Journal: PLoS ONE

Article Title: Dysregulation of microRNAs and renin-angiotensin system in high salt diet-induced cardiac dysfunction in uninephrectomized rats

doi: 10.1371/journal.pone.0180490

Figure Lengend Snippet: 60 μg of protein was loaded per each lane. To ensure the correct position of the protein of interest, a pre-stained protein marker (Invitrogen Novex ® Sharp Pre-stained Protein Standard, Cat # LC5800, Thermo Fisher Scientific) was run along with the samples in the gel. The results were expressed as -fold change over control rats. A) Immunoblots of heart. B) Quantification of western blots of SERCA2 (normalized with actin), p-AKT1 (Ser 473) (normalized with AKT) p-PTEN (Ser 380) (normalized with actin) p-AMPK (normalized with AMPK). C) Immunoblot of renal ACE2 (normalized with actin). D), E) Relative levels of miR-25, miR-155, miR-451 and miR-99b in heart normalized with sn-RNU5G; F) Relative levels of miR-25, miR-451 and miR-99b in plasma normalized with miR-30e. N = 3–5.*P<0.05, **P<0.01, ***P<0.001 vs. Ctrl; S P<0.05, SS P<0.01, SSS P<0.001 vs. HSD; U P<0.05, UU P<0.01, UUU P<0.001 vs. UNX.

Article Snippet: These were then electrotransferred to nitrocellulose membranes and were incubated with below mentioned antibodies—ACE2 (rabbit pAb, Cat# sc-20998), p-AMPK (rabbit pAb, sc-33524), AMPK (rabbit pAb, sc-25792), p-AKT (Rabbit pAb, SAB4300042), AKT (rabbit pAb, SAB4500802), actin (goat pAb, sc-1616, SantaCruz Biotechnology, Inc., CA, USA), SERCA = ATP2A2 (rabbit pAb, Cat# HPA062605-100UL, Sigma-Aldrich, MO, USA).

Techniques: Staining, Marker, Control, Western Blot, Clinical Proteomics

Journal: bioRxiv

Article Title: SARS-CoV-2 spike downregulates tetherin to enhance viral spread

doi: 10.1101/2021.01.06.425396

Figure Lengend Snippet: A) HeLa cells were transduced with ACE2 lentivirus to generate stable cell lines. Mock and ACE2 transduced cells were lysed and immunoblotted for ACE2. Tubulin was used as a loading control. (B) HeLa WT +ACE2 cells were infected with SARS-CoV-2 (MOI 0.5). Cells were fixed at 24 hpi and stained for spike (green) and DAPI (blue). (C) HeLa WT +ACE2 cells were infected with SARS-CoV-2 (MOI 0.5). Cells were fixed at 24 hpi and stained for spike (green), tetherin (red) and DAPI (blue). Uninfected cells shown with asterisk. (D) Electron micrographs showing plasma membrane associated SARS-CoV-2 virions and virus filled intracellular organelles. SARS-CoV-2 infected HeLa WT +ACE2 cells (MOI 0.5) were fixed at 24 hpi and processed for TEM. Left micrograph – plasma membrane-associated virus, middle micrograph – virus-filled tubulovesicular compartments are directed towards the plasma membrane, right micrograph – virions within DMVs. (E) Surface immunogold electron microscopy of SARS-CoV-2 infected HeLa WT +ACE2 cells. Cells were infected with SARS-CoV-2 (MOI 0.5), fixed at 24 hpi and immunogold labelled with antibodies against tetherin. (F) As (E) but labelled with antibodies against SARS-CoV-2 spike.

Article Snippet: Primary antibodies used in the study were: FLAG rat anti-DYKDDDDK (L5) (BioLegend, WB 1:1000, IF 1:200); rabbit anti-HA antibody (Cell Signalling, C29F4); rat anti-HA antibody (Roche, 3F10); rabbit monoclonal anti-tetherin antibody (Abcam, ab243230, WB 1:2000, IF 1:400, surface EM 1:200); Spike mouse anti-SARS-CoV-2 Spike antibody 1A9 (GeneTex, GTX632604, WB 1:1000, IF 1:300); rabbit anti-TGN46 (abcam, ab50595, 1:300); rabbit anti-ZFPL1 (Sigma-Aldrich, HPA014909, 1:500); rabbit anti-Beta2microglobulin (Dako 1:500); ACE2 (proteintech, 66699, WB 1:1000), Phycoerythrin-conjugated anti-human tetherin antibody (BioLegend, RS38E, FC 1:50), StrepMAB-Classic (IBA LifeSciences, IF 1:500), StrepMAB-Classic DY-549 (IBA LifeSciences, FC 1:500).

