Journal: Signal Transduction and Targeted Therapy

Article Title: Asialoglycoprotein receptor 1 promotes SARS-CoV-2 infection of human normal hepatocytes

doi: 10.1038/s41392-024-01754-y

Figure Lengend Snippet: A pooled, genome-wide CRISPR screening for candidate receptors involved in SARS-CoV-2 entry. a Outline of genome-wide activation CRISPR screen strategy. HeLa cells were infected with MPH-lentivirus able to express MS2-p65-HSF1 proteins treated with Hygromycin B for fourteen days. Then, HeLa cells expressing MS2-p65-HSF1 were infected with lentiviral dCas9 and sgRNA library targeting 20234 human genes. Fourteen days post-screening with blascicidin, genomic DNA was extracted from monoclonal cells after puromycin selection. Candidate genes were identified by Sanger sequencing. b By extracting the genomes of cells in different treatment groups and using specific primers targeting MS2-p65-HSF1 for PCR identification, it was verified that the MS2-p65-HSF1 gene was stably integrated into the infected group cells. c After infection with HeLa cells expressing MS2-p65-HSF1 with SAM library, Western blot of cell lysate with anti-Cas9 antibody was performed to identify the expression of dCas9 protein. d Statistical analysis of candidate sgRNAs. The genomes of all monoclonal cell lines were extracted, and the corresponding sgRNA sequences were amplified using primers targeting sgRNA sequences and subjected to Sanger sequencing. The candidate genes corresponding to sgRNAs were ranked according to abundance. e – g ASGR1 facilitates SARS-CoV-2 virus pseudotype with wild-type Spike infection as potently as ACE2. HEK293T cells were transfected with Flag-tagged CD134, ACE2, KREMEN1, ASGR1, CXCR2, or empty. After 24 h, the cells were infected with the GFP-labeled and luciferase SARS-CoV-2 virus pseudotype, and visualized by microscopy ( e ), expression levels of GFP by cell flow cytometer ( f ), and detection of luciferase by microplate ( g ) at 72 h post-infection. Scale bars, 1000 μm. Each data represented the mean ± SD of three independent experiments ( n = 3) and were analyzed with T-test compared with mock cells. *** p < 0.001

Article Snippet: Anti-Flag Magnetic Beads (HY-K0207) and ACE2/ANG (1-7) inhibitor-A779 (HY-P0216) were purchased from MCE.

Techniques: Genome Wide, CRISPR, Activation Assay, Infection, Expressing, Selection, Sequencing, Stable Transfection, Western Blot, Amplification, Virus, Transfection, Labeling, Luciferase, Microscopy, Flow Cytometry

Journal: Signal Transduction and Targeted Therapy

Article Title: Asialoglycoprotein receptor 1 promotes SARS-CoV-2 infection of human normal hepatocytes

doi: 10.1038/s41392-024-01754-y

Figure Lengend Snippet: SARS-CoV-2 pseudovirus can infect liver cell lines through ASGR1 in vitro. HEK293T, Huh-7, and HepG2 cells were infected with the GFP-labeled and luciferase SARS-CoV-2 pseudotype virus with the Spike protein of wild type, XBB.1.16 or B.1.617.2, detect the expression levels of GFP by cell flow cytometer ( a ), and luciferase by microplate ( b ) at 72 h post-infection. c ACE2 and ASGR1 expressions were measured by qPCR. The intracellular RNA of 293 T, Huh-7, and HepG2 cells was extracted, and after reverse transcription, the corresponding primers were used to amplify ACE2, ASGR1, and GAPDH respectively. The expression levels of ACE2 or ASGR1 were finally normalized by GAPDH. d ACE2 and ASSGR1 expressions were measured by Western blot. Western blot of cell lysate of 293T, Huh-7, and HepG2 cells with anti-ACE2 or anti-ASGR1 was performed to identify expression of ACE2 or ASGR1 protein. e ACE2 and ASSGR1 expressions were measured by Western blot in Huh-7 cells with knockout of ASGR1 , ACE2 , or both with anti-ACE2 or anti-ASGR1. f – h SARS-CoV-2 pseudovirus with wild-type Spike can infect Huh-7 cells through ACE2 or ASGR1. Huh-7 cells with knockout of ASGR1 , ACE2 , or both, were infected with the GFP-labeled and luciferase SARS-CoV-2 pseudotype virus with wild type Spike and visualized by microscopy ( f ), expression levels of GFP by cell flow cytometer ( g ), and detection of luciferase by microplate ( h ) at 72 h post-infection. Scale bars, 1000 μm. Each data represented the mean ± SD of three independent experiments ( n = 3) and were analyzed with T-test compared with 293 T or parental cells. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: Anti-Flag Magnetic Beads (HY-K0207) and ACE2/ANG (1-7) inhibitor-A779 (HY-P0216) were purchased from MCE.

Techniques: In Vitro, Infection, Labeling, Luciferase, Virus, Expressing, Flow Cytometry, Reverse Transcription, Western Blot, Knock-Out, Microscopy

Journal: Signal Transduction and Targeted Therapy

Article Title: Asialoglycoprotein receptor 1 promotes SARS-CoV-2 infection of human normal hepatocytes

doi: 10.1038/s41392-024-01754-y

Figure Lengend Snippet: SARS-CoV-2 pseudovirus infects immortalized liver cell lines and primary hepatocytes through ASGR1. a ACE2 and ASGR1 expressions were measured by qPCR. The intracellular RNA of 293T, THLE-2, or primary hepatocytes was extracted, and after reverse transcription, the corresponding primers were used to amplify ACE2, ASGR1, and GAPDH respectively. The expression levels of ACE2 or ASGR1 were finally normalized by GAPDH. b ACE2 and ASGR1 expressions were measured by Western blot. Western blot of cell lysate of THLE-2, primary hepatocytes, and Huh-7 cells with anti-ACE2 or anti-ASGR1 was performed to identify expression of ACE2 or ASGR1 protein. Each data represented the mean ± SD of three independent experiments ( n = 3) and were analyzed with T-test compared with 293T cells. c – e SARS-CoV-2 pseudovirus with wild-type Spike can infect THLE-2 cells and primary hepatocytes. THLE-2 cells and primary hepatocytes were infected with GFP-labeled and luciferase SARS-CoV-2 pseudotype virus with wild type Spike and visualized by microscopy ( c ), the expression levels of GFP by cell flow cytometer ( d ), and detection of luciferase by microplate ( e ) at 72 h post-infection. Scale bars, 1000 μm. Each data represented the mean ± SD of three independent experiments ( n = 3) and were analyzed with T-test compared with 293 T cells. f , g siRNA targeting ASGR1 prevents SARS-CoV-2 pseudovirus from infecting liver cells. THLE-2 and primary hepatocytes were treated with 100 nM control-siRNA, siRNA-1, siNRA-2, and siRNA-3 for 24 h, respectively. The intracellular RNA of THLE-2 or primary hepatocytes was extracted, and after reverse transcription, the corresponding primers were used to amplify ASGR1 and GAPDH respectively. The expression levels of ASGR1 were finally normalized by GAPDH. f , followed by infection with SARS-CoV-2 pseudotype virus, and infection efficiency was detected by luciferase ( g ). Each data represented the mean ± SD of three independent experiments ( n = 3) and were analyzed with T-test compared with Mock cells. h , i ASGR1 monoclonal antibody can prevent SARS-CoV-2 pseudovirus from infecting liver cells. THLE-2 cells and primary hepatocytes were treated with 500 μg/mL anti-ASGR1 antibody for 0 h, 1 h, 2 h, 4 h, 8 h, and 12 h (h) or with 0 μg/mL, 5 μg/mL, 50 μg/mL, 500 μg/mL, and 5000 μg/mL for 6 h ( i ) and then infected with SARS-CoV-2 pseudotype virus, and the infection efficiency was detected by luciferase. Each data represented the mean ± SD of three independent experiments ( n = 3) and were analyzed with T-test compared with 0 h or 0 μg/mL treated with cells. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: Anti-Flag Magnetic Beads (HY-K0207) and ACE2/ANG (1-7) inhibitor-A779 (HY-P0216) were purchased from MCE.

Techniques: Reverse Transcription, Expressing, Western Blot, Infection, Labeling, Luciferase, Virus, Microscopy, Flow Cytometry, Control

Journal: Signal Transduction and Targeted Therapy

Article Title: Asialoglycoprotein receptor 1 promotes SARS-CoV-2 infection of human normal hepatocytes

doi: 10.1038/s41392-024-01754-y

Figure Lengend Snippet: SARS-CoV-2 with wild-type Spike infects primary hepatocytes through ASGR1. a Primary hepatocytes were infected with SARS-CoV-2 with wild-type Spike at different pfu (pfu = 0, 5.0 ×105, 2.5 ×106, or 1 ×107) for 6 h and replaced the virus-free complete medium to continue culturing for 24 h. Then cells were immunostained with rabbit-anti-SARS-CoV-2 NP and DAPI. b Morphological detection of SARS-CoV-2 in primary hepatocytes. After cells were infected with SARS-Cov-2 with wild-type Spike and fixed with methanol overnight, they were negatively stained and observed using a projection electron microscope. c , d siRNA targeting ASGR1 prevented SARS-CoV-2 with wild-type Spike from infecting liver cells. Primary hepatocytes were treated with control-siRNA, siRNA-ACE2, or siRNA-ASGR1 for 24 h, respectively. Then primary hepatocytes were infected with SARS-CoV-2 with wild-type Spike (pfu = 1 ×107) for 6 h and replaced the virus-free complete medium to continue culturing for 24 h. After that, the primary hepatocytes were immunostained with rabbit-anti-SARS-CoV-2 NP and DAPI ( c ). The intensity of NP were quantified using Image J and shown in d , which represents the infection efficiency of the SARS-CoV-2. Each data represented the mean ± SD of three independent experiments ( n = 3) and were analyzed with T-test compared with cells treated with siRNA. *** p < 0.001

Article Snippet: Anti-Flag Magnetic Beads (HY-K0207) and ACE2/ANG (1-7) inhibitor-A779 (HY-P0216) were purchased from MCE.

Techniques: Infection, Virus, Staining, Microscopy, Control

Journal: Signal Transduction and Targeted Therapy

Article Title: Asialoglycoprotein receptor 1 promotes SARS-CoV-2 infection of human normal hepatocytes

doi: 10.1038/s41392-024-01754-y

Figure Lengend Snippet: Soluble ASGR1 protein can prevent SARS-CoV-2 pseudovirus from infecting a variety of cells. a , b Soluble ASGR1 protein could prevent SARS-CoV-2 pseudovirus from infecting liver cells. The SARS-CoV-2 pseudotype virus with wild type-Spike was incubated with 500 μg/mL soluble ASGR1 for 0 h, 1 h, 2 h, 4 h, 8 h, and 12 h ( a ) or with 0 μg/mL 5 μg/mL, 50 μg/mL and 500 μg/mL for 6 h ( b ), then infected THLE-2 cells and primary hepatocytes. The infection efficiency was detected by luciferase. c , d Soluble ASGR1 protein could prevent SARS-CoV-2 pseudovirus from infecting cells expressing ACE2. The SARS-CoV-2 pseudotype virus with wild-type Spike was incubated with 500 μg/mL soluble ASGR1 for 0 h, 1 h, 2 h, 4 h, 8 h, and 12 h ( c ) or with 0 μg/mL 5 μg/mL, 50 μg/mL and 500 μg/mL for 6 h ( d ), then infected Huh-7, Calu-3 and 293T-ACE2 cells. The infection efficiency was detected by luciferase. e Comparison of ACE2 inhibitors and ASGR1 on SARS-CoV-2 pseudovirus-infected cells. Before infection of THLE-2 cells and primary hepatocytes ( e ) or Huh-7 and Calu-3 ( f ), the SARS-CoV-2 pseudotype virus with wild type-Spike was incubated with 500 μg/mL soluble ASGR1 for 12 h or THLE-2 cells and primary hepatocytes ( e ) or Huh-7 and Calu-3 ( f ) treated with A779 (10 μM) or UDCA (10 μM) for 12 h. The infection efficiency was detected by luciferase. Each data represented the mean ± SD of three independent experiments ( n = 3) and were analyzed with T-test compared with 0 h or 0 μg/mL treated with cells. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: Anti-Flag Magnetic Beads (HY-K0207) and ACE2/ANG (1-7) inhibitor-A779 (HY-P0216) were purchased from MCE.

Techniques: Virus, Incubation, Infection, Luciferase, Expressing, Comparison

Journal: Hepatology Communications

Article Title: Mas‐related G protein‐coupled receptor type D antagonism improves portal hypertension in cirrhotic rats

doi: 10.1002/hep4.1987

Figure Lengend Snippet: In vivo hemodynamic changes in rats with cirrhotic portal hypertension induced by carbon tetrachloride intoxication or bile duct ligation surgery and noncirrhotic portal hypertension induced by partial portal vein ligation surgery compared with respective controls. Rats were given 2 weeks (CCl 4 and BDL) or 1 week (PPVL) continuous infusion of MrgD blocker D‐pro or MasR blocker A779. (A) Splanchnic vascular resistance, (B) mesenteric blood flow, (C) hepatic vascular resistance, and (D) portal pressure in three models are shown. * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001, diseased (CCl 4 or BDL or PPVL) versus olive oil‐injected/sham‐operated healthy controls. # p < 0.05, ## p < 0.01, ### p < 0.005, #### p < 0.001, saline‐infused diseased controls versus diseased rats treated with D‐pro or A779. θ p < 0.05, D‐pro versus A779‐treated diseased rats. Each bar represents the mean ± SEM profile from 10 to 15 rats per treatment or control group. A779, D‐Ala 7 ‐Ang‐(1–7); BDL, bile duct ligation; CCl 4 , carbon tetrachloride; D‐Pro, D‐Pro 7 ‐Ang‐(1–7); MasR, Mas receptor; MrgD, Mas‐related G protein‐coupled receptor type D; PPVL, partial portal vein ligation

Article Snippet: The MasR blocker D‐Ala 7 ‐Ang‐(1–7) (A779) (Mimotopes, Australia) (28 μg/kg/hour), MrgD blocker D‐Pro 7 ‐Ang‐(1–7) (D‐Pro) (Mimotopes) (28 μg/kg/hour) or saline were infused through a subcutaneously implanted osmotic minipump, as described.

Techniques: In Vivo, Ligation, Injection, Saline, Control

Journal: Hepatology Communications

Article Title: Mas‐related G protein‐coupled receptor type D antagonism improves portal hypertension in cirrhotic rats

doi: 10.1002/hep4.1987

Figure Lengend Snippet: Effects of MrgD blocker D‐pro and MasR blocker A779 on acetylcholine‐induced vasodilatory responses. (A,B) Healthy and (C,D) carbon tetrachloride‐intoxicated cirrhotic rats were analyzed for the effects of MrgD blocker D‐pro and MasR blocker A779 on acetylcholine‐induced vasodilatory responses in (A,C) proximal and (B,D) distal mesenteric resistance vessels. The A779 response curve in panel B is not shown due to a technical reason. Each data point represents mean ± SEM profile of vascular responses from n = 4–6 rats per group. * p < 0.05, D‐pro versus saline or A779 groups. A779, D‐Ala 7 ‐Ang‐(1–7); D‐Pro, D‐Pro 7 ‐Ang‐(1–7); MasR, Mas receptor; MrgD, Mas‐related G protein‐coupled receptor type D

Article Snippet: The MasR blocker D‐Ala 7 ‐Ang‐(1–7) (A779) (Mimotopes, Australia) (28 μg/kg/hour), MrgD blocker D‐Pro 7 ‐Ang‐(1–7) (D‐Pro) (Mimotopes) (28 μg/kg/hour) or saline were infused through a subcutaneously implanted osmotic minipump, as described.

Techniques: Saline

Journal: Hepatology Communications

Article Title: Mas‐related G protein‐coupled receptor type D antagonism improves portal hypertension in cirrhotic rats

doi: 10.1002/hep4.1987

Figure Lengend Snippet: Gene expression of MrgD and MasR . (A,B) MrgD and MasR were analyzed in cirrhotic mesenteric arteries isolated from carbon tetrachloride‐intoxicated and bile duct‐ligated rats, cirrhotic rats treated with the MrgD blocker D‐pro or MasR blocker A779, and healthy controls. (C–H) Protein expression of MrgD and MasR quantified by western blot and immunohistochemistry (magnification ×200), respectively, is shown. Each bar represents the mean ± SEM profile from 10 to 15 rats per group. Arrowheads show positive endothelial staining. * p < 0.05, *** p < 0.005, **** p < 0.001, diseased (CCl 4 or BDL) versus olive oil‐injected or sham‐operated healthy controls. # p < 0.05, ## p < 0.01, #### p < 0.001, diseased saline versus D‐pro or A779‐treated diseased rats. θ p < 0.05, θθ p < 0.01, D‐pro versus A779‐treated diseased rats. A779, D‐Ala 7 ‐Ang‐(1–7); BDL, bile duct ligation; CCl 4 , carbon tetrachloride; D‐Pro, D‐Pro 7 ‐ Ang‐(1–7); MasR, Mas receptor; MrgD, Mas‐related G protein‐coupled receptor type D

Article Snippet: The MasR blocker D‐Ala 7 ‐Ang‐(1–7) (A779) (Mimotopes, Australia) (28 μg/kg/hour), MrgD blocker D‐Pro 7 ‐Ang‐(1–7) (D‐Pro) (Mimotopes) (28 μg/kg/hour) or saline were infused through a subcutaneously implanted osmotic minipump, as described.

Techniques: Gene Expression, Isolation, Expressing, Western Blot, Immunohistochemistry, Staining, Injection, Saline, Ligation

Journal: Hepatology Communications

Article Title: Mas‐related G protein‐coupled receptor type D antagonism improves portal hypertension in cirrhotic rats

doi: 10.1002/hep4.1987

Figure Lengend Snippet: Gene expression of MrgD and MasR . (A,B) MrgD and MasR were analyzed in cirrhotic livers isolated from carbon tetrachloride‐intoxicated and bile duct‐ligated rats, cirrhotic rats treated with the MrgD blocker D‐pro or MasR blocker A779, and healthy controls. Up‐regulation of MasR protein expression as quantified by western blot (C,D) and immunohistochemistry (F,G) (magnification ×200) is shown. Positive staining of MasR in endothelium (large arrowhead), bile duct epithelial cells (arrow), and hepatic arterioles (small arrowhead) is shown. MrgD protein was not detectable by western blot or by immunohistochemistry (E) (magnification ×200) in cirrhotic or control livers. Each bar represents the mean ± SEM profile from 10 to 15 rats per group. * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001, diseased (CCl 4 or BDL) versus olive oil‐injected/sham‐operated healthy controls. A779, D‐Ala 7 ‐Ang‐(1–7); BDL, bile duct ligation; CCl 4 , carbon tetrachloride; D‐Pro, D‐Pro 7 ‐Ang‐(1–7); MasR, Mas receptor; MrgD, Mas‐related G protein‐coupled receptor type D

Article Snippet: The MasR blocker D‐Ala 7 ‐Ang‐(1–7) (A779) (Mimotopes, Australia) (28 μg/kg/hour), MrgD blocker D‐Pro 7 ‐Ang‐(1–7) (D‐Pro) (Mimotopes) (28 μg/kg/hour) or saline were infused through a subcutaneously implanted osmotic minipump, as described.

Techniques: Gene Expression, Isolation, Expressing, Western Blot, Immunohistochemistry, Staining, Control, Injection, Ligation

Journal: Hepatology Communications

Article Title: Mas‐related G protein‐coupled receptor type D antagonism improves portal hypertension in cirrhotic rats

doi: 10.1002/hep4.1987

Figure Lengend Snippet: Gene expression of MrgD and MasR in mesenteric arteries isolated from PPVL rats with noncirrhotic portal hypertension, PPVL rats treated with the MrgD blocker D‐pro or MasR blocker A779, and sham‐operated controls. (A) MrgD . (B) MasR . (C,D) Western blot analysis of mesenteric arterial protein expression of (C) MrgD and (D) MasR showed no difference between the four groups. (E) MrgD and MasR protein was not detected by immunohistochemical staining, likely due to very low expression levels. Each bar represents the mean ± SEM profile from 10 to 15 rats per group. A779, D‐Ala 7 ‐Ang‐(1–7); D‐Pro, D‐Pro 7 ‐Ang‐(1–7); MasR, Mas receptor; MrgD, Mas‐related G protein‐coupled receptor type D; PPVL, partial portal vein ligation

Article Snippet: The MasR blocker D‐Ala 7 ‐Ang‐(1–7) (A779) (Mimotopes, Australia) (28 μg/kg/hour), MrgD blocker D‐Pro 7 ‐Ang‐(1–7) (D‐Pro) (Mimotopes) (28 μg/kg/hour) or saline were infused through a subcutaneously implanted osmotic minipump, as described.

Techniques: Gene Expression, Isolation, Western Blot, Expressing, Immunohistochemical staining, Staining, Ligation

Journal: Hepatology Communications

Article Title: Mas‐related G protein‐coupled receptor type D antagonism improves portal hypertension in cirrhotic rats

doi: 10.1002/hep4.1987

Figure Lengend Snippet: Relationship between hemodynamic changes and the expression of MasR and MrgD in different vascular beds of healthy and cirrhotic carbon tetrachloride‐intoxicated rats. Each bar represents the mean ± SEM profile of 10–15 rats. Fold changes are shown on top of each bar of control (open bars) and cirrhotic (closed bars) rats. Thickness of receptor blocking lines indicates relative contribution by the receptor. Broken line denoting a receptor‐blocking step represents a possible pathway. A779, D‐Ala 7 ‐Ang‐(1–7); D‐Pro, D‐Pro 7 ‐Ang‐(1–7); MasR, Mas receptor; MrgD, Mas‐related G protein‐coupled receptor type D; ns, nonsignificant

Article Snippet: The MasR blocker D‐Ala 7 ‐Ang‐(1–7) (A779) (Mimotopes, Australia) (28 μg/kg/hour), MrgD blocker D‐Pro 7 ‐Ang‐(1–7) (D‐Pro) (Mimotopes) (28 μg/kg/hour) or saline were infused through a subcutaneously implanted osmotic minipump, as described.

Techniques: Expressing, Control, Blocking Assay

Journal: Frontiers in Immunology

Article Title: Angiotensin-(1-7) Promotes Resolution of Neutrophilic Inflammation in a Model of Antigen-Induced Arthritis in Mice

doi: 10.3389/fimmu.2017.01596

Figure Lengend Snippet: The Mas antagonist, A779, reverses the pro-resolution effect of angiotensin-(1-7) [Ang-(1-7)] in a model of arthritis in mice. (A,B) The number of neutrophils and the percentage of apoptotic neutrophils in the synovial cavity, respectively, in antigen-induced arthritis (AIA) mice 12 h after treatment with Ang-(1-7) alone or Ang-(1-7) and A779. (C,D) The number of neutrophils and the percentage of apoptotic neutrophils in the synovial cavity, respectively, in AIA mice 48 and 72 h after challenge treated with A779. Bars show the mean ± SEM from eight mice per group. * p ≤ 0.05 as compared with PBS-control group; # p ≤ 0.05 as compared with AIA group (one-way ANOVA followed by Newman–Keuls test).

Article Snippet: Mice were treated with an intra-articular injection (10 μl) of 100 ng of Ang-(1-7) (Bachem, Torrance, CA, USA), Mas antagonist, A779 [D-Ala 7 -Ang-(1-7), 200 ng/cavity; Bachem, Torrance, CA, USA], or the vehicle (NaCl 0.9%) 12 h after antigen challenge ( ).

Techniques: Control

Journal: Frontiers in Immunology

Article Title: Angiotensin-(1-7) Promotes Resolution of Neutrophilic Inflammation in a Model of Antigen-Induced Arthritis in Mice

doi: 10.3389/fimmu.2017.01596

Figure Lengend Snippet: Angiotensin-(1-7) [Ang-(1-7)] induces apoptosis of human neutrophils. Ang-(1-7) (30, 100, and 300 nM/well) or PBS (control, C) was incubated for 4 h with isolated human neutrophils. A779 (300 nM/well) was added 15 min before Ang-(1-7). (A) Percentage of apoptotic human neutrophils in response to increasing concentration of Ang-(1-7); (B) percentage of cells expressing Annexin V after 4 h incubation with Ang-(1-7) (100 nM/well); (C) effect of the Mas antagonist, A779, on neutrophil apoptosis induced by Ang-(1-7); (D) images of neutrophils stained for Mas receptor (green) and propidium iodide (PI, red). * p ≤ 0.05 when compared with PBS control (one-way ANOVA followed by Newman–Keuls test).

Article Snippet: Mice were treated with an intra-articular injection (10 μl) of 100 ng of Ang-(1-7) (Bachem, Torrance, CA, USA), Mas antagonist, A779 [D-Ala 7 -Ang-(1-7), 200 ng/cavity; Bachem, Torrance, CA, USA], or the vehicle (NaCl 0.9%) 12 h after antigen challenge ( ).

Techniques: Control, Incubation, Isolation, Concentration Assay, Expressing, Staining

Journal: Oncotarget

Article Title: Angiotensin-(1-7) counteracts the transforming effects triggered by angiotensin II in breast cancer cells

doi: 10.18632/oncotarget.19290

Figure Lengend Snippet: Western Blot analyses from the non-tumorigenic mammary cell line NMuMG were performed for p-AKT and AKT ( A-B ) and p-ERK1/2 and ERK1/2 ( C-D ). Cells were pre-incubated for 5 min with Irbesartan (10 -6 M), PD123319 (10 -6 M), A779 (10 -6 M), or D-Pro (10 -6 M), and then stimulated with AngII (10 -7 M) (A-C) or ( B-D ) Ang-(1-7) (10 -7 M) for the indicated time. Blots show representative Western blots. N of 3 independent experiments; values shown in bar represent mean±SEM quantified by densitometry and relative to control-untreated cells. *P<0.05, **P<0.01, ***P<0.001 vs untreated control cells; # P<0.05, ## P<0.01, ### P<0.001 vs AngII or §§ P<0.01, §§§ P<0.001 vs Ang-(1-7) treated cells.

Article Snippet: D-Ala 7 -Ang-(1-7) (A779), Irbesartan, AngII and Ang-(1-7) were purchased from Bachem AG, Bubendorf, Switzerland, Losartan from Sigma-Aldrich (St. Louis, MO, USA), and PD123319 (PD) from Parke-Davis Pharmaceutical Research (Detroit, MI, USA).

Techniques: Western Blot, Incubation, Control