 independent experiments (*P < 0.05).

Article Snippet: The following primary antibodies were used: p66SHC (AM00143PU-N; Acris Antibodies), pSer p66SHC (566807; EMD Millipore), SHC (610878; BD Biosciences), HA-tag (MMS-101P; Covance), PDH (ab110334; abcam), Actin (sc-47778; Santa Cruz), pser 232 PDH (AP1063; EMD Millipore), LDHA (#2012; Cell Signalling), PDK1 (ADI-KAP-PK112-F; Enzo Life Sciences), and PKM2 (#3198; Cell Signalling).

Techniques: Activation Assay, Western Blot

Journal: Scientific Reports

Article Title: p66Shc activation promotes increased oxidative phosphorylation and renders CNS cells more vulnerable to amyloid beta toxicity

doi: 10.1038/s41598-018-35114-y

Figure Lengend Snippet: Aβ exposure promotes activation of ectopically expressed p66Shc in HT22 cells and a reduction in aerobic glycolysis. ( A ) Immunoblot analysis of extracts from HT22 cells transfected with the indicated plasmids and treated with Aβ 1–42 (20 µM) for 24 hours. ( B ) Densitometric analysis of immunoblots revealed that Aβ exposure promoted a significant increase in p66Shc phosphorylation while repressing PDH phosphorylation. Aβ treatment also promoted a significant decrease in the levels of PDK1, LDHA, and PKM2 in HT22 cells ectopically expressing p66Shc compared to pcDNA transfected control cells. Data presented are the mean ± SEM of 3 independent experiments (*P < 0.05; **P < 0.01; ***P < 0.001).

Article Snippet: The following primary antibodies were used: p66SHC (AM00143PU-N; Acris Antibodies), pSer p66SHC (566807; EMD Millipore), SHC (610878; BD Biosciences), HA-tag (MMS-101P; Covance), PDH (ab110334; abcam), Actin (sc-47778; Santa Cruz), pser 232 PDH (AP1063; EMD Millipore), LDHA (#2012; Cell Signalling), PDK1 (ADI-KAP-PK112-F; Enzo Life Sciences), and PKM2 (#3198; Cell Signalling).

Techniques: Activation Assay, Western Blot, Transfection, Expressing

Journal: Scientific Reports

Article Title: p66Shc activation promotes increased oxidative phosphorylation and renders CNS cells more vulnerable to amyloid beta toxicity

doi: 10.1038/s41598-018-35114-y

Figure Lengend Snippet: p66Shc activation enhances Aβ toxicity. ( A ) Treatment of B12 cells with both Aβ 1–42 (20 µM) and DOPPA (100 nM) was significantly more toxic than Aβ treatment alone. ( B ) Silencing of p66Shc expression in B12 cells led to reduced Aβ-induced toxicity compared to B12 cells transfected with control siRNA and treated with Aβ. ( C ) HT22 cells ectopically expressing p66Shc and treated with DOPPA (100 nM) exhibited significantly decreased viability following Aβ treatment compared to pcDNA control cells treated with both agents. ( D ) DOPPA induced activation of p66Shc exacerbated Aβ toxicity in mouse primary cortical neurons. Data presented are the mean ± SEM of 3 independent experiments (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

Article Snippet: The following primary antibodies were used: p66SHC (AM00143PU-N; Acris Antibodies), pSer p66SHC (566807; EMD Millipore), SHC (610878; BD Biosciences), HA-tag (MMS-101P; Covance), PDH (ab110334; abcam), Actin (sc-47778; Santa Cruz), pser 232 PDH (AP1063; EMD Millipore), LDHA (#2012; Cell Signalling), PDK1 (ADI-KAP-PK112-F; Enzo Life Sciences), and PKM2 (#3198; Cell Signalling).

Techniques: Activation Assay, Expressing, Transfection

Journal: Cell Death & Disease

Article Title: BAG3 promotes autophagy and glutaminolysis via stabilizing glutaminase

doi: 10.1038/s41419-019-1504-6

Figure Lengend Snippet: a HepG2 or MCF7 cells were infected with lentivirus containing empty or BAG3 construct. Total protein was extracted and protein expression of indicated autophagy-related genes were investigated by immunoblotting. b HepG2 or MCF7 cells infected with lentivirus containing empty or BAG3 construct were treated with vehicle, CQ and E64D plus pepstatin A respectively, protein expression levels of LC3, p62 and BAG3 were analyzed using Western blot analysis. c HepG2 or MCF7 cells stably overexpressing EGFP-LC3B were infected with lentivirus containing empty or BAG3 construct. Cells were treated with vehicle, CQ or E64D plus pepstatin A, and the punctate distribution of EGFP-LC3B was visualized under the florescence microscopy. d Quantitative analysis of ( c ). Results shown represent mean ± SD from five representative microscopic fields and three independent experiments were performed. e HepG2 or MCF7 cells transduced with lentivirus containing empty or BAG3 construct, and ultrastructure was analyzed using transmission electron microscopy. * P < 0.05. Error bars indicate means ± SD

