Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

Article Title: Investigation of an 18 F-labelled Imidazopyridotriazine for Molecular Imaging of Cyclic Nucleotide Phosphodiesterase 2A

doi: 10.3390/molecules23030556

Figure Lengend Snippet: IC 50 values of the novel 2-fluoroethoxy derivative TA5 for the inhibition of human PDE2A and human PDE10A compared to our already published data for the lead compound TA1 and the PDE2A ligands TA2 – 4 [ 13 , 19 , 20 ].

Article Snippet: The inhibitory potencies of TA1 – 5 for human recombinant PDE2A and PDE10A proteins were determined by BioCrea GmbH (Radebeul, Germany) [ ].

Techniques: Inhibition

Journal: eLife

Article Title: PDE2A2 regulates mitochondria morphology and apoptotic cell death via local modulation of cAMP/PKA signalling

doi: 10.7554/eLife.21374

Figure Lengend Snippet: ( A ) Localisation of PDE2A1-GFP, PDE2A2-GFP and PDE2A3-GFP in neonatal rat ventricular myocytes (NRVM) labelled with mitotracker red. Scale bar: 10 µm. ( B ) Localisation of wild type (PDE2A2wt-RFP) or catalytically inactive (PDE2A2dn-RFP) PDE2A2 in NRVM. Scale bar: 10 µm. ( C ) NRVM treated with the indicated drugs (F/I: Forskolin/IBMX) and incubated with mitotracker red to stain mitochondria. Scale bar: 10 µm. ( D ) Quantitative analysis of mitochondrial length on cells as shown in C . n = 40 cells from three biological replicates. ( E ) Quantitative analysis of mitochondrial length in cells transfected with a control siRNA sequence (siGLO), a specific siRNA for PDE2A (siRNA PDE2A ) alone or in combination with a plasmid carrying a siRNA-resistant PDE2A2 sequence. n = 30 cells from 3 biological replicates. ( F ) Western blotting analysis of cell lysates obtained from NRVM treated with the indicated drugs and probed for phospho-DRP1 (ser637), total DRP1 and GAPDH, as indicated. Representative of 5 biological replicates. ( G ) Quantification of the western blotting analysis as shown in F ). ( H ) Quantitative analysis of mitochondrial length in MEFs wt and MEFs Drp1KO stable clones treated with DMSO, BAY60 and Cilostamide, and MEFs Drp1KO overexpressing Drp1 wt-GFP or Drp1 mut -GFP and treated with the same drugs. n = 30 cells from three biological replicates. ANOVA test with Bonferroni correction was used for statistical analysis. *0.01≤p≤0.05, **0.001≤p<0.01, ***p<0.001. DOI: http://dx.doi.org/10.7554/eLife.21374.002

Article Snippet: PDE2A1 (RG235036), PDE2A2 (RG226806) and PDE2A3 (RG207219) in pCMV6-AC-GFP were from Origene (MD, USA).

Techniques: Incubation, Staining, Transfection, Control, Sequencing, Plasmid Preparation, Western Blot, Clone Assay

Journal: eLife

Article Title: PDE2A2 regulates mitochondria morphology and apoptotic cell death via local modulation of cAMP/PKA signalling

doi: 10.7554/eLife.21374

Figure Lengend Snippet: ( A ) Localisation of PDE2A1-GFP, PDE2A2-GFP and PDE2A3-GFP in neonatal rat ventricular myocytes (NRVM) labelled with mitotracker red. Scale bar: 10 µm. ( B ) Localisation of wild type (PDE2A2wt-RFP) or catalytically inactive (PDE2A2dn-RFP) PDE2A2 in NRVM. Scale bar: 10 µm. ( C ) NRVM treated with the indicated drugs (F/I: Forskolin/IBMX) and incubated with mitotracker red to stain mitochondria. Scale bar: 10 µm. ( D ) Quantitative analysis of mitochondrial length on cells as shown in C . n = 40 cells from three biological replicates. ( E ) Quantitative analysis of mitochondrial length in cells transfected with a control siRNA sequence (siGLO), a specific siRNA for PDE2A (siRNA PDE2A ) alone or in combination with a plasmid carrying a siRNA-resistant PDE2A2 sequence. n = 30 cells from 3 biological replicates. ( F ) Western blotting analysis of cell lysates obtained from NRVM treated with the indicated drugs and probed for phospho-DRP1 (ser637), total DRP1 and GAPDH, as indicated. Representative of 5 biological replicates. ( G ) Quantification of the western blotting analysis as shown in F ). ( H ) Quantitative analysis of mitochondrial length in MEFs wt and MEFs Drp1KO stable clones treated with DMSO, BAY60 and Cilostamide, and MEFs Drp1KO overexpressing Drp1 wt-GFP or Drp1 mut -GFP and treated with the same drugs. n = 30 cells from three biological replicates. ANOVA test with Bonferroni correction was used for statistical analysis. *0.01≤p≤0.05, **0.001≤p<0.01, ***p<0.001. DOI: http://dx.doi.org/10.7554/eLife.21374.002

Article Snippet: PDE2A1 (RG235036), PDE2A2 (RG226806) and PDE2A3 (RG207219) in pCMV6-AC-GFP were from Origene (MD, USA).

Techniques: Incubation, Staining, Transfection, Control, Sequencing, Plasmid Preparation, Western Blot, Clone Assay

Journal: eLife

Article Title: PDE2A2 regulates mitochondria morphology and apoptotic cell death via local modulation of cAMP/PKA signalling

doi: 10.7554/eLife.21374

Figure Lengend Snippet: ( A ) Localisation of PDE2A1-GFP, PDE2A2-GFP and PDE2A3-GFP in neonatal rat ventricular myocytes (NRVM) labelled with mitotracker red. Scale bar: 10 µm. ( B ) Localisation of wild type (PDE2A2wt-RFP) or catalytically inactive (PDE2A2dn-RFP) PDE2A2 in NRVM. Scale bar: 10 µm. ( C ) NRVM treated with the indicated drugs (F/I: Forskolin/IBMX) and incubated with mitotracker red to stain mitochondria. Scale bar: 10 µm. ( D ) Quantitative analysis of mitochondrial length on cells as shown in C . n = 40 cells from three biological replicates. ( E ) Quantitative analysis of mitochondrial length in cells transfected with a control siRNA sequence (siGLO), a specific siRNA for PDE2A (siRNA PDE2A ) alone or in combination with a plasmid carrying a siRNA-resistant PDE2A2 sequence. n = 30 cells from 3 biological replicates. ( F ) Western blotting analysis of cell lysates obtained from NRVM treated with the indicated drugs and probed for phospho-DRP1 (ser637), total DRP1 and GAPDH, as indicated. Representative of 5 biological replicates. ( G ) Quantification of the western blotting analysis as shown in F ). ( H ) Quantitative analysis of mitochondrial length in MEFs wt and MEFs Drp1KO stable clones treated with DMSO, BAY60 and Cilostamide, and MEFs Drp1KO overexpressing Drp1 wt-GFP or Drp1 mut -GFP and treated with the same drugs. n = 30 cells from three biological replicates. ANOVA test with Bonferroni correction was used for statistical analysis. *0.01≤p≤0.05, **0.001≤p<0.01, ***p<0.001. DOI: http://dx.doi.org/10.7554/eLife.21374.002

Article Snippet: PDE2A1 (RG235036), PDE2A2 (RG226806) and PDE2A3 (RG207219) in pCMV6-AC-GFP were from Origene (MD, USA).

Techniques: Incubation, Staining, Transfection, Control, Sequencing, Plasmid Preparation, Western Blot, Clone Assay

Journal: eLife

Article Title: PDE2A2 regulates mitochondria morphology and apoptotic cell death via local modulation of cAMP/PKA signalling

doi: 10.7554/eLife.21374

Figure Lengend Snippet: ( A ) Wild type (MEF wt ) and PDE2KO (MEF PDE2KO ) mouse embryonic fibroblasts stained with mitotracker green. Scale bar: 10 µm. ( B ) MEF wt and MEF PDE2KO expressing catalytically inactive (PDE2A2dn-RFP) or wild type (PDE2A2wt-RFP) PDE2A2, respectively and stained with mitotracker green. The overlay of the RFP and mitotracker signal is also shown. Panels on the right show the fluorescence intensity profile for the mitotracker (red line) and PDE2A2-RFP proteins (green line) along with the line shown in the overlay images. Scale bar: 10 µm. ( C ) Quantitative analysis of mitochondria morphology on images shown in B . n = 35 cells from three biological replicates. ( D ) Quantitative analysis of mitochondria morphology in MEF cells treated with the PKA inhibitor H89. n = 25 cells from two biological replicates. ANOVA test with Bonferroni correction was used for statistical analysis. *0.01≤p≤0.05, **0.001≤p<0.01, ***p<0.001. DOI: http://dx.doi.org/10.7554/eLife.21374.005

