Journal: Dyes and Pigments

Article Title: Potent inhibition of Severe Acute Respiratory Syndrome Coronavirus 2 by photosensitizers compounds

doi: 10.1016/j.dyepig.2021.109570

Figure Lengend Snippet: Screening of two photosensitizers by SARS-CoV-2 pseudoviruses entry assay. (A) Dose-responses of the inhibition of viral infectivity were also measured for photosensitizer ZnPc5K and ce6, giving the EC 50 and EC 90 . The photosensitizers completely inhibited the viral infectivity at 1 μM concentration upon LED illumination. The EGFP intensities of all fields in a microplate well at different concentrations were summed to represent the virus infected into the cells. Error bars represent the SD of duplicates in one experiment. Scale bar, 500 μm. (B) Photocytotoxicity of ZnPc5k and ce6 on hACE2-293T cells was measured by CCK-8 assay and showed almost no phototoxicity up to 5 μM with a light dose of 0.48 J/cm 2 . (C) Photosensitizers inactivated the SARS-CoV-2 pseudoviruses and reduced virus load inside ACE2-293T cells upon LED illumination. Serially diluted ZnPc5K or ce6 and pseudoviruses were incubated with ACE2-293T cells for 4 h, and illuminated by LED to a light dose of 0.48 J/cm 2 . After further 44 h incubation, the fluorescent images (green for EGFP for virus and blue for Hoechst for nucleus staining) were recorded and analyzed. Error bars represent the SD of triplicates in one experiment. Scale bar, 500 μm.

Article Snippet: The pLVX-hACE2 plasmid was constructed by cloning the coding region from Human ACE2 cDNA ORF Clone with N-terminal Flag tag (SinoBiological, HG10108-NF, China) into pLVX-IRES-Puro Lentiviral vector between Xho I and Not I sites.

Techniques: Inhibition, Infection, Concentration Assay, Virus, CCK-8 Assay, Incubation, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: Rosiglitazone treatment restores renal responsiveness to atrial natriuretic peptide in rats with congestive heart failure

doi: 10.1111/jcmm.14366

Figure Lengend Snippet: Probes used for validation of PPARgamma‐regulated gene targets

Article Snippet: Ace2 , Angiotensin I converting enzyme 2 , Rn01416293_m1.

Techniques: Biomarker Discovery, Membrane

Journal: Journal of Cellular and Molecular Medicine

Article Title: Rosiglitazone treatment restores renal responsiveness to atrial natriuretic peptide in rats with congestive heart failure

doi: 10.1111/jcmm.14366

Figure Lengend Snippet: Summary of ANP signalling‐related gene expression altered in the renal tissue of CHF rats compared with controls

Article Snippet: Ace2 , Angiotensin I converting enzyme 2 , Rn01416293_m1.

Techniques: Gene Expression, Expressing, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Rosiglitazone treatment restores renal responsiveness to atrial natriuretic peptide in rats with congestive heart failure

doi: 10.1111/jcmm.14366

Figure Lengend Snippet: Summary of ANP signalling‐related gene expression altered in the renal tissue of CHF rats treated with RGZ compared with the vehicle

Article Snippet: Ace2 , Angiotensin I converting enzyme 2 , Rn01416293_m1.

Techniques: Gene Expression, Expressing

Journal: Small Methods

Article Title: Virus‐Free and Live‐Cell Visualizing SARS‐CoV‐2 Cell Entry for Studies of Neutralizing Antibodies and Compound Inhibitors

doi: 10.1002/smtd.202001031

Figure Lengend Snippet: Establishment of the CSBT and CRBT assays. A) Schematics of the constructs of ACE2hR and ACE2iRb3 for generations of ACE2‐overexpressing cell lines. EF1αp, human EF‐1 alpha promoter; hACE2, human ACE2; IRES, internal ribosome entry site; H2BmRb3, H2B‐fused mRuby3; BsR, blasticidin S‐resistance gene; 2A, P2A peptide; ins, insulator; hCMVmie, a modified CMV promoter derived from pEE12.4 vector; hACE2‐mRb3, human ACE2 with C‐terminal fusing of mRuby3; H2BiRFP, H2B‐fused iRFP670; PuR, puromycin resistance gene. B) Western blot analyses of expressions of ACE2 in 293T and H1299 cells stably transfected with different constructs. NT cell, nontransfected cells. C) Fluorescence confocal images of 293T‐ACE2iRb3 cells incubated with SARS‐CoV2‐RBG and SARS‐CoV2‐STG for different times. The nucleus H2B‐iRFP670 was pseudocolored blue. The scale bar was 10 µm. D) Schematic illustration of the procedures of cell‐based high‐content imaging assay using fluorescent RBG or STG viral entry sensors. E) Dose‐dependent fluorescence responses (cMFI) of various probes derived from different CoVs on 293T‐ACE2iRb3 cells. SARS‐CoV2‐RBD488 was a dylight488‐conjugated SARS‐CoV2‐RBD protein, and SARS‐CoV2‐ST488 was a dylight488‐conjugated SARS‐CoV2‐ST protein. Each probe was tested at 500, 250, 125, 62.5, and 31.25 × 10 −9 m , respectively. F) Comparisons of the fluorescence response (cMFI) of various SARS‐CoV‐2 probes on 293T‐ACE2iRb3 cells. For panels (E) and (F), cell images were obtained for 25 different views for each test, and the data were expressed as mean ± SD. G) Dose‐dependent cMFI inhibition of recombinant ACE2, SARS‐CoV2‐RBD, and SARS‐CoV2‐S1 proteins for the binding and uptake of SARS‐CoV2‐STG (upper panel) and SARS‐CoV2‐RBG (lower panel). The experiments were performed following the procedure as described in panel (D). The data were mean ± SD. CSBT, cell‐based spike function blocking test; CRBT, cell‐based RBD function blocking test.

Article Snippet: The ACE2 cDNA fragment (Sino Biological, HG10108‐ACG) linking with an IRES‐H2BmRuby3‐P2A‐BsR DNA fragment (synthesized by Generalbiol, Anhui, China) was cloned in‐frame into the XbaI/SalI sites of pLV‐EF1α‐MCS‐IRES‐Bsd to obtain the pLVEF1αIHRB‐ACE2hR vector.

Techniques: Construct, Modification, Derivative Assay, Plasmid Preparation, Western Blot, Stable Transfection, Transfection, Fluorescence, Incubation, Imaging, Inhibition, Recombinant, Binding Assay, Blocking Assay

Journal: Small Methods

Article Title: Virus‐Free and Live‐Cell Visualizing SARS‐CoV‐2 Cell Entry for Studies of Neutralizing Antibodies and Compound Inhibitors

doi: 10.1002/smtd.202001031

Figure Lengend Snippet: The 83H7 mAb inhibits SARS‐CoV‐2 via the intracellular neutralization pathway. The 293T‐ACE2iRb3 cells were incubated with 20 × 10 −9 m of dylight633‐labeled mAbs (Ab633) of 36H6, 53G2, 83H7, and 8H6 and an irrelevant control antibody (ctrAb), in the presence or absence of STG (2.5 × 10 −9 m ). Live‐cell fluorescence image dynamically tracked using a 63× water immersion objective. Five replicate wells were measured for each group, and 16 fields of each well were imaged. Time‐series (at 10 min, 1 h, 2 h, 3 h, 5 h, 7 h, 9 h, 11 h, and 13 h) analyses of the A) STG‐IVNs, B) STG‐IVpMFI, C) Ab633‐IVNs, and D) Ab633‐IVpMFI and E) the percentage of STG/Ab633 colocalized vesicles to total internalized STG vesicles. IVNs, average internalized vesicle numbers; IVpMFI, the average peak MFI of internalized vesicles. F) Comparisons of the STG‐IVA of the internalized STG vesicles among groups co‐incubated with various mAbs at 5 h postincubation. ** indicates p < 0.01; IVA, average area ( px 2 ) of internalized STG vesicles. G) Confocal images of STG (green channel), Ab633 (red channel), and ACE2‐mRuby3 (white channel) in 293T‐ACE2iRb3 cells at 5 h post STG/Ab633 coincubation. Scale bar, 20 µm.

Article Snippet: The ACE2 cDNA fragment (Sino Biological, HG10108‐ACG) linking with an IRES‐H2BmRuby3‐P2A‐BsR DNA fragment (synthesized by Generalbiol, Anhui, China) was cloned in‐frame into the XbaI/SalI sites of pLV‐EF1α‐MCS‐IRES‐Bsd to obtain the pLVEF1αIHRB‐ACE2hR vector.

Techniques: Neutralization, Incubation, Labeling, Fluorescence

Journal: Small Methods

Article Title: Virus‐Free and Live‐Cell Visualizing SARS‐CoV‐2 Cell Entry for Studies of Neutralizing Antibodies and Compound Inhibitors

doi: 10.1002/smtd.202001031

Figure Lengend Snippet: Detection of compound‐induced influence on SARS‐CoV‐2 S‐mediated cellular entry. A) Schematic summary of the possible mechanisms of 11 compound inhibitors involved in the study. CytD, cytochalasin D; MDC, dansylcadaverine; Baf.A1, bafilomycin A1; vRNA, viral RNA. B) Dose‐dependent inhibitions of 11 compounds against SARS‐CoV‐2 LVpp infection on H1299‐ACE2hR cells. All compounds were tested in a 2‐fold dilution series, and the initial drug concentrations were begun at their maximal noncytotoxic concentrations. The initial concentrations were 200 × 10 −6 m for amiloride, MDC and DMSO (as a solvent control); 100 × 10 −6 m for dynasore; 10 × 10 −6 m for filipin, APY0201, YM201636, and tetrandrine; 4 × 10 −6 m for nystatin; 100 × 10 −9 m for Baf.A1 and apilimod. ND, not detected. C) Confocal images of STG (green channel), ACE2‐mRuby3 (red channel), and nucleus (blue channel) in 293T‐ACE2iRb3 cells at 5 h post STG incubation. The cells were pretreated with compounds for 1 h before STG loading. These pictures were obtained by using Leica gSTED confocal microscopy on cells treated with compounds at their respective initial concentrations as above‐mentioned. Scale bar, 10 µm. D) Quantitative analysis of the influence of entry inhibitors on STG internalization. Dose‐dependent influence of various compounds on STG internalization characteristics on 293T‐ACE2iRb3 cells at 1 h (left panels) and 5 h (right panels) after incubation. All compounds were tested in a 4‐fold dilution series (4 gradients for DMSO control, and 5 gradients for others), and the initial drug concentrations were identical with as (B). Three replicate wells were measured for each group, and 16 fields of each well were imaged. For each compound, 5 colored bars from left‐to‐right orderly displayed the values measured from cells treated with 4‐fold serial high‐to‐low concentrations of compounds. STG‐IFR, internalized STG fluorescence intensity ratio; STG‐IVA, average area (µm 2 ) of internalized STG vesicles; STG‐IVNs, average numbers of internalized STG vesicles per cell; * p < 0.05.

Article Snippet: The ACE2 cDNA fragment (Sino Biological, HG10108‐ACG) linking with an IRES‐H2BmRuby3‐P2A‐BsR DNA fragment (synthesized by Generalbiol, Anhui, China) was cloned in‐frame into the XbaI/SalI sites of pLV‐EF1α‐MCS‐IRES‐Bsd to obtain the pLVEF1αIHRB‐ACE2hR vector.

Techniques: Infection, Incubation, Confocal Microscopy, Fluorescence

Journal: Journal of Molecular and Cellular Cardiology

Article Title: Sex- and age-specific regulation of ACE2: Insights into severe COVID-19 susceptibility

doi: 10.1016/j.yjmcc.2021.11.003

Figure Lengend Snippet: Assessment of ACE2 and TMPRSS2 across sex and aging. A. ACE2 mRNA expression ( n = 36, biological replicates), protein levels ( n = 11, biological replicates), and activity (n = 36, biological replicates) are positively correlated across all organs in male and female animals (B . ). C. TMPRSS2 mRNA (n = 36, biological replicates) and protein levels (n = 11, biological replicates). Data from A-C was generated from C57BL6/J mice using a pooled analysis of males and females. Levels are compiled from all sexes and age groups for expression and activity; however, protein levels are compiled from representative and three replicate gels sampled from a subset of all age groups (Supplementary Fig. S1). All organs were run on the same gel for comparison. Immunoblots were over-exposed (O.E.) to visualize low levels in the lung and heart. Expression and activity data were run one time in the same plate for quantification. Data are represented as median with interquartile range (IQR) and were analyzed as independent samples with the Kruskal-Wallis test with pairwise comparison adjusted by Bonferroni correction; * indicates differences from the heart; # indicates differences from the lung; and † indicates differences from the kidney. * p < 0.05; ** p < 0.01, *** p < 0.001 where the number of symbols indicates the level of significance. Analysis of mRNA expression (D.), protein levels (E.), and ACE2 activity (F.) of ACE2 and TMPRSS2 across sex and age in the heart (magenta), lung (green), kidney (yellow), and small intestine (SI) (blue) ( n = 6, biological replicates/group). ACE2 global knockout mice were analyzed as a negative control (KO) for western blots. Raw images are provided in Supplementary Fig. S2-S3. Data are represented as the mean ± SEM and analyzed by two-way ANOVA with Tukey post hoc test (mRNA and activity), or unpaired student's t- test (protein levels). For all experiments, young animals ( n = 12) were obtained from four litters and each litter was collected over two days. Adult and aged animals (n = 12 /group) were obtained from five and three litters respectively, and each litter was collected over two days. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Real-time quantitative PCR was performed with TaqMan premixed assays (ThermoFisher Scientific) for gene expression in mice for TMPRSS2 (Mm00443687_m1) and ACE2 (Mm01159009_m1), and in humans for ACE2 (Hs00222343_m1).

Techniques: Expressing, Activity Assay, Generated, Comparison, Western Blot, Knock-Out, Negative Control

Journal: Journal of Molecular and Cellular Cardiology

Article Title: Sex- and age-specific regulation of ACE2: Insights into severe COVID-19 susceptibility

doi: 10.1016/j.yjmcc.2021.11.003

Figure Lengend Snippet: Sex- and age-differences in ACE2 levels in mouse and human hearts. A. Representative immunoblot of mouse hearts for ACE2 protein levels ( n = 6 biological replicates/group). Three gels were compiled for quantification (Supplementary Fig. S4). Data are represented as the mean ± SEM and were analyzed by two-way ANOVA with Tukey post hoc test; * p < 0.05; ** p < 0.01, *** p < 0.001. For all experiments, young animals ( n = 12) were obtained from four litters and each litter was collected over two days. Adult and aged animals ( n = 12 /group) were obtained from five and three litters respectively, and each litter was collected over two days. B. Immunofluorescence of ACE2 (green) and pericyte marker NG2 (red). Wheat Germ Agglutinin (WGA) (blue) was used to delineate the cell membrane. Qualitative images were captured from n = 2 biological replicates and n = 16 (8 images/animal) technical replicates for each age group. C. Representative immunoblot for ACE2 in human hearts ( n = 6, biological replicates/group). Two gels were compiled for quantification (Supplementary Fig. S4). Quantification represents the combined result from two western blots. Data are represented as the mean ± SEM and were analyzed by two-way ANOVA with Tukey post hoc test. * indicates differences from young males, and # indicates differences between aged males and aged females; * p < 0.05; ** p < 0.01, *** p < 0.001 where the number of symbols indicates the level of significance. D. Immunofluorescence of ACE2 (green) and NG2 (red). WGA (blue) was used to delineate the cell membrane. Qualitative images were captured from n = 2 biological replicates and n = 16 (8 images/donor) technical replicates for each age group. E. ACE2 protein levels and activity are positively correlated, but not mRNA expression. * indicates differences from young males, and # indicates differences between aged males and aged females. * p < 0.05; ** p < 0.01, *** p < 0.001 where the number of symbols indicates the level of significance. F. Schematic figure showing the multi-organ impact of sex and aging on ACE2 levels as a potential contributor to the increased male susceptibility to severe COVID-19. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Real-time quantitative PCR was performed with TaqMan premixed assays (ThermoFisher Scientific) for gene expression in mice for TMPRSS2 (Mm00443687_m1) and ACE2 (Mm01159009_m1), and in humans for ACE2 (Hs00222343_m1).

Techniques: Western Blot, Immunofluorescence, Marker, Membrane, Activity Assay, Expressing

Journal: Journal of Molecular and Cellular Cardiology

Article Title: Sex- and age-specific regulation of ACE2: Insights into severe COVID-19 susceptibility

doi: 10.1016/j.yjmcc.2021.11.003

Figure Lengend Snippet:

Article Snippet: Real-time quantitative PCR was performed with TaqMan premixed assays (ThermoFisher Scientific) for gene expression in mice for TMPRSS2 (Mm00443687_m1) and ACE2 (Mm01159009_m1), and in humans for ACE2 (Hs00222343_m1).

Techniques: