Journal: bioRxiv

Article Title: Alzheimer’s Aβ assembly binds sodium pump and blocks endothelial NOS activity via ROS-PKC pathway

doi: 10.1101/2020.12.10.419879

Figure Lengend Snippet: A,C: Effect of 46 nM ASPD (A) or 100 nM ouabain (C) on carbachol-induced relaxation of ex-vivo rat aortic rings (n = 3 for mASD3-preincubated ASPD-treated group, and n = 5 for other groups). B: Representative images of immunohistochemical multiple staining of NAKα3 and vWF in the endothelial layer of rat aortic rings. The arrowheads indicate NAKα3 on the apical surface of endothelium. Scale bars: 1 μm. In (A,C), data are presented as means ± S.E. ** P < 0.01 (ANOVA with Scheffé’s method (A) and Welch’s t -test (C)).

Article Snippet: These sections were incubated overnight at 4°C with primary antibody against NAKα3 (ANP-003, 1:200; Alomone Labs, Jerusalem, Israel) and von Willebrand factor glycoprotein (sc-365712, 1:50; Santa Cruz Biotechnology, Dallas, TX) in the presence of normal goat serum in PBS, and then incubated with the appropriate Alexa Fluorconjugated secondary antibody (1:1000, Molecular Probes, Waltham, MA) for 1 hr at r.t. Counterstaining was carried out with 4’,6-diamidino-2-phenylindole (DAPI, 1:500; Dojindo Molecular Technologies, Kumamoto, Japan).

Techniques: Ex Vivo, Immunohistochemical staining, Staining

Journal: bioRxiv

Article Title: Alzheimer’s Aβ assembly binds sodium pump and blocks endothelial NOS activity via ROS-PKC pathway

doi: 10.1101/2020.12.10.419879

Figure Lengend Snippet: A: Representative 2D images of immunocytochemical staining of NAKα3 and nuclei (DAPI) on human primary brain microvessel endothelial cells (left) and vertical section image prepared from z-stack 3D image (right). Scale bars: 5 μm. B: Western blotting for NAKα3 in the endothelial cells (left) and agarose electrophoresis of RT-PCR products for ATP1A3 mRNA (right). C: Binding ratio of ASPD signal to total NAKα3 signal (the representative images in upper panels and the quantification in bottom, n = 5). Scale bars: 5 μm. D: High-power representative 2D images of multiple immunocytochemical staining of ASPD (ASPD-specific, mASD3 antibody), NAKα3, and nuclei (DAPI) on 30 nM ASPD-treated endothelial cells (left). The vertical section image prepared from z-stack 3D image (right upper) and its line-scan analysis of fluorescence intensities of NAKα3 (green line) and ASPD (red line) (right bottom). Scale bars: 5 μm for solid line and 1 μm for hatched line. E: Western blotting for NAKα3 in the siRNA-transfected endothelial cells (left, n = 3) and quantification of number of punctate NAKα3 signal on the endothelial cells (right, n = 5). Scale bars: 5 μm. F: Effect of siRNA transfection on the binding ratio of ASPD signal to total NAKα3 signal on the endothelial cells (n = 5). The transfection of ATP1A3 siRNA decreased the ASPD binding ratio to 37 ± 12%, 11 ± 2%, 42 ± 2%, and 66.2 ± 2% in the 3 nM, 10 nM, 30 nM, and 100 nM ASPD-treated groups, respectively. In (C,E,F), data are presented as means ± S.E. * P < 0.05 (ANOVA with Scheffé’s method).

Article Snippet: These sections were incubated overnight at 4°C with primary antibody against NAKα3 (ANP-003, 1:200; Alomone Labs, Jerusalem, Israel) and von Willebrand factor glycoprotein (sc-365712, 1:50; Santa Cruz Biotechnology, Dallas, TX) in the presence of normal goat serum in PBS, and then incubated with the appropriate Alexa Fluorconjugated secondary antibody (1:1000, Molecular Probes, Waltham, MA) for 1 hr at r.t. Counterstaining was carried out with 4’,6-diamidino-2-phenylindole (DAPI, 1:500; Dojindo Molecular Technologies, Kumamoto, Japan).

Techniques: Staining, Western Blot, Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Fluorescence, Transfection

Journal: bioRxiv

Article Title: Alzheimer’s Aβ assembly binds sodium pump and blocks endothelial NOS activity via ROS-PKC pathway

doi: 10.1101/2020.12.10.419879

Figure Lengend Snippet: High-power representative 2D images of immunocytochemical multiple staining of ASPD (ASPD-specific, mASD3 antibody), NAKα3, and nuclei (DAPI) on 30 nM ASPD-treated human primary brain microvessel endothelial cells with or without siRNA transfection (Upper, middle, and bottom panels show non-treated, mock siRNA-treated, and ATP1A3 siRNA-treated cells, respectively). Scale bars: 5 μm.

Article Snippet: These sections were incubated overnight at 4°C with primary antibody against NAKα3 (ANP-003, 1:200; Alomone Labs, Jerusalem, Israel) and von Willebrand factor glycoprotein (sc-365712, 1:50; Santa Cruz Biotechnology, Dallas, TX) in the presence of normal goat serum in PBS, and then incubated with the appropriate Alexa Fluorconjugated secondary antibody (1:1000, Molecular Probes, Waltham, MA) for 1 hr at r.t. Counterstaining was carried out with 4’,6-diamidino-2-phenylindole (DAPI, 1:500; Dojindo Molecular Technologies, Kumamoto, Japan).

Techniques: Staining, Transfection

Journal: bioRxiv

Article Title: Alzheimer’s Aβ assembly binds sodium pump and blocks endothelial NOS activity via ROS-PKC pathway

doi: 10.1101/2020.12.10.419879

Figure Lengend Snippet: ASPD bind to cell-surface NAKα3 on cerebral microvessel endothelial cells, promote mitochondrial ROS production, activate PKC, increase eNOS-Thr 495 phosphorylation, and attenuate NO release, resulting in suppression of blood microvessel relaxation response.

Article Snippet: These sections were incubated overnight at 4°C with primary antibody against NAKα3 (ANP-003, 1:200; Alomone Labs, Jerusalem, Israel) and von Willebrand factor glycoprotein (sc-365712, 1:50; Santa Cruz Biotechnology, Dallas, TX) in the presence of normal goat serum in PBS, and then incubated with the appropriate Alexa Fluorconjugated secondary antibody (1:1000, Molecular Probes, Waltham, MA) for 1 hr at r.t. Counterstaining was carried out with 4’,6-diamidino-2-phenylindole (DAPI, 1:500; Dojindo Molecular Technologies, Kumamoto, Japan).

Techniques: Phospho-proteomics

Journal: Scientific reports

Article Title: A novel immune score model predicting the prognosis and immunotherapy response of breast cancer.

doi: 10.1038/s41598-023-31153-2

Figure Lengend Snippet: Figure 4. Weighted gene co-expression network analysis (WGCNA) and identification of candidate immune-related hub genes. (A) Analysis of network topology for various soft-thresholding powers. (B) Gene dendrogram and module colours. (C) Immune-related genes (IRGs) in the brown, green, and yellow modules were significantly correlated with BC progression. (D) Venn diagram results confirmed that IRGs ANO6, NPR3, CCL24, FLT3, and ULBP2 were also recruited to construct our prognostic risk model. (E) Module membership (MM) vs. gene significance (GS) analysis of 7 modules showed that MM was significantly correlated with GS in brown, yellow, blue, and red modules. WGCNA weighted gene co-expression network analysis, IRG immune- related gene, MM, module membership, GS gene significance.

Article Snippet: The mammary sections were incubated overnight at 4 °C with a cocktail of primary antibodies against human NPR3 (1:100; Proteintech, China), followed by incubation with a cocktail of secondary antibodies (Life Technologies, CA, USA) for 1 h at room temperature on the next day.

Techniques: Expressing, Construct

Journal: Scientific reports

Article Title: A novel immune score model predicting the prognosis and immunotherapy response of breast cancer.

doi: 10.1038/s41598-023-31153-2

Figure Lengend Snippet: Figure 7. Genetic alteration of immune-related genes (IRGs). In this study, we explored Tumour mutation burden (TMB), somatic mutation, and copy number variations (CNVs) between high- and low-risk groups using the “maftools” R package. Moreover, the cBio Cancer Genomics Portal database (cBioPortal) was performed to assess mutations and CNVs in breast cancer (BC) tissues. (A) Analysis of the mutation status of the 7 IRGs in our risk model. (B) The most frequent genetic alterations of NPR3 were missense mutation and amplification. (C) TMB comparison between the high- and low-risk groups. (D) The different survival probabilities for high-risk score BC patients with high TMB and BC patients with both low-risk score and low TMB. (E) The mutation frequency and classification of the top 20 gene mutations in the high-risk group. (F) The mutation frequency and classification of the top 20 gene mutations in the low-risk group. IRG immune-related gene, TMB tumour mutation burden, CNV copy number variation, cBioPortal cBio Cancer Genomics Portal database, BC breast cancer.

Article Snippet: The mammary sections were incubated overnight at 4 °C with a cocktail of primary antibodies against human NPR3 (1:100; Proteintech, China), followed by incubation with a cocktail of secondary antibodies (Life Technologies, CA, USA) for 1 h at room temperature on the next day.

Techniques: Mutagenesis, Amplification, Comparison

Journal: Scientific reports

Article Title: A novel immune score model predicting the prognosis and immunotherapy response of breast cancer.

doi: 10.1038/s41598-023-31153-2

Figure Lengend Snippet: Figure 8. Patients with high- and low-risk scores had different immune statuses. Comparison of the single- sample gene set enrichment analysis (ssGSEA) scores of 22 types of immune cells (A) and 13 immune-related pathways (B) between low- (blue box) and high-risk (red box) groups in The Cancer Genome Atlas (TCGA) project. (C) Immune-related gene prognostic index (IRGPI) differed among subtypes in the high and low-risk groups. (D) The tumour stemness of breast cancer (BC) patients between the high- and low-risk groups. (E) The relationship between copy number variations (CNVs) of 4 immune-related genes (IRGs) (ANO6, CCL24, FLT3, and NPR3) and the level of infiltration of B cells, T cells, and macrophages in BC. *P < 0.05; **P < 0.01; ***P < 0.001. ssGSEA single-sample gene set enrichment analysis, TCGA The Cancer Genome Atlas, IRGPI immune-related gene prognostic index, BC breast cancer, CNV copy number variation, IRG immune-related gene.

Article Snippet: The mammary sections were incubated overnight at 4 °C with a cocktail of primary antibodies against human NPR3 (1:100; Proteintech, China), followed by incubation with a cocktail of secondary antibodies (Life Technologies, CA, USA) for 1 h at room temperature on the next day.

Techniques: Comparison

Journal: Scientific reports

Article Title: A novel immune score model predicting the prognosis and immunotherapy response of breast cancer.

doi: 10.1038/s41598-023-31153-2

Figure Lengend Snippet: Figure 11. Validation of the predictive ability of the risk model in an external cohort. (A) The relative expression of the 7 immune-related genes (IRGs) in breast cancer (BC) samples through quantitative reverse transcription polymerase chain reaction (qRT‒PCR) (n = 30). * indicated P < 0.05, ** indicated P < 0.01, P value based on t-test. (B) The expression levels of PD-1, PD-L1, and CTLA-4 were decreased in the high-risk group, as evaluated by qRT‒PCR (n = 30). *** indicated P < 0.001, P value based on t-test. The immunohistochemistry (IHC) results revealed that the expression of NPR3 (C) was increased but that of PD-1 (D), PD-L1 (E), and CTLA-4 (F) was decreased in the high-risk group. (G) The immunofluorescence (IF) assay demonstrated that M2 macrophages were more abundant in high-risk patients. IRG immune-related gene, BC breast cancer, qRT‒PCR quantitative reverse transcription polymerase chain reaction, IHC immunohistochemistry, IF immunofluorescence.

Article Snippet: The mammary sections were incubated overnight at 4 °C with a cocktail of primary antibodies against human NPR3 (1:100; Proteintech, China), followed by incubation with a cocktail of secondary antibodies (Life Technologies, CA, USA) for 1 h at room temperature on the next day.

Techniques: Biomarker Discovery, Expressing, Reverse Transcription, Polymerase Chain Reaction, Immunohistochemistry, Immunofluorescence

Journal: Scientific reports

Article Title: A novel immune score model predicting the prognosis and immunotherapy response of breast cancer.

doi: 10.1038/s41598-023-31153-2

Figure Lengend Snippet: Figure 12. The effect of NPR3 on the proliferation, migration, and apoptosis of breast cancer (BC) cells. The results of quantitative reverse transcription polymerase chain reaction (qRT‒PCR) showed that si-NPR3-1 successfully knocked down the expression of NPR3 in MDA-MB-231 (A) and MCF-7 (B) cells (n = 3). * indicated P < 0.05, ** indicated P < 0.01, ns indicated no significance, P value based on t-test. The depletion of NPR3 observably suppressed the proliferation ability in both MDA-MB-231 (C) and MCF-7 (D) cells compared with the si-NC group (n = 3). *** indicated P < 0.001, P value based on t-test. The knockdown of NPR3 significantly decreased the migration of MDA-MB-231 (E,G) and MCF-7 (F, H) cells (n = 3). ** indicated P < 0.01, *** indicated P < 0.001, P value based on t-test. (I) Flow cytometry indicated that NPR3 deletion could increase apoptosis in MDA-MB-231 and MCF-7 cells (n = 3). ** indicated P < 0.01, *** indicated P < 0.001, P value based on t-test. All experiments were implemented separately in triplicate. BC breast cancer, qRT‒PCR quantitative reverse transcription polymerase chain reaction.

Article Snippet: The mammary sections were incubated overnight at 4 °C with a cocktail of primary antibodies against human NPR3 (1:100; Proteintech, China), followed by incubation with a cocktail of secondary antibodies (Life Technologies, CA, USA) for 1 h at room temperature on the next day.

Techniques: Migration, Reverse Transcription, Polymerase Chain Reaction, Expressing, Knockdown, Flow Cytometry

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: Biomedicines

Article Title: HIF-2α Controls Expression and Intracellular Trafficking of the α2-Subunit of Na,K-ATPase in Hypoxic H9c2 Cardiomyocytes.

doi: 10.3390/biomedicines11112879

Figure Lengend Snippet: Figure 3. Effects of hypoxia and HIF silencing on α2-NKA mRNA expression. H9c2 cells were infected with 100 MOI shHIF-1α and shHIF-2α containing adenoviral vectors and kept in normoxia (19% O2) or hypoxia (1% O2) for 24 h. mRNA expression was normalized to 28SrRNA, and the results are given as the normalized values of scr.co. Mean values ± SD of four independent experiments as shown. The level of significance was set to p < 0.05: * effect of hypoxia compared to normoxia, + effect of HIF-2α silencing in hypoxic cells compared to hypoxic scr.co. scr.co: scrambled control; shHIF-1α: HIF-1α-silenced cells; shHIF-2α: HIF-2α-silenced cells.

Article Snippet: The antibody against α2-NKA (rabbit, #ANP-002, 1:1000 dilution) was from Alomone Labs and beta-actin, used to normalize the band densities as a housekeeping gene (mouse, #A2228, 1:10000 dilution), was from Sigma.

Techniques: Expressing, Infection, Control