primary antibodies against ace2 nbpi-76614  (Novus Biologicals)

Journal: Frontiers in Immunology

Article Title: The purinergic receptor P2X7 and the NLRP3 inflammasome are druggable host factors required for SARS-CoV-2 infection

doi: 10.3389/fimmu.2023.1270081

Figure Lengend Snippet: NLRP3 inflammasome activation dictates permissiveness of epithelial cells to SARS-CoV-2 infection. (A-D) Caco-2 (A, B) or ACE2-A549 (C, D) cells were infected with SARS-CoV-2 for 48 hours at indicated MOI (A, C) or during indicated hours post-infection (hpi) (MOI = 4) (B, D) and IL-1β secretion was monitored. E ACE2-A549 cells were infected during 48 hours with SARS-CoV-2 or with SARS-CoV-2 neutralized with convalescent COVID-19 patient serum. IL-1β secretion was then monitored. (F, G) Caco-2 (F) and ACE2-A549 (G) cells were infected (or not) with SARS-CoV-2 (MOI = 2) during 48 hours in presence of indicated concentrations of Tranilast and evaluated for Spike (S) and β-actin expressions. (H, I) Caco-2 (H) or ACE2-A549 (I) cells were infected for 48 hours with SARS-CoV-2 (MOI = 0.2) in presence of 100 µM Tranilast, and analyzed for SARS-CoV-2 E RNA expression using quantitative RT-PCR. Fold changes (FC) are indicated. (J, K) ACE2-A549 cells were infected for 48 hours with SARS-CoV-2 (MOI = 2) in presence of 20 µM MCC950, 100 µM Tranilast or with SARS-CoV-2 neutralized with convalescent COVID-19 patient serum and analyzed for spike (S) expression by fluorescence microscopy. Representative images (bar, 20 µm) (J) and percentages of spike (S)-positive (S + ) cells (K) are shown. DNA is detected using Hoechst 33342. (L-O) Caco-2 (L, M) or ACE2-A549 (N, O) cells were subjected to siRNA against NLRP3 for 48 hours and analyzed for NLRP3 mRNA expression (L, N) or infected with SARS-CoV-2 for 48 hours and evaluated for SARS-CoV-2 RdRp RNA expression, by quantitative RT-PCR (M, O) . RT-PCR samples were first normalized by β-actin mRNA level in each sample, and then normalized to the control condition. The data are presented as means ± SEM from at least 3 independent experiments. p values (* p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001) were determined using one-way ANOVA Tukey’s multiple comparisons test (A-E, K) and unpaired t-test (H, I, L-O) .

Article Snippet: Primary antibodies against ACE2 (#AF933) and Spike (S) (#GTX632604) were from R&D Systems and Genetex, respectively.

Techniques: Activation Assay, Infection, RNA Expression, Quantitative RT-PCR, Expressing, Fluorescence, Microscopy, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Frontiers in Immunology

Article Title: The purinergic receptor P2X7 and the NLRP3 inflammasome are druggable host factors required for SARS-CoV-2 infection

doi: 10.3389/fimmu.2023.1270081

Figure Lengend Snippet: Caspase-1 activity regulates SARS-CoV-2 replication. (A-D) Caco-2 (A, C) or ACE2-A549 (B, D) were infected with SARS-CoV-2 (MOI = 2) in presence of 50 µM of caspase-1 inhibitor, Ac-YVAD-cmk (YVAD), for 48 hours and analyzed for spike (S) expression by fluorescence microscopy. Representative images (bar, 20 µm) (A, B) and percentages of spike (S)-positive (S + ) cells (C, D) are shown. DNA is detected using Hoechst 33342. (E-H) Caco-2 (E, F) and ACE2-A549 (G, H) were treated with siRNA against caspase-1 (CASP-1) for 48 hours and analyzed for CASP-1 mRNA quantification by RT-PCR (E, G) , or infected with SARS-CoV-2 at MOI = 0.2 (F) or MOI = 2 (H) during 48 hours, and analyzed for SARS-CoV-2 RdRp RNA expression by quantitative RT-PCR (F) or for spike (S) and β-actin expressions (H) . The data are presented as means ± SEM from at least 3 independent experiments. p values (* p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001) were determined using one-way ANOVA Tukey’s multiple comparisons test (C, D) and unpaired t-test (E-G) .

