Journal: Journal of Cell Science

Article Title: Direct targeting of host microtubule and actin cytoskeletons by a chlamydial pathogenic effector protein

doi: 10.1242/jcs.263450

Figure Lengend Snippet: CPn0572 associates with the interphase and mitotic MT cytoskeleton. (A) Schematic representation of the Ctr TarP protein and full-length Cpn CPn0572 protein. Previously identified domains are shown: phosphorylation domain (phos), dark gray boxes; proline-rich domain (PRD), orange box; G-actin binding domain (ABD), light blue box; F-actin-binding domain (FAB), dark blue box; and vinculin-binding site (VBS), white box ( ; ). (B) Representative confocal images of HEp-2 cells expressing GFP-CPn0572 (green) for 24 h prior to fixation. Actin was visualized with Rhodamine–phalloidin staining (red) and MTs with anti-α-tubulin antibody. As secondary antibody anti-mouse-IgG conjugated to Alexa Fluor 647 was used (magenta). (C) Merged images of indicated single channels shown in (B). White boxes show enlargements. (D) Representative confocal images of HEp-2 cells transfected with plasmid expressing GFP–CPn0572 (green) for 24 h, prior to fixation. Shown is an exclusive predominant association of GFP–CPn0572 with actin. Actin was visualized with Rhodamine-phalloidin staining (red). (E) Shown is a predominant association of GFP–CPn0572 with interphase MTs visualized with anti-α-tubulin antibody (red). DAPI was used to visualize DNA (blue). (F) Quantification of HEp-2 cells transfected with a GFP–CPn0572 plasmid 24 h prior to fixation. Exclusive colocalization of GFP–CPn0572 with actin was scored in 81% (black) cells, whereas in 11% cells analyzed GFP–CPn0572 localized to both MTs and actin structures (dotted). In 7.7% cells, an exclusive GFP–CPn0572 localization to MT structures was observed (striped). Data represent a mean of three experiments, n =100 cells/experiment. (G) Diagrammatic representation of CPn0572 variants. Quantification of cells expressing GFP–CPn0572 variants and association with the MT cytoskeleton as a percentage (%) of cells counted. Association with the actin cytoskeleton is depicted as+(actin association) or−(no actin association). When no actin association was observed (four bottom CPn0572 variants), CPn0572 was either associated with MT structures (percentage given) or showed a non-specific cytoplasmic staining. * marks deletion variant with MT and actin phenotypes differing from the other protein variants (visualized in Fig. S1 , dot-like and curved-fiber phenotypes concentrated in proximity to the nucleus). (H) Schematic representation of full-length CPn0572 including the newly defined MT-binding region from aa 595–755 (purple box). (I) Confocal images of U2OS cells transfected with GFP–CPn0572 595-755 plasmid for 18 h. Mitotic metaphase is shown diagrammatically on the left. MTs were visualized by anti-α-tubulin antibody staining (red) and DNA with DAPI (blue). (J) Representative confocal image of mitotic U2OS cell expressing full length GFP-CPn0572 (green) full length for 18 h. MTs were visualized by using anti-α-tubulin antibody (magenta), actin with rhodamine-phalloidin staining (red) and DNA with DAPI. In A and G, the numbers indicate amino acid positions. For B–E, I and J images shown are representative of three or more repeats. Scale bars: 10 µm.

Article Snippet: MTs were stained with anti-α-tubulin antibody (Origene Technologies, Inc.; #BM753S; 1:150) and DNA was visualized with DAPI (Merck KGaA, Darmstadt, Germany; 1:500).

Techniques: Binding Assay, Expressing, Staining, Transfection, Plasmid Preparation, Variant Assay

Journal: Journal of Cell Science

Article Title: Direct targeting of host microtubule and actin cytoskeletons by a chlamydial pathogenic effector protein

doi: 10.1242/jcs.263450

Figure Lengend Snippet: Cpn TarP proteins from human and animal isolates can associate with the MT cytoskeleton. (A) Phylogenetic tree of members of the TarP protein family from different chlamydial species with the corresponding isolate indicated. Asterisks mark the TarP members selected for transfection experiment show in C. Phylogenetics were undertaken with Clustal Omega. (B) Schematic illustration of TarP family members and the C-terminus (blue) used for generation of GFP-fusion variants. (C) Representative confocal fluorescence images of HEp-2 cells expressing GFP-tagged versions of C-terminal fragments of the indicated TarP family members. Determination of colocalization with MTs is shown on the right. Cells were transfected with indicated plasmids for 18 h. MTs were visualized using anti-α-tubulin antibody (red) and DNA with DAPI (blue). White boxes show enlargements. Images shown are representative of three repeats. Scale bars: 10 µm.

Article Snippet: MTs were stained with anti-α-tubulin antibody (Origene Technologies, Inc.; #BM753S; 1:150) and DNA was visualized with DAPI (Merck KGaA, Darmstadt, Germany; 1:500).

Techniques: Transfection, Fluorescence, Expressing

Journal: Journal of Cell Science

Article Title: Direct targeting of host microtubule and actin cytoskeletons by a chlamydial pathogenic effector protein

doi: 10.1242/jcs.263450

Figure Lengend Snippet: Severity of alteration of MT structure correlates with time of CPn0572 595-755 expression. (A) Representative confocal fluorescence images of HEp-2 cells transfected with a plasmid encoding GFP for 18 h (top panels) or GFP–CPn0572 595-755 for the indicated times prior to fixation. MTs were visualized with anti-α-tubulin antibody (red) and DNA with DAPI (blue). Scale bars: 10 µm. (B) Western blot analysis of HEp-2 cells expressing GFP-CPn0572 595-755 for the indicated time points. Protein extracts were separated on a 10% SDS-PAGE followed by Western blot analysis. Western blot was probed with anti-GFP or anti-GAPDH antibodies. Blot shown is representative of two repeats. (C) Explanatory cartoon to show co-dependency of GFP–CPn0572 595-755 expression levels and the increase of aberrant MTs (only the 0 h and 18 h time points represent actual measurements). (D) Quantification (mean±s.e.m.) of MT phenotypes shown in A. Typical examples of MTs phenotypes are shown for (i) GFP-expressing control cells (18 h), (ii) GFP–CPn0572 595-755 -expressing cells (6 h) and (iii) GFP–CPn0572 595-755 -expressing cells (18 h). MT thickness was determined by calculating the average diameter measured at three different positions of a single MT bundle using ImageJ. Thin MTs were defined as MTs with <0.302 µm in diameter; thick MTs were defined as MTs with ≥0.302 µm in diameter. n =3 cells (30 MTs measured per condition). *** P <0.001; ** P <0.005; ns, not significant (two-tailed unpaired Student's t -test). (E) Quantification (mean±s.e.m.) of the percentage of GFP- or GFP–CPn0572 595-755 -expressing mitotic U2OS cells with spindle defects. n =3 independent experiments each representing 50 cells. *** P <0.001 (two-tailed unpaired Student's t -test). (F) Confocal images of U2OS cells transfected with a plasmid encoding GFP or GFP-CPn0572 595-755 for 18 h. Top images show a GFP-expressing mitotic cell in anaphase. Bottom images show a representative example of the main spindle defect observed in GFP–CPn0572 595-755 - expressing cells. MTs were visualized by using an α-tubulin antibody (red) and DNA was stained with DAPI (blue). Images shown are representative of three repeats. Scale bars: 10 µm.

Article Snippet: MTs were stained with anti-α-tubulin antibody (Origene Technologies, Inc.; #BM753S; 1:150) and DNA was visualized with DAPI (Merck KGaA, Darmstadt, Germany; 1:500).