Techniques: Transduction, Stable Transfection, Control, Infection, Staining, Clinical Proteomics, Membrane, Virus, Electron Microscopy

Journal: bioRxiv

Article Title: SARS-CoV-2 spike downregulates tetherin to enhance viral spread

doi: 10.1101/2021.01.06.425396

Figure Lengend Snippet: (A) A549 cells were transduced with ACE2 lentivirus to generate stable cell lines. Mock and ACE2 transduced cells were lysed and immunoblotted for ACE2. Tubulin served as a loading control. (B) A549+ACE2 cells were infected with SARS-CoV-2 (MOI 0.5). Cells were fixed at 24 hpi and stained for spike (green) and DAPI (blue). Uninfected cells shown with asterisk (C) A549+ACE2 cells were treated with IFNα (1000 U/ml, 24 hours) to upregulate tetherin expression. Cells were infected with SARS-CoV-2 (MOI 0.5). Cells were fixed at 24 hpi and stained for spike (green), tetherin (red) and DAPI (blue). Uninfected cells shown with asterisk. (D) A549+ACE2 cells were treated with IFNα (1000 U/ml) and infected with SARS-CoV-2 (MOI 0.5), fixed at 24 hpi and processed for TEM. Infected cells display very few virions on their plasma membrane (left inset) but significant DMV formation (right inset). (E) Electron micrograph of the perinuclear region of A549+ACE2 mock infected cells. Zoomed area shows typical Golgi morphology. (F) Electron microscopy of the perinuclear region of SARS-CoV-2 infected A549+ACE2 cells. Cells were infected at an MOI of 0.5 and fixed at 24 hpi. Zoomed area shows membrane rearrangements and DMVs. (G) T84 cells were infected with SARS-CoV-2 (MOI 0.5) and fixed at 24 hpi and stained for spike (green) and tetherin (red). (H) T84 cells were infected with SARS-CoV-2 (MOI 0.5) and fixed at 24 hpi and processed for TEM. Tethered virions were frequently present at the plasma membrane.

Article Snippet: Primary antibodies used in the study were: FLAG rat anti-DYKDDDDK (L5) (BioLegend, WB 1:1000, IF 1:200); rabbit anti-HA antibody (Cell Signalling, C29F4); rat anti-HA antibody (Roche, 3F10); rabbit monoclonal anti-tetherin antibody (Abcam, ab243230, WB 1:2000, IF 1:400, surface EM 1:200); Spike mouse anti-SARS-CoV-2 Spike antibody 1A9 (GeneTex, GTX632604, WB 1:1000, IF 1:300); rabbit anti-TGN46 (abcam, ab50595, 1:300); rabbit anti-ZFPL1 (Sigma-Aldrich, HPA014909, 1:500); rabbit anti-Beta2microglobulin (Dako 1:500); ACE2 (proteintech, 66699, WB 1:1000), Phycoerythrin-conjugated anti-human tetherin antibody (BioLegend, RS38E, FC 1:50), StrepMAB-Classic (IBA LifeSciences, IF 1:500), StrepMAB-Classic DY-549 (IBA LifeSciences, FC 1:500).

Techniques: Transduction, Stable Transfection, Control, Infection, Staining, Expressing, Clinical Proteomics, Membrane, Electron Microscopy

Journal: bioRxiv

Article Title: SARS-CoV-2 spike downregulates tetherin to enhance viral spread

doi: 10.1101/2021.01.06.425396

Figure Lengend Snippet: (A) Lentiviral ACE2 was used to generate stable HeLa Bst2KO +ACE2 cells, and ACE2 expression was verified by Western blotting. GAPDH served as a loading control. (B) HeLa WT +ACE2 and HeLa Bst2KO +ACE2 cells were infected with SARS-CoV-2 (MOI 0.5) and fixed at 24 hpi. Cells were stained for spike (green) to demonstrate infection with SARS-CoV-2, and tetherin (red). (C) High MOI viral growth curves were performed by infecting HeLa WT +ACE2 and HeLa Bst2KO +ACE2 cells with SARS-CoV-2 at MOI (5). Titres were measured by plaque assays. (D) Low MOI viral growth curves were performed by infecting HeLa WT +ACE2 and HeLa Bst2KO +ACE2 cells with SARS-CoV-2 at MOI (1). Titres were measured by plaque assays.

Article Snippet: Primary antibodies used in the study were: FLAG rat anti-DYKDDDDK (L5) (BioLegend, WB 1:1000, IF 1:200); rabbit anti-HA antibody (Cell Signalling, C29F4); rat anti-HA antibody (Roche, 3F10); rabbit monoclonal anti-tetherin antibody (Abcam, ab243230, WB 1:2000, IF 1:400, surface EM 1:200); Spike mouse anti-SARS-CoV-2 Spike antibody 1A9 (GeneTex, GTX632604, WB 1:1000, IF 1:300); rabbit anti-TGN46 (abcam, ab50595, 1:300); rabbit anti-ZFPL1 (Sigma-Aldrich, HPA014909, 1:500); rabbit anti-Beta2microglobulin (Dako 1:500); ACE2 (proteintech, 66699, WB 1:1000), Phycoerythrin-conjugated anti-human tetherin antibody (BioLegend, RS38E, FC 1:50), StrepMAB-Classic (IBA LifeSciences, IF 1:500), StrepMAB-Classic DY-549 (IBA LifeSciences, FC 1:500).

