Journal: Frontiers in Cell and Developmental Biology

Article Title: An MRTF-A–Sp1–PDE5 Axis Mediates Angiotensin-II-Induced Cardiomyocyte Hypertrophy

doi: 10.3389/fcell.2020.00839

Figure Lengend Snippet: Cardiomyocyte deletion of myocardin-related transcription factor A (MRTF-A) attenuates Ang-II-induced cardiac hypertrophy in mice. MRTF-A conditional knockout (CKO) mice and wild-type (WT) mice were induced to develop cardiac hypertrophy by Ang II infusion as described in section “Materials and Methods.” (A) Immunofluorescence staining was performed with anti-MRTF-A and anti-actinin. (B) Heart weight vs. body weight ratio. (C) Heart weight vs. tibia bone length ratio. (D) Echocardiographic measurements of left ventricular systolic dimension (LVSd). (E) Echocardiographic measurements of left ventricular posterior wall dimension (LVPWd). (F) Echocardiographic measurements of ejection fraction (EF). (G) Echocardiographic measurements of fractional shortening (FS). (H) Cardiac sections were stained with wheat germ agglutinin (WGA). Cross-sectional areas were quantified by Image Pro. (I) Immunohistochemistry (IHC) staining of cardiac sections with anti-MRTF-A, antibrain natriuretic peptide (anti-BNP), and anti-β-major histocompatibility complex (anti-β-MHC) antibodies, Cross-sectional areas were quantified by Image Pro. N = 3–5 mice for the saline groups and N = 5 mice for the Ang II groups. Error bars represent SD. * p < 0.05; ** p < 0.01; *** p < 0.001. Scale bar: 50 μm.

Article Snippet: Western analyses were performed with anti-α-tubulin (Proteintech, Wuhan, China 11224-1, 1:2,000), anti-MRTF-A (Proteintech, China 21166-1, 1:1,000), anti-PDE5 (Proteintech, China 22624-1, 1:1,000), anti-BNP primary antibody (BOSTER, China, 1:1,000 dilution), anti-β-MHC primary antibody (Proteintech, China, 1:1,000 dilution), and anti-Sp1 (Abcam, United Kingdom ab13370, 1:1,000).

Techniques: Knock-Out, Immunofluorescence, Staining, Immunohistochemistry

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: 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

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Discovery of another mechanism for the inhibition of particulate guanylyl cyclases by the natriuretic peptide clearance receptor.

doi: 10.1073/pnas.2307882120

Figure Lengend Snippet: Fig. 2. NPRC immunoprecipitates with NPRA and NPRB. HEK 293FT cells were transfected with plasmids expressing Flag-tagged natriuretic peptide receptors. Twenty-four hours after transfection, cell lysates were immunoprecipitated with anti-Flag Affinity agarose. (A) Endogenous NPRC coimmunoprecipitated with hNPRA-Flag, hNPRB-Flag, or VEGFR1-Flag. (B) Endogeneous NPRC coimmunoprecipitated with GC-C-Flag. (C) NPRC band intensity to input of NPRC was calculated through Image J software was presented in the right panel. Šídák’s multiple comparisons test for one-way ANOVA comparing all samples to the negative control VEGFR1 for western blots in A and B are indicated on graph. *P < 0.05, ** P < 0.01, *** P < 0.001. (D) hNPRA-Flag expressing HEK 293FT cells were treated with ANP (ANP1-28, 200 nM) or a truncated form of ANP that preferentially binds NPRC (ANP4-23, 200 nM) for 30 min. Neither ANP1-28 or ANP4-23 affected the ability of endogenous NPRC to coimmunoprecipitate with hNPRA-Flag. (E) Endogenous NPRC coimmunoprecipitated with Flag-rNPRA and a truncated version of Flag- rNPRA lacking the c-terminal amino acids 528-1057 [Flag-rNPRA(1-527)].

Article Snippet: For western blotting the following primary antibodies were used: Flag (Sigma #F1804), NPRA (Novus Biologicals #NBP1- 31333), NPRB (Proteintech #55113- 1- AP), NPRC (Novus Biologicals #NBP1- 31365), NanoLuc® Luciferase (R&D Systems #MAB10026), β- actin (Cell Signaling #4967).

Techniques: Transfection, Expressing, Immunoprecipitation, Software, Negative Control, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Discovery of another mechanism for the inhibition of particulate guanylyl cyclases by the natriuretic peptide clearance receptor.

doi: 10.1073/pnas.2307882120

Figure Lengend Snippet: Fig. 3. Endogenous natriuretic peptide receptor expression in cell models used. Expression of NPRA, NPRB, and NPRC were detected by western blot in HeLa, HEK293FT, and HAP-1 cells. β-actin was used as the loading control.

Article Snippet: For western blotting the following primary antibodies were used: Flag (Sigma #F1804), NPRA (Novus Biologicals #NBP1- 31333), NPRB (Proteintech #55113- 1- AP), NPRC (Novus Biologicals #NBP1- 31365), NanoLuc® Luciferase (R&D Systems #MAB10026), β- actin (Cell Signaling #4967).

Techniques: Expressing, Western Blot, Control

Journal: Science advances

Article Title: NPRC deletion attenuates cardiac fibrosis in diabetic mice by activating PKA/PKG and inhibiting TGF-β1/Smad pathways.

doi: 10.1126/sciadv.add4222

Figure Lengend Snippet: Fig. 9. Activation of cAMP/PKA or cGMP/PKG signaling increased TGIF1 expression, but cAMP/PKA activation inhibited TGF-β1 expression. (A) Representative Western blot images of p-PKA substrates and p-CREB expression in NRCFs, which were treated with either forskolin or forskolin combined with H89 followed by HG treatment. (B) Quantification of the protein expression of p-PKA substrates and p-CREB in (A). n = 6 per group. (C) Representative Western blot images of TGF-β1, TGIF1, p-Smad2, and p-Smad3 expression in NRCFs, which were treated with either forskolin or forskolin combined with H89 followed by HG treatment. (D) Quantification of the protein expression of TGF-β1, TGIF1, p-Smad2, and p-Smad3 in (C). n = 6 per group. (E) Representative Western blot images of TGF-β1, TGIF1, and p-VASP expression in NRCFs transfected with Ad-GFP, Ad-ANP, Ad-BNP, or Ad-CNP. (F) Quantification of the protein expression of TGF-β1, TGIF1, and p-VASP in (E). n = 6 per group. (G) Relative mRNA level of tgif1 in NRCFs transfected with Ad-GFP, Ad-ANP, Ad-BNP, or Ad-CNP. n = 6 per group. (H) Representative Western blot images of p-VASP in NRCFs treated with either 8-br-cGMP or 8-br-cGMP combined with KT5823 followed by HG treatment. (I) Quantification of the protein expression of p-VASP in (H). n = 6 per group. (J) Representative Western blot images of TGF-β1, TGIF1, p-Smad2, and p-Smad3 expression in NRCFs treated with either 8-br-cGMP or 8-br-cGMP combined with KT5823 followed by HG treatment. (K) Quantification of the protein expression of TGF-β1, TGIF1, p-Smad2, and p-Smad3 in (J). n = 6 per group. Normal distributions were tested by Shapiro-Wilk method. One-way ANOVA was used in (B), (D), (I), and (K). One-way ANOVA with Dunnett’s post hoc test was used in (F) and (G).

Article Snippet: A continuous subcutaneous infusion of saline was conducted in the DM + saline and DM + NPRC−/− groups, respectively, and infusion of ANP (ANP1-28, MCE, USA; 0.5 μg/kg per minute), BNP (BNP1-32, MCE, USA; 0.03 μg/kg per minute), and CNP (CNP1-22, TargetMol, USA; 0.1 μg/kg per minute) was performed in the DM + ANP, DM + BNP, and DM + CNP groups, respectively.

Techniques: Activation Assay, Expressing, Western Blot, Transfection