Techniques: Expressing, Fluorescence, Transfection, Plasmid Preparation, Western Blot, SDS Page, Control, Two Tailed Test, Staining

Journal: Journal of Cell Science

Article Title: Direct targeting of host microtubule and actin cytoskeletons by a chlamydial pathogenic effector protein

doi: 10.1242/jcs.263450

Figure Lengend Snippet: CPn0572 associated with MTs during chlamydial infection impacts infection efficiency. (A) PCR and (B) western blot verification of Ctr expressing CPn0572–FLAG. For B cells infected with Ctr or Ctr transformed with plasmid encoding CPn0572–FLAG (pMH16) for 48 h were lysed and proteins were separated on 10% SDS-PAGE. For following western blot analysis anti-CPn0572 (generated in our lab), anti-FLAG and anti-DnaK antibodies were used. Images shown are representative of one (A) or two (B) repeats. (C) Confocal images of HEp-2 cells infected with Ctr or Ctr transformed with plasmid encoding CPn0572–FLAG for 15 min. CPn0572–FLAG was visualized with anti-FLAG antibody (green) and DNA with DAPI (blue). (D) Quantification (mean±s.e.m.) of inclusion size [µm 2 ] 24 hpi of Ctr or Ctr transformed with plasmid encoding CPn0572–FLAG. n =2, each representing 30 cells. * P <0.05 (two-tailed unpaired Student's t -test). (E) Representative images from three independent experiments showing single focal planes of HEp-2 cells infected with Ctr or Ctr transformed with plasmid encoding CPn0572–FLAG (MOI=0.5, 48 hpi). Cells were treated with 0.5% Triton X-100 for 30 s and fixed with 0.5% glutaraldehyde. Images show a focal plane between the inclusion and the plasma membrane. (F) Quantification (mean±s.e.m.) of the diameter of MTs measured at the inclusion or distal of the inclusion in cells infected with Ctr - or CPn0572-expressing Ctr . MT diameter was measured by measuring the diameter at three different positions of one MT fiber using ImageJ. Data represent measurements of MT diameter for 10 MTs ( n =4 cells). *** P <0.001; ns, not significant (two-tailed unpaired Student's t -test). (G) Representative images from three repeats of Caco-2 cells infected with Ctr transformed with plasmid encoding CPn0572–FLAG for 48 h. For E and G, CPn0572–FLAG was visualized with anti-FLAG (green), MTs with anti-α-tubulin antibody (red) and eukaryotic and chlamydial DNA with DAPI (blue). For C, E and G, white boxes show enlargements (zoom). Scale bars: 10 µm.

Article Snippet: MTs were stained with anti-α-tubulin antibody (Origene Technologies, Inc.; #BM753S; 1:150) and DNA was visualized with DAPI (Merck KGaA, Darmstadt, Germany; 1:500).

Techniques: Infection, Western Blot, Expressing, Transformation Assay, Plasmid Preparation, SDS Page, Generated, Two Tailed Test, Membrane

Journal: Journal of Cell Science

Article Title: Direct targeting of host microtubule and actin cytoskeletons by a chlamydial pathogenic effector protein

doi: 10.1242/jcs.263450

Figure Lengend Snippet: CPn0572 595-755 protects MTs against cold-induced MT depolymerization. (A,B) Representative confocal images of HEp-2 cells transfected with plasmids encoding GFP (A) or GFP–CPn0572 595-755 (B) for 18 h. MTs were wild-type (after incubation at 37°C) or depolymerized through incubation on ice for 15 or 60 min and then visualized by anti-α-tubulin antibody (red) and DAPI to stain DNA (blue). Boxed regions correspond to the enlarged images shown on the right. Scale bars: 10 µm. (C) Confocal images of HEp-2 cells transfected with a plasmid encoding GFP or GFP–CPn0572 595-755 for 18 h. MTs were visualized with an anti-acetylated-α-tubulin antibody (red) and DNA with DAPI (blue). Images shown in A–C are representative of three or more repeats. Scale bars: 10 µm. (D) A typical example of a Western blot analysis of HEp-2 cells expressing GFP or GFP-CPn0572 595-755 for 18 h. Protein extracts were separated on a 10% SDS-PAGE followed by western blot analysis. Western blot was probed with anti-GFP, anti-acetylated-α-tubulin, anti-α-tubulin or anti-GAPDH antibodies. (E) Quantification of relative acetylated-α-tubulin levels shown in D. Band intensities from Western blots of acetylated-α-tubulin in cells expressing GFP–CPn0572 595-755 were determined and compared relative to control cells (GFP alone=1). ImageJ 1.47v was used for band intensity quantification. Error bars denote ±s.e.m., n =3 independent experiments. * P <0.05 (two-tailed unpaired Student's t -test).

