Journal: Frontiers in Cardiovascular Medicine

Article Title: Sacubitril/valsartan attenuates atrial conduction disturbance and electrophysiological heterogeneity with ameliorating fibrosis in mice

doi: 10.3389/fcvm.2024.1341601

Figure Lengend Snippet: BNP and CNP suppressed the activation of primary adult cardiac fibroblasts. ( A ) The protocol of the in vitro assay. ( B ) Effects of ANP (left), BNP (middle) or CNP (right) (at 1.0 µ M each) against the pro-fibrotic alteration of Ccn2 and Acta2 induced by TGFβ ( n = 5–8). ( C ) Additional effect of LBQ657 (50 µ M) on BNP (left) or CNP (right) (5.0 µ M each) ( n = 6–7). ( D ) Antagonizing actions of P19 (1.0 µ M) on CNP (1.0 µ M) added with LBQ657 (10 µ M) ( n = 6). ( E ) Effects of AP811 (1.0 µ M) on CNP (1.0 µ M) with LBQ657 (10 µ M) ( n = 7). Multiple comparison was performed using one-way ANOVA with Tuckey's post-hoc test. Error bars, SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. ANP, atrial natriuretic peptide; BNP, B-type natriuretic peptide; CNP, C-type natriuretic peptide; TGFβ, transforming growth factor β.

Article Snippet: At the same time, a pre-treatment with Atrial Natriuretic Peptide (ANP) (Phoenix Pharmaceuticals, Inc., Burlingame, California, USA), B-type Natriuretic Peptide (BNP) (Bachem, Bubendorf, Switzerland) or C-type Natriuretic Peptide (CNP) (MedChemExpress, Monmouth Junction, New Jersey, USA) at 1.0 or 5.0 μ M was initiated with or without antagonists of their cognate receptors; A71915 (Sigma Aldrich, Burlington, Vermont, USA) antagonizing natriuretic peptide receptor-A (NPR-A); P19 (Phoenix Pharmaceuticals, Inc.) antagonizing natriuretic peptide receptor-B (NPR-B); AP811 (R&D Systems, Minneapolis, Minnesota, USA) antagonizing natriuretic peptide receptor-C (NPR-C), at 1.0 μ M each, as well as LBQ657 (sacubitrilat; Cayman Chemical, Ann Arbor, Michigan, USA) at 10 or 50 μ M. Transforming growth factor β (TGFβ; PeproTech, Cranbury, New Jersey, USA) (1.0 ng/ml) was added 30 min after the application of NPs, followed by another incubation for 24 h. After incubation with the drugs, RNA was extracted from the cultured cells and reverse transcription was performed as described above.

Techniques: Activation Assay, In Vitro, Comparison

Journal: Cells

Article Title: Rab6A as a Pan-Astrocytic Marker in Mouse and Human Brain, and Comparison with Other Glial Markers (GFAP, GS, Aldh1L1, SOX9)

doi: 10.3390/cells10010072

Figure Lengend Snippet: Primary antibodies.

Article Snippet: CNPase , rabbit , Synaptic Systems, 355 002, RRID:AB_2620111 , 1:500.

Techniques: Concentration Assay, Plasmid Preparation

Journal: PLoS ONE

Article Title: 2’,3’-Cyclic Nucleotide 3’-Phosphodiesterases Inhibit Hepatitis B Virus Replication

doi: 10.1371/journal.pone.0080769

Figure Lengend Snippet: (A) The expression of the CNP proteins was studied by immunofluorescence microscopy using the anti-CNP polyclonal rabbit antibody and Alexa Fluor 488-anti-rabbit IgG. The nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI). (B) Huh7 and HepG2 cells were cultured without or with 300, 1000 IU/ml of IFNα-2b for 2 days. Cells were harvested and the cell lysates were determined by Western blot analysis. Actin served as a loading control (protein panel). (C) For CNP mRNA quantitative analysis, cDNA pools were synthesized from the 1 μg of total RNA of cells and diluted cDNA was amplified by quantitative real-time PCR. Actin was served as reverse transcription and PCR control. The mRNA% presents the ratio of values of total CNP (tCNP) to that of naive HepG2 cells (n=3).

Article Snippet: Membranes were blocked with 0.05% TBST (Tris-Buffered Saline Tween-20) containing 5% nonfat dry milk and probed with antibodies against CNP (Proteintech 1:3000), β-actin (Abcam, 1:5000), secondary antibodies conjugated to horseradish peroxidase were used and followed by ECL (Merck Millipore) detection.