Techniques: Expressing, Western Blot, Control, Infection, Staining

Journal: bioRxiv

Article Title: SARS-CoV-2 spike downregulates tetherin to enhance viral spread

doi: 10.1101/2021.01.06.425396

Figure Lengend Snippet: (A) Schematic diagram to illustrate the domain organization of SARS-CoV-1 ORF7a and SARS-CoV-2 ORF7a. SP = signal peptide. TM = transmembrane domain. Expanded region below shows regions flanking the transmembrane domain with amino acid differences (*) and deletions (-) between SARS-CoV-1 ORF7a and SARS-CoV-2 ORF7a. (B) Representative confocal immunofluorescence images of HeLa cells transiently transfected with SARS-CoV-1 ORF7a-FLAG or SARS-CoV-2 ORF7a-FLAG. SARS-CoV-1 ORF7a-FLAG predominately colocalizes with TGN46 (red), while SARS-CoV-2 ORF7a-FLAG shows additional staining outside that colocalizing with TGN46 (arrowheads). (C) Manders’ coefficients were calculated to measure the ORF7a-FLAG overlap with TGN46. n = 3 independent experiments. Means of each independent experiment are plotted. Two-tailed, unpaired t-tests were performed. *** p < 0.001. (D) SARS-CoV-2 infected HeLa WT +ACE2 cells display fragmentation of Golgi markers. HeLa WT +ACE2 cells were infected with SARS-CoV-2 (MOI 0.5) and fixed at 24 hpi. Infected cells were identified by spike staining (green) and cells were costained with Golgi markers TGN46 (top) and ZFPL1 (below). Areas of TGN46 and ZFPL1 are enlarged (right) to highlight Golgi fragmentation in SARS-CoV-2 infected cells.

Article Snippet: Primary antibodies used in the study were: FLAG rat anti-DYKDDDDK (L5) (BioLegend, WB 1:1000, IF 1:200); rabbit anti-HA antibody (Cell Signalling, C29F4); rat anti-HA antibody (Roche, 3F10); rabbit monoclonal anti-tetherin antibody (Abcam, ab243230, WB 1:2000, IF 1:400, surface EM 1:200); Spike mouse anti-SARS-CoV-2 Spike antibody 1A9 (GeneTex, GTX632604, WB 1:1000, IF 1:300); rabbit anti-TGN46 (abcam, ab50595, 1:300); rabbit anti-ZFPL1 (Sigma-Aldrich, HPA014909, 1:500); rabbit anti-Beta2microglobulin (Dako 1:500); ACE2 (proteintech, 66699, WB 1:1000), Phycoerythrin-conjugated anti-human tetherin antibody (BioLegend, RS38E, FC 1:50), StrepMAB-Classic (IBA LifeSciences, IF 1:500), StrepMAB-Classic DY-549 (IBA LifeSciences, FC 1:500).

Techniques: Immunofluorescence, Transfection, Staining, Two Tailed Test, Infection

Journal: Nutrients

Article Title: Hypertension Programmed by Perinatal High-Fat Diet: Effect of Maternal Gut Microbiota-Targeted Therapy

doi: 10.3390/nu11122908

Figure Lengend Snippet: Effect of perinatal high-fat (HF) diet, prebiotic inulin (PRE), and probiotic Lactobacillus casei (PRO) on the RAS components in male offspring at 16 weeks of age. Representative western blots, Ponceau red staining, and relative abundance of ( A ) angiotensin-converting enzyme 2 (ACE2, 90 kDa), ( B ) angiotensin type 1 receptor (AT1R, 43 kDa), ( C ) angiotensin type 2 receptor (AT2R, 50 kDa), and ( D ) angiotensin (1-7) receptor MAS (37 kDa) bands in offspring kidneys at 16 weeks of age. ( E ) Gene expression of renin-angiotensin system components Ren, Agt , and Ace in male offspring at 16 weeks of age. n = 8/group. * p < 0.05 versus CON; # p < 0.05 versus HF.

Article Snippet: We used the following antibodies: for ACE2, rabbit anti-rat ACE2 (1:1000, overnight incubation; Santa Cruz Biotechnology, Santa Cruz, California, USA); for AT1R, a rabbit anti-rat AT1R antibody (1:500, overnight incubation; AB15552, Millipore, Billerica, MA, USA); for AT2R, a rabbit anti-rat AT2R antibody (1:250, overnight incubation; sc-9040, Santa Cruz Biotechnology, Santa Cruz, CA, USA); and for angiotensin (1-7) receptor MAS, a rabbit anti-rat MAS antibody (1:1000, overnight incubation; Santa Cruz Biotechnology).

Techniques: Western Blot, Staining, Expressing

Journal: Cell Reports

Article Title: Limited extent and consequences of pancreatic SARS-CoV-2 infection

doi: 10.1016/j.celrep.2022.110508

Figure Lengend Snippet: Stringent ACE2 requirement for pancreatic islet cell infection with SARS-CoV-2 (A) Representative contour plots gated on live α, β, and “other” cells pre-treated with IgG (irrelevant polyclonal goat antibody AF7197) or the anti-ACE2 blocking antibody AF933 prior to SARS-CoV-2 infection (48 h). (B) Summary of SARS-CoV-2 NP expression by live islet cell subsets as a function of IgG treatment or ACE2 blockade (n = 6 donors). (C) Percent infection inhibition for β and “other” cells (inhibition for α cells is not shown because the very low extent of α cell infection in IgG-treated cultures for 2 of 6 donors substantially skews such calculations). (D) Infectious SARS-CoV-2 titers and extent of infection inhibition following ACE2 blockade (n = 3 donors). (E) Quantification of chemokines and cytokines in UV-inactivated TCS of SARS-CoV-2-infected islet cell cultures under conditions of IgG treatment or ACE2 blockade (48-h infection, n = 3 donors). (F) Infectious SARS-CoV-2 titers in TCS as a function of glucose concentration in islet culture medium (n = 3 donors). (G) Quantification of CXCL10 and CXCL11 in TCS as a function of glucose concentration. All summary bar diagrams represent mean ± SD and scatter for the indicated number of donors; statistical analyses were conducted by paired t test or repeated-measures ANOVA with Tukey’s multiple comparisons where applicable (∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001). All summary bar diagrams represent mean ± SD and scatter for the indicated number of donors; statistical analyses were conducted by paired t test or repeated-measures ANOVA with Tukey’s multiple comparisons where applicable.

Article Snippet: Anti-human ACE2 (polyclonal goat IgG Poly5036) - AF647 (in-house conjugation) , Biolegend , Cat# 503602; RRID: AB_2892475.

Techniques: Infection, Blocking Assay, Expressing, Inhibition, Concentration Assay

Journal: Cell Reports

Article Title: Limited extent and consequences of pancreatic SARS-CoV-2 infection

doi: 10.1016/j.celrep.2022.110508

Figure Lengend Snippet:

Article Snippet: Anti-human ACE2 (polyclonal goat IgG Poly5036) - AF647 (in-house conjugation) , Biolegend , Cat# 503602; RRID: AB_2892475.

Techniques: Conjugation Assay, Purification, Blocking Assay, Recombinant, Control, Virus, Saline, Modification, Staining, Library Quantification, Antibody Labeling, Flow Cytometry, Software, Cytometry, Sequencing

Journal: Nature Communications

Article Title: Morphogenesis and cytopathic effect of SARS-CoV-2 infection in human airway epithelial cells

doi: 10.1038/s41467-020-17796-z

Figure Lengend Snippet: a SARS-CoV-2 replication kinetics in HAE from different donors, HCoV-NL63 was used as a control ( n = 3). b Transepithelial electrical resistance (TEER in Ω cm 2 ) between the apical and basal poles was measured at each time point ( n = 3). c SARS-CoV-2 infected both ciliated cells (72 h pi) and secretory cells (72 h pi). arrows: virus particles, arrowhead: cilium, asterisk: secretory vesicle, insets dashed-line squares indicate magnification of arrowed areas. d Costaining of SARS-CoV-2 N protein (green) with ciliated cell marker β-tubulin-IV (red), goblet cell marker Muc5AC (red), club cell marker CCSP (red), and ACE2 (red) positive cells. HCoV-NL63 N protein (green) staining was used as a control (72 h pi). Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) (blue). Data a , b are the means ± s.d. of three independent biological replicates. Source data a – d are provided as a Source Data file.

Article Snippet: ACE2 , Sino biologicals (10108-T56), rabbit polyclona (1:100).

Techniques: Infection, Marker, Staining

Journal: Nature Communications

Article Title: Morphogenesis and cytopathic effect of SARS-CoV-2 infection in human airway epithelial cells

doi: 10.1038/s41467-020-17796-z

Figure Lengend Snippet: Source of antibodies and dyes with work concentration for immunofluorescence.

Article Snippet: ACE2 , Sino biologicals (10108-T56), rabbit polyclona (1:100).

Techniques: Concentration Assay, Immunofluorescence