Journal: Cardiovascular research

Article Title: HIF-1-mediated up-regulation of cardiotrophin-1 is involved in the survival response of cardiomyocytes to hypoxia.

doi: 10.1093/cvr/cvr202

Figure Lengend Snippet: Figure 6 CT-1 inhibition increased hypoxia-induced apoptosis in HL-1. Cells were infected with control siRNA lentiviral particles (siControl) or siRNA lentiviral particles against CT-1 (siCT-1) and exposed to normoxia or hypoxia for 16 h. (A) Analysis of apoptosis by flow cytometry after 7-Amino-Actinomycin (AAD)/PE-Annexin V staining. The flow cytometry profile of one representative experiment is shown. Data were gated for damaged cells (PE-Annexin V2 and 7-AAD+, top left quadrant), necrotic cells or late apoptosis (PE-Annexin V+ and 7-AAD+, top right quadrant), viable cells (PE-Annexin V2 and 7-AAD2, bottom left quadrant), and early apoptotic cells (PE-Annexin V+ and 7-AAD2, bottom right quadrant). (B) Bar graph showing the percentage of early apoptotic cells determined by flow cytometry. Non-infected cells (C). n ¼ 3. *P , 0.05 vs. cells transfected with the same siRNA and exposed to normoxia; #P , 0.05 vs. cells exposed to the same treatment but transfected with siControl. (C) Representative fluorescence microscope images for PE-Annexin V staining (red fluorescence) and nuclear staining (DAPI, blue fluorescence). (D) Caspase 3/7 activity was analysed as described in the Methods section. n ¼ 3, in triplicate. *P , 0.05 vs. cells transfected with the same siRNA and exposed to normoxia; #P , 0.05 vs. cells exposed to the same treatment but transfected with siControl.

Article Snippet: HL-1 cells were transfected with siRNAs using Lipofectamine-RNAiMAX (Invitrogen), as discussed previously.27 After transfection, cells were kept in complete medium for 24 h, arrested, and then exposed to hypoxia for 16 h. In additional experiments, HL-1 cells were infected with siRNA lentiviral particles, siCT-1 (CT-1 shRNA-mouse Lentiviral Particles, sc-39328-V) or siControl (Control shRNA Lentiviral Particles, sc-108080) from Santa Cruz, grown in complete medium for 48 h and selected with puromycin.

Techniques: Inhibition, Infection, Control, Cytometry, Staining, Transfection, Microscopy, Activity Assay

Journal: Nature

Article Title: Rare coding variants in Phospholipase D3 (PLD3) confer risk for Alzheimer's disease

doi: 10.1038/nature12825

Figure Lengend Snippet: The diagram represents the different steps to filter the variants identified by exome-sequencing, which lead to the identification of the PLD3-V232M variant. The diagram also shows the subsequent genetic analyses in large case-control datasets that validated the association of the V232M variant and PLD3 with risk for AD.

Article Snippet: The following plasmids were used in this study: pCMV6-XL5 human PLD3 wild-type (Origene), pCS2-myc human APP695 wild-type 43 , pCGN-PLD1b WT 44 and K758R 45 , pCGN-PLD2 WT 46 and K898R 45 , pGFL GFP 47 , pGFP-V-RS-PLD3-shRNA-GI548821 (Origene), and pGFP-V-RSScr-shRNA-TR30013 (Origene).

Techniques: Sequencing, Variant Assay

Journal: Nature

Article Title: Rare coding variants in Phospholipase D3 (PLD3) confer risk for Alzheimer's disease

doi: 10.1038/nature12825

Figure Lengend Snippet: Association of the PLD3-V232M variant in seven independent case-control datasets

Article Snippet: The following plasmids were used in this study: pCMV6-XL5 human PLD3 wild-type (Origene), pCS2-myc human APP695 wild-type 43 , pCGN-PLD1b WT 44 and K758R 45 , pCGN-PLD2 WT 46 and K898R 45 , pGFL GFP 47 , pGFP-V-RS-PLD3-shRNA-GI548821 (Origene), and pGFP-V-RSScr-shRNA-TR30013 (Origene).

Techniques: Variant Assay

Journal: Nature

Article Title: Rare coding variants in Phospholipase D3 (PLD3) confer risk for Alzheimer's disease

doi: 10.1038/nature12825

Figure Lengend Snippet: Association between PLD3 -V232M (rs145999145) and Alzheimer's Disease risk in individuals of European-descent.

Article Snippet: The following plasmids were used in this study: pCMV6-XL5 human PLD3 wild-type (Origene), pCS2-myc human APP695 wild-type 43 , pCGN-PLD1b WT 44 and K758R 45 , pCGN-PLD2 WT 46 and K898R 45 , pGFL GFP 47 , pGFP-V-RS-PLD3-shRNA-GI548821 (Origene), and pGFP-V-RSScr-shRNA-TR30013 (Origene).

Techniques:

Journal: Nature

Article Title: Rare coding variants in Phospholipase D3 (PLD3) confer risk for Alzheimer's disease

doi: 10.1038/nature12825

Figure Lengend Snippet: . Age at onset was analyzed for association with the PLD3 V232M variant in 2,220 cases and 1,841 controls from the Knight-ADRC and NIA-LOAD, by the Kaplan-Meier method and tested for significant differences using the Log-rank test. A) Case only analysis. The carriers of the minor allele (AG) have an AAO 3 years lower than the non-carriers (69 vs 73; p=3×10-3). B) Controls were included as censored data. The carriers of the minor allele (AG) have an AAO 8 years lower than the non-carriers (70 vs 78; p=3×10-3).

Article Snippet: The following plasmids were used in this study: pCMV6-XL5 human PLD3 wild-type (Origene), pCS2-myc human APP695 wild-type 43 , pCGN-PLD1b WT 44 and K758R 45 , pCGN-PLD2 WT 46 and K898R 45 , pGFL GFP 47 , pGFP-V-RS-PLD3-shRNA-GI548821 (Origene), and pGFP-V-RSScr-shRNA-TR30013 (Origene).

Techniques: Variant Assay

Journal: Nature

Article Title: Rare coding variants in Phospholipase D3 (PLD3) confer risk for Alzheimer's disease

doi: 10.1038/nature12825

Figure Lengend Snippet: Sequence variants found in PLD3 in the NIA-LOAD, Knight-ADRC and NIA-UK datasets.

Article Snippet: The following plasmids were used in this study: pCMV6-XL5 human PLD3 wild-type (Origene), pCS2-myc human APP695 wild-type 43 , pCGN-PLD1b WT 44 and K758R 45 , pCGN-PLD2 WT 46 and K898R 45 , pGFL GFP 47 , pGFP-V-RS-PLD3-shRNA-GI548821 (Origene), and pGFP-V-RSScr-shRNA-TR30013 (Origene).

Techniques: Sequencing

Journal: Nature

Article Title: Rare coding variants in Phospholipase D3 (PLD3) confer risk for Alzheimer's disease

doi: 10.1038/nature12825

Figure Lengend Snippet: A) Schematic representation of PLD3 and the relative position of the PLD3 variants. PLD3 has two PLD phosphodiesterase domains, which contain an HKD signature motif (H-x-K-x(4)-D-x(6)-G-T-x-N, where x represents any amino acid residue). The scheme also shows the exon composition of the longest PLD3 mRNA and the position of the variants found in this study. Variants highlighted in red and noted with an “*” are significantly associated with AD risk. Variants noted with a “†” were found only in AD cases. Variants noted with a “ɣ” are more frequent in AD cases compared to controls. B) PLD3 neuronal gene expression is significantly lower in AD cases compared to controls. We used the GEO dataset GSE528127, in which neurons were laser-captured to analyze whether PLD3 mRNA expression levels are different between AD cases and cognitively normal elderly individuals.. C-D) The PLD3 A442A variant is associated with lower total PLD3 mRNA expression and lower levels of exon11 containing transcripts. C) Primers specific to exons 7, to 11 (two pairs of primers) were designed with PrimerExpress. cDNA from eight PLD3 A442A carriers and ten age, gender, APOE, CDR and PMI-matched individuals were obtained from parietal lobe. Relative expression of exon 11 compared to the other exons was calculated by the ΔCt method. Exon 11 containing transcripts in relation to exon 7-10 containing transcripts were 20% lower in A442A carriers (P<0.05). Graphs represent the mean±SEM. D). Real-time PCR was used to quantify total PLD3 mRNA and standardized using GADPH mRNA as a reference. P-value is for the gene-expression levels of major allele carriers vs. minor allele carriers after correcting for dementia severity.

