Journal: Hypertension

Article Title: Clinical Effects of Phosphodiesterase 3A Mutations in Inherited Hypertension With Brachydactyly

doi: 10.1161/hypertensionaha.115.06000

Figure Lengend Snippet: Figure 3. A, Diagram represents the location of the mutations and the phosphodiesterase 3A (PDE3A) isoforms A1–A3. The epitopes of the used antibodies are illustrated: Y(1), Y(2), Y(3), and Y(4). B, The PDE3A isoforms PDE3A1, PDE3A2, and PDE3A3 were detected in human heart tissue and in PDE3A-transfected HeLa cells. In platelets, PDE3A2 was preferentially observed (upper panels 1 and 2). Platelet activation using thrombin receptor activator peptide (TRAP)-6 and collagen increased slightly phosphorylation of Ser428 (lower panel 3). Platelet PDE3A2 showed strong phosphorylation of Ser438 particularly upon stimulation (lower panel 4). NHR indicates N-terminal hydrophobic region 1; and ORF, open reading frame.

Article Snippet: A commonly used, cancer-derived cell line named after Henrietta Lacks (HeLa) cells were transiently transfected with an empty vector (sc300-w/o) or a vector encoding full-length wild-type PDE3A cDNA (Origene, SC300151, NM_000921), and 48 hours after transfection, cells were stimulated for 1 hour at CONS CALIFORNIA DIG LIB on November 19, 2015http://hyper.ahajournals.org/Downloaded from 802 Hypertension October 2015 with 20 μmol/L Forskolin and 100 ng/mL phorbol 12-myristate 13-acetate (PMA).

Techniques: Transfection, Activation Assay, Phospho-proteomics

Journal: Hypertension

Article Title: Clinical Effects of Phosphodiesterase 3A Mutations in Inherited Hypertension With Brachydactyly

doi: 10.1161/hypertensionaha.115.06000

Figure Lengend Snippet: Figure 4. A, Hela cells were transfected with empty vector (sc300-w/o, blue line), wild-type (red line), and mutated PDE3A expression vectors (gray and black lines). Hydrolysis of cAMP was monitored as cAMP responsive element (CRE) luciferase activity. With incremental forskolin concentrations, the PDE3A mutations showed a significant reduction of the CRE-mediated luciferase activity as a result of the higher cAMP hydrolysis compared with the wild-type PDE3A.14 Cilostazol showed no effect abrogating the PDE3A mutations gain-of- function effects. B, Milrinone decreased the enhanced cAMP hydrolysis more effectively than cilostazol with increasing cAMP levels. C, cGMP stimulation with increasing l-arginine concentrations and cilostazol had light effects blocking the enhanced cAMP hydrolysis. D, cGMP in combination with milrinone determined that cGMP competitively inhibited cAMP hydrolysis. E, The soluble guanylate cyclase stimulator BAY 41–8543 reduced the enhanced cAMP hydrolysis of the PDE3A mutations compared with the wild-type PDE3A. **P<0.002, mean±SD; n=3; Wilcoxon and Mann–Whitney test).

Article Snippet: A commonly used, cancer-derived cell line named after Henrietta Lacks (HeLa) cells were transiently transfected with an empty vector (sc300-w/o) or a vector encoding full-length wild-type PDE3A cDNA (Origene, SC300151, NM_000921), and 48 hours after transfection, cells were stimulated for 1 hour at CONS CALIFORNIA DIG LIB on November 19, 2015http://hyper.ahajournals.org/Downloaded from 802 Hypertension October 2015 with 20 μmol/L Forskolin and 100 ng/mL phorbol 12-myristate 13-acetate (PMA).

Techniques: Transfection, Plasmid Preparation, Expressing, Luciferase, Activity Assay, Blocking Assay, MANN-WHITNEY

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

Journal: Cell Metabolism

Article Title: The Polycomb-Dependent Epigenome Controls β Cell Dysfunction, Dedifferentiation, and Diabetes

doi: 10.1016/j.cmet.2018.04.013

Figure Lengend Snippet:

Article Snippet: Twist1 (NM_011658) Mouse Tagged ORF Clone , Origene , MR227370.

Techniques: Immunofluorescence, Recombinant, Enzyme-linked Immunosorbent Assay, Sample Prep, TUNEL Assay, Software

Journal: International Journal of Molecular Sciences

Article Title: Protective Effect of Paeoniae Radix Alba Carbonisata on Hepatic Amyloidosis by Regulating Calcium Homeostasis

doi: 10.3390/ijms27062582

Figure Lengend Snippet: PRAC-E modulates calcium homeostasis via the cGMP/PKG/ATP2A1 signaling axis, thereby alleviating inflammation and amyloid deposition. ( A , B ) The dose-dependent ( A ) and time-dependent ( B ) effects of LYSO-6 on NCTC1469 cells. ( C ) Screening of the effective concentration of PRAC-E on LYSO-6-stimulated cells. ( D – F ) The levels of IL-6, TNF-α, and IL-8 in the cell culture supernatant. ( G – I ) The levels of SAA, SAP ( G ), and APO-E ( H ) in NCTC1469 cells. ( J ) CR staining of NCTC1469 cells. ( K ) The dose-dependent effects of BAPTA-AM on NCTC1469 cells. ( L , M ) Representative Fluo-4 AM fluorescence staining and semi-quantification of NCTC1469 cells. ( N ) Ca 2+ -ATPase activity in NCTC1469 cells. ( O ) Intracellular cGMP levels in NCTC1469 cells were measured by ELISA. ( P – R ) Western blotting and semi-quantitative analysis of PDE5A, PKG, and ATP2A1 in NCTC1469 cells. Data are presented as the mean ± SD ( n = 6). # p < 0.05, ## p < 0.01, ### p < 0.001 vs. solvent control group; * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. LYSO-6; ns, no significance.

Article Snippet: Rabbit antibodies against β-actin (Cat# 20536-1-AP), PDE5A (Cat# 22624-1-AP), and ATP2A1 (Cat# 22361-1-AP) were obtained from Proteintech Group, Inc. (Wuhan, China).

Techniques: Concentration Assay, Cell Culture, Staining, Fluorescence, Activity Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Solvent, Control

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