Article Snippet: MTs were stained with anti-α-tubulin antibody (Origene Technologies, Inc.; #BM753S; 1:150) and DNA was visualized with DAPI (Merck KGaA, Darmstadt, Germany; 1:500).

Techniques: Transfection, Incubation, Staining, Plasmid Preparation, Western Blot, Expressing, SDS Page, Control, Two Tailed Test

Journal: Molecular Metabolism

Article Title: Mammalian D-Cysteine controls insulin secretion in the pancreas

doi: 10.1016/j.molmet.2024.102043

Figure Lengend Snippet: SR controls global and Ins1 promoter methylation . (A) Expression of DNMT1, DNMT3A and DNMT3B in pancreatic nuclear extracts of WT and SR −/− mice. Laminin A/C was a nuclear loading control. Data are representative of 3–5 independent experiments. (B) Total DNMT activity in nuclear extracts of pancreas of age matched WT and SR −/− mice. Nuclear extracts (20 μg) was added to each well of a total DNMT activity assay 96 well plate. The percent reduction in total DNMT activity was measured relative to WT. ∗p < 0.0001 relative to WT. ( one way ANOVA ) (C) Whole genome bisulfite sequencing of Ins1 promoter of WT and SR −/− mice (region 2500 bp). Data show CpG methylation at 7 different CpG sites. p values indicate differences between WT and SR −/− Ins1 promoter at the different sites. ( t-test ) (D–E) Global methyl cytosine (mC) and hydroxymethyl cytosine (hmC) expression in WT and SR −/− pancreas following DNA dot blot on a nitrocellulose membrane and probed with mouse monoclonal mC and hmC antibody (1:2000 dilution) respectively. Data are representative of 3 independent experiments. (F) Expression of CREB and p-CREB (S133) in pancreatic lysates of WT and SR −/− mice. Data are representative of 3–5 independent experiments. (G) Expression of CREB and p-CREB (S133) in pancreatic nuclear lysates of WT and SR −/− mice. Data are representative of 3–5 experiments. (H) Schematic of the promoter region of DNMT1 gene and CREB occupancy in the pancreas of WT and SR −/− mice. D1-D5 indicate the different regions on the promoter that were analyzed following ChIP with CREB antibody. Numbers designate regions upstream of the transcription start site (dark arrow). (I) ChIP-qPCR of region D1-D5 of DNMT1 promoter immunoprecipitated with CREB antibody and fold change in DNMT1 expression determined by real time PCR using SYBR green. Region D1-D5 span the promoter region of DNMT1 including 183 bases into exon1 (D5). Fold change in expression of the promoter regions is indicated relative to WT. Region D3 (159 bp) was not plotted due to no change in expression. Data are mean of 3 independent experiments. Error Bars are SD. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article).

Article Snippet: Anti cAMP mouse monoclonal antibody (MAB2146-SP; Novus Biologicals).

Techniques: Methylation, Expressing, Control, Activity Assay, Methylation Sequencing, CpG Methylation Assay, Dot Blot, Membrane, Immunoprecipitation, Real-time Polymerase Chain Reaction, SYBR Green 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: 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