Article Snippet: Antibodies were used against the following: GLS (Sigma-Aldrich, WH0002744M1), GLS (Abcam, ab156876), GAPDH (Merck-Millipore, AB2302), LC3B (Origene, am20213pu), P62 (BD Biosciences, 610833), Beclin1 (Cell Signaling Technology, 3495), ATG3 (Cell Signaling Technology, 3415), ATG5 (Cell Signaling Technology, 12994), ATG7 (Cell Signaling Technology, 8558), ATG12 (Cell Signaling Technology, 4180), SIRT5 (Cell Signaling Technology, 8779), c-Myc (Invitrogen, R950-25), DYKDDDDK Tag (Cell Signaling Technology, 14793), HA-Tag (Cell Signaling Technology, 3724), pan-succinylation (PTM Biolabs, Hangzhou).

Techniques: Infection, Construct, Expressing, Western Blot, Stable Transfection, Microscopy, Transduction, Transmission Assay, Electron Microscopy

Journal: Cell Death & Disease

Article Title: BAG3 promotes autophagy and glutaminolysis via stabilizing glutaminase

doi: 10.1038/s41419-019-1504-6

Figure Lengend Snippet: a Control or BAG3-overexpressing HepG2 and MCF7 cells were transfected with scramble shRNA or shRNA specific against Beclin1 (shBeclin1), and Western blot analysis was performed using the indicated antibodies. b HepG2 or MCF7 cells transduced with lentivirus containing empty or BAG3 construct were treated with vehicle, 3-MA or WM respectively, protein expression levels of LC3, p62 and BAG3 were analyzed using Western blot analysis. c HepG2 or MCF7 cells stably overexpressing EGFP-LC3B were infected with lentivirus containing empty or BAG3 construct. Then cells were treated with vehicle, 3-MA or WM, and the punctate distribution of EGFP-LC3B was visualized under the florescence microscopy. d Quantitative analysis of ( c ). Results shown represent mean ± SD from five representative microscopic fields and three independent experiments were performed. * P < 0.05. N.S. not significant. Error bars indicate means ± SD

Article Snippet: Antibodies were used against the following: GLS (Sigma-Aldrich, WH0002744M1), GLS (Abcam, ab156876), GAPDH (Merck-Millipore, AB2302), LC3B (Origene, am20213pu), P62 (BD Biosciences, 610833), Beclin1 (Cell Signaling Technology, 3495), ATG3 (Cell Signaling Technology, 3415), ATG5 (Cell Signaling Technology, 12994), ATG7 (Cell Signaling Technology, 8558), ATG12 (Cell Signaling Technology, 4180), SIRT5 (Cell Signaling Technology, 8779), c-Myc (Invitrogen, R950-25), DYKDDDDK Tag (Cell Signaling Technology, 14793), HA-Tag (Cell Signaling Technology, 3724), pan-succinylation (PTM Biolabs, Hangzhou).

Techniques: Transfection, shRNA, Western Blot, Transduction, Construct, Expressing, Stable Transfection, Infection, Microscopy

Journal: PLoS Neglected Tropical Diseases

Article Title: Resource Planning for Neglected Tropical Disease (NTD) Control Programs: Feasibility Study of the Tool for Integrated Planning and Costing (TIPAC)

doi: 10.1371/journal.pntd.0002619

Figure Lengend Snippet: Tanzania drug acquisition (in drug units, * FY Oct. 2010–Sept. 2011). **

Article Snippet: ZMAX POS bottles , Pfizer , ITI , 328,104 , 0 , 328,104 , 0.

Techniques:

Journal: Sensors (Basel, Switzerland)

Article Title: Methodology for Verifying the Indication Correctness of a Vessel Compass Based on the Spectral Analysis of Heading Errors and Reliability Theory

doi: 10.3390/s22072530

Figure Lengend Snippet: The accuracy and mean error of the compasses, taking into account the latitude of 54.6 degrees N.

Article Snippet: POS MV Ocean Master Applanix Trimble , 0.02 , 0.02.

Techniques: