Journal: Cell Research

Article Title: Coinfection with influenza A virus enhances SARS-CoV-2 infectivity

doi: 10.1038/s41422-021-00473-1

Figure Lengend Snippet: a, b A549 cells were mock-infected or infected with WSN at an MOI of 0.1. At 12 h.p.i., total RNA was extracted from cells, and ACE2, TMPRSS2, Furin, and CatL mRNA levels ( a ) or NP, Mx1, and ISG54 mRNA levels ( b ) were evaluated via qRT-PCR using the SYBR green method. The data are expressed as fold changes relative to the mock infections. c A549 cells were infected with WSN at an MOI of 0.1. IAV NP protein (red) and ACE2 (green) were detected with an immunofluorescence assay at 12 h.p.i. Scale bars are shown. A549 ( d ), Calu-3 ( e ), and NHBE ( f ) cells were preinfected with WSN at an MOI of 0.1 for 12 h. Cells were then infected with live SARS-CoV-2 at an MOI of 0.01 for another 48 h. Total RNA was extracted from cells, and ACE2 mRNA was evaluated via qRT-PCR using the SYBR green method. The protein expression levels of ACE2, SARS-CoV-2 N gene, IAV NP, and β-actin were measured via western blotting assay. * means increased exposure to visualize ACE2. ( g ) The relative mRNA levels of ACE2 were measured in lung homogenates from the indicated groups, and the protein expression of IAV NP and ACE2 was detected via western blotting. ( d – g ) The data are expressed as fold changes relative to the non-IAV infection control. ( h – i ) To establish ACE2 knockdown cells, A549 cells were transduced with lentivirus encoding the CRISPR-Cas9 system with two guide RNAs targeting ACE2 (sgRNA1 and sgRNA2) or control guide RNA. Cells were infected with live SARS-CoV-2 at an MOI of 0.01 with or without IAV infection using the same procedure described above. The ACE2 (qRT-PCR) ( h ) and SARS-CoV-2 E gene (Taqman-qRT-PCR) ( i ) mRNA levels were detected. The data are expressed as the fold change relative to the non-IAV infection control. Values represent means ± SD of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The primary antibodies used in this study were rabbit polyclonal antibody against ACE2 for immunofluorescence (Sino Biological, 10108-T26) and anti-influenza virus-NP antibody (kindly provided by Professor Ningshao Xia).

Techniques: Infection, Quantitative RT-PCR, SYBR Green Assay, Immunofluorescence, Expressing, Western Blot, Transduction, CRISPR

Journal: Research in Veterinary Science

Article Title: Elevated angiotensin-converting enzyme 2 (ACE2) expression in cats with hypertrophic cardiomyopathy

doi: 10.1016/j.rvsc.2022.09.024

Figure Lengend Snippet: ACE2 immunohistochemical analysis of the cat lung, heart, and kidneys from hypertrophic cardiomyopathy (HCM) and non-HCM cases. (a) Total score of abundance and intensity of ACE2 immunolabelling, expressed as sum of bronchiole, alveoli, heart, and kidney scores for each animal, were compared between the two groups. (b, c) Semi-quantitative and qualitative assessment were made for the abundance or intensity of ACE2, respectively. Score as follow: abundance - 0 (no positive staining), 1 (0 to <25% positive cells), 2 (≥25% to <50%), 3 (≥50% to <75%) and 4 (≥75% to 100%); intensity 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). Ten cases were reviewed for both HCM and non-HCM. Height of bars (a, b, c) represent the mean value and the error bars represent standard deviation. Mann-Whitney test. * p < 0.05.

Article Snippet: Briefly, this involved antigen retrieval of FFPE sections in pH 6 buffer, immunolabelling with rabbit polyclonal antibody against ACE2 (Abcam) and Envision flex (Dako) and visualisation with DAB chromogen.

Techniques: Immunohistochemical staining, Staining, Standard Deviation, MANN-WHITNEY

Journal: Research in Veterinary Science

Article Title: Elevated angiotensin-converting enzyme 2 (ACE2) expression in cats with hypertrophic cardiomyopathy

doi: 10.1016/j.rvsc.2022.09.024

Figure Lengend Snippet: Immunohistochemical labelling of ACE2 in the heart and lung of cats from non-HCM (a, c) and HCM (b, d) cases. Low levels of ACE2 immunolabelling in the heart and lung of non-HCM cats, limited to the vascular endothelium (arrow head) and type I pneumocytes (arrow), respectively (a, c). ACE2 immunolabelling was more abundant and intense in the vasculature (arrowhead) within the heart (b), and more abundant and intense in the alveoli typically in the type I pneumocytes (arrow) and occasionally in type II pneumocytes (arrowhead) of cats with HCM (d). Images taken with 200× objectives.

Article Snippet: Briefly, this involved antigen retrieval of FFPE sections in pH 6 buffer, immunolabelling with rabbit polyclonal antibody against ACE2 (Abcam) and Envision flex (Dako) and visualisation with DAB chromogen.

Techniques: Immunohistochemical staining

Journal: Bioscience Reports

Article Title: Sex differences in the lung ACE/ACE2 balance in hypertensive rats

doi: 10.1042/BSR20211201

Figure Lengend Snippet: The gene expression of ( A ) ACE and ( B ) ACE2 was quantitatively measured by RT-PCR and normalized to cyclophilin and is expressed as the % of male Wistar rats. ( C ) Lung ACE/ACE2 gene expression ratio was expressed as 2 −Δ C t /2 −Δ C t . The protein expression of ( D ) ACE and ( E ) ACE2 was determined by immunoblotting of lung membrane proteins and normalized to β-actin and is expressed as the % of male Wistar rats (Supplementary Figures S3 and S4). The values represent individual measurements and the means ± SEMs. * P <0.05; ** P <0.01; *** P <0.001.

Article Snippet: Then, the PVDF membranes were incubated with blocking solution (5% nonfat dry milk or 5% bovine serum albumin and 0.1% Tween 20 in PBS, pH 7.4) for 1 h and overnight (4°C) with specific primary antibodies: a rabbit monoclonal antibody against ACE (1:1000; ab254222, Abcam, Cambridge, MA), a rabbit polyclonal antibody against ACE2 (1:1000; ab15348, Abcam) or a mouse monoclonal antibody against β-actin (1:5000; ab6276, Abcam).

Techniques: Gene Expression, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Membrane

Journal: Bioscience Reports

Article Title: Sex differences in the lung ACE/ACE2 balance in hypertensive rats

doi: 10.1042/BSR20211201

Figure Lengend Snippet: The activities of ( A ) ACE and ( B ) ACE2 were assayed by a fluorogenic method. ( C ) Lung ACE/ACE2 activity ratio. Lung ( D ) Ang II and ( E ) Ang-(1-7) concentrations were determined by ELISA. ( F ) Lung Ang II/Ang-(1-7) peptide concentration ratio. The values represent individual measurements and the means ± SEMs. * P <0.05; ** P <0.01; *** P <0.001.

Article Snippet: Then, the PVDF membranes were incubated with blocking solution (5% nonfat dry milk or 5% bovine serum albumin and 0.1% Tween 20 in PBS, pH 7.4) for 1 h and overnight (4°C) with specific primary antibodies: a rabbit monoclonal antibody against ACE (1:1000; ab254222, Abcam, Cambridge, MA), a rabbit polyclonal antibody against ACE2 (1:1000; ab15348, Abcam) or a mouse monoclonal antibody against β-actin (1:5000; ab6276, Abcam).

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Bioscience Reports

Article Title: Sex differences in the lung ACE/ACE2 balance in hypertensive rats

doi: 10.1042/BSR20211201

Figure Lengend Snippet: Serum ( A ) ACE and ( B ) ACE2 concentrations were determined by ELISA. The values represent individual measurements and the means ± SEMs. ** P <0.01 and *** P <0.001. Correlations between the serum ( C ) ACE and ( D ) ACE2 concentrations and tail-cuff blood pressure. The correlation coefficients ( r ) and P -values were obtained by Pearson’s correlation test, and lines were obtained by linear regression plotting.

Article Snippet: Then, the PVDF membranes were incubated with blocking solution (5% nonfat dry milk or 5% bovine serum albumin and 0.1% Tween 20 in PBS, pH 7.4) for 1 h and overnight (4°C) with specific primary antibodies: a rabbit monoclonal antibody against ACE (1:1000; ab254222, Abcam, Cambridge, MA), a rabbit polyclonal antibody against ACE2 (1:1000; ab15348, Abcam) or a mouse monoclonal antibody against β-actin (1:5000; ab6276, Abcam).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Bioscience Reports

Article Title: Sex differences in the lung ACE/ACE2 balance in hypertensive rats

doi: 10.1042/BSR20211201

Figure Lengend Snippet: ( A ) Lung ADAM17 activity was determined by a fluorescent enzymatic assay in the presence or absence of TAPI 0. The TAPI 0 sensitive enzymatic activity was expressed as RFUs/µg/h. The values represent individual measurements and the means ± SEMs. The absence of a correlation between lung ADAM17 activity and ( B ) lung ACE2 protein or ( C ) serum ACE2 concentration. The correlation coefficients ( r ) and P -values were obtained by Pearson’s correlation test, and lines were obtained by linear regression plotting.

Article Snippet: Then, the PVDF membranes were incubated with blocking solution (5% nonfat dry milk or 5% bovine serum albumin and 0.1% Tween 20 in PBS, pH 7.4) for 1 h and overnight (4°C) with specific primary antibodies: a rabbit monoclonal antibody against ACE (1:1000; ab254222, Abcam, Cambridge, MA), a rabbit polyclonal antibody against ACE2 (1:1000; ab15348, Abcam) or a mouse monoclonal antibody against β-actin (1:5000; ab6276, Abcam).

Techniques: Activity Assay, Enzymatic Assay, Concentration Assay

Journal: Bioscience Reports

Article Title: Sex differences in the lung ACE/ACE2 balance in hypertensive rats

doi: 10.1042/BSR20211201

Figure Lengend Snippet: ( A ) Correlation between serum ACE concentration and lung ACE mRNA level. ( B ) The absence of a correlation between serum ACE concentration and lung ACE protein abundance. ( C ) The absence of a correlation between lung ACE protein abundance and lung ACE mRNA level. ( D ) The absence of a correlation between serum ACE2 concentration and lung ACE2 mRNA level. ( E ) The absence of a correlation between serum ACE2 abundance and lung ACE2 protein abundance. ( F ) Correlation between lung ACE2 protein abundance and lung ACE2 mRNA level. The correlation coefficients ( r ) and P -values were obtained by Pearson’s correlation test, and lines were obtained by linear regression plotting.

Article Snippet: Then, the PVDF membranes were incubated with blocking solution (5% nonfat dry milk or 5% bovine serum albumin and 0.1% Tween 20 in PBS, pH 7.4) for 1 h and overnight (4°C) with specific primary antibodies: a rabbit monoclonal antibody against ACE (1:1000; ab254222, Abcam, Cambridge, MA), a rabbit polyclonal antibody against ACE2 (1:1000; ab15348, Abcam) or a mouse monoclonal antibody against β-actin (1:5000; ab6276, Abcam).

Techniques: Concentration Assay, Quantitative Proteomics

Journal: Bioscience Reports

Article Title: Sex differences in the lung ACE/ACE2 balance in hypertensive rats

doi: 10.1042/BSR20211201

Figure Lengend Snippet: Normalized ( A ) ACE mRNA and ( B ) ACE2 mRNA expression data and the ( C) ACE to ACE2 mRNA ratio from normotensive and hypertensive patients whose lung samples were available in the GTEx database. Boxplots show the minimum, first quartile, median, third quartile, and maximum values. Each dot represents an individual data point.

Article Snippet: Then, the PVDF membranes were incubated with blocking solution (5% nonfat dry milk or 5% bovine serum albumin and 0.1% Tween 20 in PBS, pH 7.4) for 1 h and overnight (4°C) with specific primary antibodies: a rabbit monoclonal antibody against ACE (1:1000; ab254222, Abcam, Cambridge, MA), a rabbit polyclonal antibody against ACE2 (1:1000; ab15348, Abcam) or a mouse monoclonal antibody against β-actin (1:5000; ab6276, Abcam).

Techniques: Expressing