Techniques: Expressing, Immunofluorescence, Microscopy, Cell Culture, Western Blot, Control, Synthesized, Amplification, Real-time Polymerase Chain Reaction, Reverse Transcription

Journal: PLoS ONE

Article Title: 2’,3’-Cyclic Nucleotide 3’-Phosphodiesterases Inhibit Hepatitis B Virus Replication

doi: 10.1371/journal.pone.0080769

Figure Lengend Snippet: (A) Huh7 cells were transfected with pCDNA5-CNP1 or pCDNA5-CNP2 and harvested at day 2 post transfection. The expression of CNP and tubulin was revealed by immunofluorescence microscopy using the anti-CNP polyclonal rabbit antibody and anti-tublin monoclonal mice antibody, the secondary antibodies were Alexa Fluor 594-conjugated goat anti-rabbit IgG Alexa Fluor 488-conjugated goat anti-mouse or rabbit IgG. The nuclei were counterstained with DAPI. Mitochondria were stained using Mitocapture Tm kit and indicated by red color. White arrow indicates a CNP1-transfected Huh7 cell undergone a morphological change. (B) Huh7 ells in 12-well plate were transfected with 2 μg of plasmids pCDNA5-CNP1 or pCDNA5-CNP2. Cell lysates were determined by Western blot analysis. Actin served as a loading control.

Article Snippet: Membranes were blocked with 0.05% TBST (Tris-Buffered Saline Tween-20) containing 5% nonfat dry milk and probed with antibodies against CNP (Proteintech 1:3000), β-actin (Abcam, 1:5000), secondary antibodies conjugated to horseradish peroxidase were used and followed by ECL (Merck Millipore) detection.

Techniques: Transfection, Expressing, Immunofluorescence, Microscopy, Staining, Western Blot, Control

Journal: PLoS ONE

Article Title: 2’,3’-Cyclic Nucleotide 3’-Phosphodiesterases Inhibit Hepatitis B Virus Replication

doi: 10.1371/journal.pone.0080769

Figure Lengend Snippet: Panels show micrographs of liver biopsy specimens from three HBV-infected patients. Specimens were costained with antibodies against HBV X protein (red) and CNP (green); nuclei were stained with DAPI (blue). These images were collected at 100 magnification and show the representative images. Control micrograph was collected from the non-infectious area. The dashed rectangle in P2 (Patient 2) indicated the HBx positive but CNP negative cells; the real line indicated the both positive area. The enlarge panel exhibits the subcellular distribution of HBx and CNP from the real line area of P3 panel.

Article Snippet: Membranes were blocked with 0.05% TBST (Tris-Buffered Saline Tween-20) containing 5% nonfat dry milk and probed with antibodies against CNP (Proteintech 1:3000), β-actin (Abcam, 1:5000), secondary antibodies conjugated to horseradish peroxidase were used and followed by ECL (Merck Millipore) detection.

Techniques: Infection, Staining, Control

Journal: JCI Insight

Article Title: Collagen-binding C-type natriuretic peptide enhances chondrogenesis and osteogenesis

doi: 10.1172/jci.insight.198959

Figure Lengend Snippet: ( A ) Collagen-binding ability and thermal stability of CBD-CNP. Unbound CBD-CNP was assessed by SDS-PAGE. M, molecular weight marker; B, buffer control; No heat, no preheating; Heat, preheating at 56°C 30 minutes; (–), no collagen powder was added; (+), collagen powder was added. ( B ) Surface plasmon resonance analysis of CBD-CNP binding to recombinant NPR-B immobilized on a CM5 chip. The Kd value was calculated from sensorgrams as described under the experimental procedures. ( C ) Ligand-receptor binding assay with CBD-CNP and NPR-B, the receptor for CNP. ( D ) Inhibitory effect of FGF2-FGFR3 downstream signaling pathway of RCS cells owing to the binding of CNP to NPR-B. Phosphorylation of ERK1/2 in RCS cells was assessed by FGF2 alone and combined treatment with CNP-22 or CBD-CNP. ( E ) Cell proliferation assessment of RCS cells after treatment with FGF2 alone or in combination with CNP-22 or CBD-CNP ( n = 5). The cell proliferation rate was calculated based on the cell count with no drug treatment as 100%. ( F ) Alcian blue staining of RCS cells in each group. Scale bars: 200 μm in F . Statistical analysis was performed by using 1-way ANOVA followed by Tukey post hoc tests. SDS-PAGE, sodium dodecyl sulfate polyacrylamide electrophoresis; RU, response unit; IP, immunoprecipitation; IB, immunoblot; NPR-B, natriuretic peptide receptor B; CNP, C-type natriuretic peptide; FGF2, fibroblast growth factor 2; FGFR3, fibroblast growth factor receptor 3; RCS, rat chondrosarcoma; ERK, extracellular signal-regulated kinase; P-ERK, phospho-extracellular signal-regulated kinase.

Article Snippet: The persistence of CNP and CBD-CNP in articular cartilage was assessed by DAB staining using ImmPRESS Horse Anti-Rabbit IgG Plus Polymer Kit (Vector Laboratories) and anti-CNP antibody (abx214951, Abbexa) at 1:100 dilution according to the manufacturer’s protocol.

Techniques: Binding Assay, SDS Page, Molecular Weight, Marker, Control, SPR Assay, Recombinant, Reporter Assay, Phospho-proteomics, Cell Characterization, Staining, Electrophoresis, Immunoprecipitation, Western Blot

Journal: JCI Insight

Article Title: Collagen-binding C-type natriuretic peptide enhances chondrogenesis and osteogenesis

doi: 10.1172/jci.insight.198959

Figure Lengend Snippet: ( A ) Representative DAB staining images of fetal murine tibiae (E16.5, FVB background) treated with FGF2 in the presence of CNP-22 or CBD-CNP with daily medium changes. CNP-22 was detected with anti-CNP antibody, and CBD-CNP was detected with anti-CBD antibody. ( B ) Fetal murine tibiae were cultured and treated with FGF2 in the presence of CNP-22 or CBD-CNP with daily medium changes. After 4 days, the tibial lengths were measured and compared with those of contralateral untreated tibiae in the same patients ( n = 9). ( C ) To assess the prolonged effects, only a single administration of CNP-22 or CBD-CNP was performed on the first day, whereas FGF2 was added daily. The tibial lengths were measured after 4 days from the first dosage ( n = 7 in FGF2 and CBD-CNP groups, and n = 6 in CNP-22 group). Scale bars: 50 μm in A ; 1 mm in B and C . The tibial lengths within patients were compared using a paired t test. DAB, 3,3′-diaminobenzidine.

Article Snippet: The persistence of CNP and CBD-CNP in articular cartilage was assessed by DAB staining using ImmPRESS Horse Anti-Rabbit IgG Plus Polymer Kit (Vector Laboratories) and anti-CNP antibody (abx214951, Abbexa) at 1:100 dilution according to the manufacturer’s protocol.

Techniques: Staining, Cell Culture

Journal: JCI Insight

Article Title: Collagen-binding C-type natriuretic peptide enhances chondrogenesis and osteogenesis

doi: 10.1172/jci.insight.198959

Figure Lengend Snippet: ( A ) Representative DAB staining images of knee joint tissues from the mouse model of knee OA following intraarticular injection of PBS, CNP-22, or CBD-CNP. CNP-22 was detected with anti-CNP antibody, and CBD-CNP was detected with anti-CBD antibody. ( B ) Representative μCT images of the mouse model of knee OA with intraarticular injection of PBS, CNP-22, or CBD-CNP. The images of control and OA with PBS were obtained from the same patient. ( C ) Quantitative analyses of subchondral bone plate thickness and trabecular thickness using μCT images ( n = 10 in control/OA with PBS, and OA with CBD-CNP groups, and n = 9 in OA with CNP-22 group). The data of control and OA with PBS were obtained from the same patients. Scale bars: 100 μm in A ; 500 μm in B . Statistical analysis was performed using the Student’s t test. OA, osteoarthritis.

Article Snippet: The persistence of CNP and CBD-CNP in articular cartilage was assessed by DAB staining using ImmPRESS Horse Anti-Rabbit IgG Plus Polymer Kit (Vector Laboratories) and anti-CNP antibody (abx214951, Abbexa) at 1:100 dilution according to the manufacturer’s protocol.

Techniques: Staining, Injection, Control

Journal: BMC Pregnancy and Childbirth

Article Title: Maternal plasma pro-atrial and C-type natriuretic peptide levels and their associations with cardiovascular and renal function in the second half of normal pregnancy: a longitudinal study

doi: 10.1186/s12884-021-03824-3

Figure Lengend Snippet: Percentiles of maternal plasma C-type natriuretic peptide (pg/mL) at 22–40 weeks of gestational age (GA)

Article Snippet: The human C-type natriuretic peptide quantification kit (CSB-E08909h, CUSABIO, Lab-Tech AS, Norway) had an inter-assay CV of ≤10% and an intra-assay CV of ≤8%, and LOD was 15.6 pg/mL.

Techniques: Clinical Proteomics