Article Snippet: PDE2A1 (RG235036), PDE2A2 (RG226806) and PDE2A3 (RG207219) in pCMV6-AC-GFP were from Origene (MD, USA).

Techniques: Staining, Expressing, Fluorescence

Journal: eLife

Article Title: PDE2A2 regulates mitochondria morphology and apoptotic cell death via local modulation of cAMP/PKA signalling

doi: 10.7554/eLife.21374

Figure Lengend Snippet: ( A ) Representative Western blotting analysis of cytosolic and mitochondrial sub-fractions obtained from NRVM lysates not treated and treated with Proteinase K (10 µM). PDE2A was probed with a PDE2A specific antibody. To assess fraction purity, antibody for OXPHOS subunits (black arrowheads), TOM20 and Cytochrome C were used. GAPDH was used to assess contamination of cytosol in the mitochondrial fraction. The panel on the right shows the quantification from four biological replicates. Student t -test was used for statistical analysis. * = p<0.05. ( B ) Representative Western blot of subcellular fractions obtained from Hela cells expressing PDE2A2-GFP. The mitochondrial fraction was either non treated or treated with Proteinase K. PDE2A2 was assessed with a GFP-specific antibody. Mitofilin was probed here in addition to the mitochondrial markers used in A ). This experiment was repeated twice with similar results. ( C ) Representative Western blot of cytosolic, mitochondrial, mitoplasts and post-mitoplast fractions obtained from NRVM lysates. Samples were either untreated or treated with Proteinase K (PK, 10 µM) or Triton-X plus PK. PDE2A was probed with a PDE2A specific antibody. Cytochrome c oxidase subunit II (COX2) is a marker for mitochondrial matrix; Tubulin is marker for cytosol; TIM23 is marker for IMM; cytochrome-c is a marker for IMS. Blot is representative of three independent experiments. ( D ) Confocal and STED image (first and second column, respectively) of a HeLa cell expressing PDE2A2-GFP and labelled with antibodies to cytochrome c and GFP. Third and fourth columns show magnification of the boxed areas. Plots on the right show average intensity profiles across the indicated mitochondrial tubule section. Cytochrome c profile is in green and PDE2A2 profile is in red. Scale bar: 2 µm. ( E ) Representative electron microscopy image of NRVM probed with PDE2A antibody detected by protein A conjugated with 10 nm gold beads. The count of relative mitochondrial distribution of the immunogold particles is shown on the right (n = 25 mitochondria). Magnification 100X, scale bar: 200 nm. DOI: http://dx.doi.org/10.7554/eLife.21374.009

Article Snippet: PDE2A1 (RG235036), PDE2A2 (RG226806) and PDE2A3 (RG207219) in pCMV6-AC-GFP were from Origene (MD, USA).

Techniques: Western Blot, Expressing, Marker, Electron Microscopy

Journal: eLife

Article Title: PDE2A2 regulates mitochondria morphology and apoptotic cell death via local modulation of cAMP/PKA signalling

doi: 10.7554/eLife.21374

Figure Lengend Snippet: ( A ) Representative confocal and STED images of a HeLa cell labelled with antibodies specific to cytochrome c and TOM20. ( B ) Representative confocal and STED images of HeLa cell expressing PDE2A2-GFP and labelled with antibodies specific to TOM20 and GFP. ( C ) In both panels, a magnification of the boxed areas is also shown. Panels on the right show fluorescence intensity profiles across the indicated mitochondrial tubule section. Scale bar: 2 µm. DOI: http://dx.doi.org/10.7554/eLife.21374.010

Article Snippet: PDE2A1 (RG235036), PDE2A2 (RG226806) and PDE2A3 (RG207219) in pCMV6-AC-GFP were from Origene (MD, USA).

Techniques: Expressing, Fluorescence