Journal: Molecular Medicine Reports

Article Title: Piperazine ferulate attenuates high glucose-induced mesangial cell injury via the regulation of p66 Shc

doi: 10.3892/mmr.2021.12013

Figure Lengend Snippet: Specific details of the antibodies used in western blot analysis.

Article Snippet: p66 Shc , Novus Biological, LLC (NBP2-20352) , Polyclonal Ab. , Rabbit , 1:500.

Techniques: Western Blot

Journal: Molecular Medicine Reports

Article Title: Piperazine ferulate attenuates high glucose-induced mesangial cell injury via the regulation of p66 Shc

doi: 10.3892/mmr.2021.12013

Figure Lengend Snippet: PF inhibits the expression of p-p66 Shc in HG-exposed mesangial cells. (A) Western blot analysis of p-p66 shc and total p66 shc . (B) Densitometric analyses of p-p66 shc /p66 shc and (C) p-p66 shc . (D) Western blot analysis of IKKα/β, p-IKKα/β, NF-κB p65 and p-NF-κB p65. (E) Densitometric analyses of p-IKKα/β/IKKα/β and (F) p-NF-κB p65/NF-κB p65. (G) Fluorescence image of ROS detected using dihydroethidium; ×400 magnification. Data are expressed as the mean ± SEM, n=3. *P<0.05, **P<0.01, ***P<0.001 vs. control group; # P<0.05, ### P<0.001 vs. HG group. PF, piperazine ferulate; HG, high glucose; p-, phosphorylated; Shc, Src homology/collagen; +PF, HG + PF.

Article Snippet: p66 Shc , Novus Biological, LLC (NBP2-20352) , Polyclonal Ab. , Rabbit , 1:500.

Techniques: Expressing, Western Blot, Fluorescence, Control

Journal: Molecular Medicine Reports

Article Title: Piperazine ferulate attenuates high glucose-induced mesangial cell injury via the regulation of p66 Shc

doi: 10.3892/mmr.2021.12013

Figure Lengend Snippet: PF regulates HG-induced mesangial cell injury by inhibiting p66 shc . (A) Effect of pcDNA-p66 shc (1 ng/µl or 2 ng/µl) on the expression of p66 shc . (B) Western blot analysis verified the validity of the pcDNA-p66 Shc . (C) Level of IL-6 in the cell culture supernatant. (D) Western blot analysis of caspase-3, p-IKKα/β, total IKKα/β, Bcl2, Bax, fibronectin and β-actin. Densitometric analyses of (E) caspase-3, (F) p-IKK/IKK, (G) Bcl2, (H) Bax and (I) fibronectin. (J) Representative image of mesangial cells stained with JC-1 (red for aggregate form of JC-1 and green for monomeric form); ×400 magnification. Data are expressed as the mean ± SEM, n=3. **P<0.01, ***P<0.001 vs. vector group; # P<0.05, ## P<0.01, ### P<0.001 vs. vector + HG group; $ P<0.01 vs. vector + HG + PF group. PF, piperazine ferulate; HG, high glucose; vector, empty vector control; p-, phosphorylated; Shc, Src homology/collagen.

Article Snippet: p66 Shc , Novus Biological, LLC (NBP2-20352) , Polyclonal Ab. , Rabbit , 1:500.

Techniques: Expressing, Western Blot, Cell Culture, Staining, Plasmid Preparation, Control

Journal: Molecular Medicine Reports

Article Title: Piperazine ferulate attenuates high glucose-induced mesangial cell injury via the regulation of p66 Shc

doi: 10.3892/mmr.2021.12013

Figure Lengend Snippet: PF attenuates kidney injury in diabetic mice. (A) Western blot analysis of p-p66 shc , total p66 shc , IκBα, p-NF-κB p65, total NF-κB p65, p-IKKα/β, total IKKα/β and β-actin. (B) Densitometric analysis of p-p66 shc and total p66 shc . (C) Densitometric analyses of p-p66 shc /p66 shc and p-NF-κB p65/NF-κB p65. (D) Densitometric analyses of IκBα and p-IKKα/β/IKKα/β. (E) Representative image of the TUNEL staining; ×400 magnification; white arrowheads indicate TUNEL-positive cells. Data are expressed as the mean ± SEM, n=3. *P<0.05, **P<0.01 vs. control group; # P<0.05, ## P<0.01 vs. HG group. PF, piperazine ferulate; HG, high glucose; p-, phosphorylated; Shc, Src homology/collagen; +PF, diabetic nephropathy + PF.

Article Snippet: p66 Shc , Novus Biological, LLC (NBP2-20352) , Polyclonal Ab. , Rabbit , 1:500.

Techniques: Western Blot, TUNEL Assay, Staining, Control

Journal: Molecular Medicine Reports

Article Title: Piperazine ferulate attenuates high glucose-induced mesangial cell injury via the regulation of p66 Shc

doi: 10.3892/mmr.2021.12013

Figure Lengend Snippet: Schematic diagram of pathways involved in the protective effects of PF against HG-induced mesangial cell injury. In the diabetic environment, HG induces glomerular mesangial cell injury, which is characterized by an increase in apoptosis, inflammatory cytokine release and mesangial matrix synthesis. PF attenuated HG-induced mesangial cell injury by inhibiting p66 Shc . PF, piperazine ferulate; HG, high glucose; Shc, Src homology/collagen; ROS, reactive oxygen species; DN, diabetic nephropathy.

Article Snippet: p66 Shc , Novus Biological, LLC (NBP2-20352) , Polyclonal Ab. , Rabbit , 1:500.

Techniques:

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, Control, Plasmid Preparation, Phospho-proteomics

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, Phospho-proteomics, Control

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: Phospho-proteomics, 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, Membrane, Staining, Fluorescence, Microscopy, Control

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, Membrane, Transfection, Plasmid Preparation, Staining, Fluorescence, Microscopy, Control

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, Knockdown, Phospho-proteomics, Staining, Control

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, Phospho-proteomics

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, Phospho-proteomics, Expressing, Control

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, Control