Article Snippet: The following plasmids were used in this study: pCMV6-XL5 human PLD3 wild-type (Origene), pCS2-myc human APP695 wild-type 43 , pCGN-PLD1b WT 44 and K758R 45 , pCGN-PLD2 WT 46 and K898R 45 , pGFL GFP 47 , pGFP-V-RS-PLD3-shRNA-GI548821 (Origene), and pGFP-V-RSScr-shRNA-TR30013 (Origene).

Techniques: Expressing, Variant Assay, Real-time Polymerase Chain Reaction

Journal: Nature

Article Title: Rare coding variants in Phospholipase D3 (PLD3) confer risk for Alzheimer's disease

doi: 10.1038/nature12825

Figure Lengend Snippet: Association analysis for PLD3 A442A in four European-descent datasets

Article Snippet: The following plasmids were used in this study: pCMV6-XL5 human PLD3 wild-type (Origene), pCS2-myc human APP695 wild-type 43 , pCGN-PLD1b WT 44 and K758R 45 , pCGN-PLD2 WT 46 and K898R 45 , pGFL GFP 47 , pGFP-V-RS-PLD3-shRNA-GI548821 (Origene), and pGFP-V-RSScr-shRNA-TR30013 (Origene).

Techniques:

Journal: Nature

Article Title: Rare coding variants in Phospholipase D3 (PLD3) confer risk for Alzheimer's disease

doi: 10.1038/nature12825

Figure Lengend Snippet: PLD3 is associated with risk for AD in African-Americans

Article Snippet: The following plasmids were used in this study: pCMV6-XL5 human PLD3 wild-type (Origene), pCS2-myc human APP695 wild-type 43 , pCGN-PLD1b WT 44 and K758R 45 , pCGN-PLD2 WT 46 and K898R 45 , pGFL GFP 47 , pGFP-V-RS-PLD3-shRNA-GI548821 (Origene), and pGFP-V-RSScr-shRNA-TR30013 (Origene).

Techniques: Variant Assay

Journal: Nature

Article Title: Rare coding variants in Phospholipase D3 (PLD3) confer risk for Alzheimer's disease

doi: 10.1038/nature12825

Figure Lengend Snippet: PLD3 (probe 201050_at) and APP (probe 211277_x_at) expression levels were extracted from the GSE5281 dataset. PLD3 mRNA levels are significantly lower in AD cases compared to controls (p=8.10×10-10), but APP is higher in AD cases (p=7.88×10-8). PLD3 mRNA levels are inversely correlated with APP mRNA expression levels (p=1.00×10-16). The correlation is stronger in AD cases (Person correlation coefficient = -0.55), than in controls (Person correlation coefficient = -0.44), but in both scenarios the correlation is highly significant.

Article Snippet: The following plasmids were used in this study: pCMV6-XL5 human PLD3 wild-type (Origene), pCS2-myc human APP695 wild-type 43 , pCGN-PLD1b WT 44 and K758R 45 , pCGN-PLD2 WT 46 and K898R 45 , pGFL GFP 47 , pGFP-V-RS-PLD3-shRNA-GI548821 (Origene), and pGFP-V-RSScr-shRNA-TR30013 (Origene).

Techniques: Expressing

Journal: Nature

Article Title: Rare coding variants in Phospholipase D3 (PLD3) confer risk for Alzheimer's disease

doi: 10.1038/nature12825

Figure Lengend Snippet: A-B. Overexpression and knockdown of PLD3 produce opposing effects on extracellular Aβ levels. N2A cells stably expressing hAPP695-WT were transiently transfected with vectors containing no insert (pcDNA3), human PLD3-WT, scrambled shRNA (Origene), or mouse PLD3 shRNA (Origene) for 48 hours. Cell media were analyzed with Aβ40 and Aβ42 ELISAs and corrected for total intracellular protein. Aβ levels were then expressed relative to pcDNA3. Graphs represent the mean±SEM. A) Overexpression of human PLD3 produces significantly less extracellular Aβ42 and Aβ40. “*”, p<0.0001. B). Knockdown of endogenous PLD3 cells produces significantly more extracellular Aβ42 and Aβ40. “*”, p<0.002. C) Members of the PLD protein family have different effects on APP processing. HEK293T cells were transiently transfected with vectors containing hAPP-WT and an empty vector (pcDNA3), PLD1, PLD2, or PLD3-WT or PLD1, PLD2, PLD3 carrying a dominant negative mutation.. Left panel, PLD3 affects full-length APP levels. Cell lysates were extracted in non-ionic detergent, analyzed by SDS-PAGE and immunoblotting with antibodies to the myc-tag on APP (9E10) or β-tubulin. Middle (Aβ42) and right (Aβ40) panel, cell media were analyzed with Aβ40 and Aβ42 ELISAs and corrected for total intracellular protein. Graphs represent the mean±SEM. “*”, p<0.01, different from pcDNA3; “ο”, p=0.002, different from PLD1-WT; “•”, p<0.0001, different from PLD2-WT. Images are representative of at least three replicate experiments.

Article Snippet: The following plasmids were used in this study: pCMV6-XL5 human PLD3 wild-type (Origene), pCS2-myc human APP695 wild-type 43 , pCGN-PLD1b WT 44 and K758R 45 , pCGN-PLD2 WT 46 and K898R 45 , pGFL GFP 47 , pGFP-V-RS-PLD3-shRNA-GI548821 (Origene), and pGFP-V-RSScr-shRNA-TR30013 (Origene).

Techniques: Over Expression, Stable Transfection, Expressing, Transfection, shRNA, Plasmid Preparation, Dominant Negative Mutation, SDS Page, Western Blot

Journal: Nature

Article Title: Rare coding variants in Phospholipase D3 (PLD3) confer risk for Alzheimer's disease

doi: 10.1038/nature12825

Figure Lengend Snippet: HEK293T cells were transiently transfected with vectors containing APP WT and an empty vector (pcDNA3) or PLD3 WT for 48 hours. Cell lysates were extracted in non-ionic detergent, pre-cleared with Protein A beads, and immunoprecipitated with an antibody to the myc-tag on APP (9E10). Immunoblots were probed with an antibody specific to human PLD3. PLD1 and PLD2 reportedly do not inmunoprecipitate with APP17,16.

Article Snippet: The following plasmids were used in this study: pCMV6-XL5 human PLD3 wild-type (Origene), pCS2-myc human APP695 wild-type 43 , pCGN-PLD1b WT 44 and K758R 45 , pCGN-PLD2 WT 46 and K898R 45 , pGFL GFP 47 , pGFP-V-RS-PLD3-shRNA-GI548821 (Origene), and pGFP-V-RSScr-shRNA-TR30013 (Origene).

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: PLoS ONE

Article Title: A Conserved Function in Phosphatidylinositol Metabolism for Mammalian Vps13 Family Proteins

doi: 10.1371/journal.pone.0124836

Figure Lengend Snippet: Plasmids.

Article Snippet: pRFP-C-RS-scrambled shRNA 15 , RFP, scrambled shRNA , OriGene TR30015.

Techniques: shRNA