Article Snippet: Primary antibodies against ACE2 (#AF933) and Spike (S) (#GTX632604) were from R&D Systems and Genetex, respectively.

Techniques: Activity Assay, Infection, Expressing, Fluorescence, Microscopy, Reverse Transcription Polymerase Chain Reaction, RNA Expression, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: The purinergic receptor P2X7 and the NLRP3 inflammasome are druggable host factors required for SARS-CoV-2 infection

doi: 10.3389/fimmu.2023.1270081

Figure Lengend Snippet: Purinergic receptor P2X7 modulates SARS-CoV-2 replication through NLRP3 inflammasome activation. (A-D) Vero E6 (A, C) or ACE2-A549 (B, D) cells were infected with SARS-CoV-2 at MOI = 0.1 for 24 hours (A, C) or at MOI = 2 for 48 hours (B, D) , with 10 µM OxATP and 50 µM PPADS (A, C) or with 100 µM OxATP and 100 µM BzATP (B, D) and analyzed for spike (S) expression by fluorescence microscopy. Representative images (bar, 20 µm) (A, B) and percentages of spike (S)-positive (S + ) cells (C, D) are shown. DNA is detected using Hoechst 33342. (E) ACE2-A549 cells were infected with SARS-CoV-2 (MOI = 0.2) for 48 hours in presence of 100 µM OxATP and analyzed for SARS-CoV-2 E RNA expression by quantitative RT-PCR. (F) ACE2-A549 cells were treated with siRNA against P2X7 for 48 hours and analyzed for spike (S), P2X7 and β-actin expression. (G) ACE2-A549 cells were infected with SARS-CoV-2 (MOI = 0.2) for 48 hours in presence of 100 µM BzATP and analyzed for SARS-CoV-2 E RNA expression by quantitative RT-PCR. (H) Primary salivary gland epithelial cells were infected with SARS-CoV-2 (MOI = 0.5) for 48 hours in presence of 100 µM OxATP and analyzed for SARS-CoV-2 RdRp RNA expression by quantitative RT-PCR. (I) Huh7 cells were infected with SARS-CoV-2 Alpha variant in presence of 100 µM OxATP for 48 hours. Then, the viral yielding was determined by detecting RdRp RNA expression by quantitative RT-PCR in the supernatant (SN) of control and SARS-CoV-2 Alpha variant infected Huh7 cells. Fold changes (FC) are indicated in (E, G-I) . (J) ACE2-A549 cells were infected with SARS-CoV-2 during 48 hours (MOI = 2) in presence of control, 50 or 100 µM BzATP, 100 µM Tranilast, or the combination of either 50 µM BzATP and 100 µM Tranilast or 100 µM BzATP and 100 µM Tranilast, and analyzed for spike (S) and β-actin expression. All experiments are representative of at least 3 independent experiments. The data are presented as means ± SEM from at least 3 independent experiments. p values (* p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001) were determined using one-way ANOVA Tukey’s multiple comparisons test (C, D) and unpaired t-test (E, G-I ).

Article Snippet: Primary antibodies against ACE2 (#AF933) and Spike (S) (#GTX632604) were from R&D Systems and Genetex, respectively.

Techniques: Activation Assay, Infection, Expressing, Fluorescence, Microscopy, RNA Expression, Quantitative RT-PCR, Variant Assay, Control

Journal: Frontiers in Immunology

Article Title: The purinergic receptor P2X7 and the NLRP3 inflammasome are druggable host factors required for SARS-CoV-2 infection

doi: 10.3389/fimmu.2023.1270081

Figure Lengend Snippet: Purinergic receptor P2X7 and NLRP3 inflammasome control SARS-CoV-2 replication without affecting viral entry. (A-H) ACE2-TMPRSS2-A549 (A-G) or Vero E6 (H) cells were incubated during 4 hours with control (A, B) , 100 µM OxATP (C) , 100 µM BzATP (D) , and infected for 48 hours (B-F) or 4 hours (G, H) with green fluorescent protein (GFP)-tagged HIV-1NL4-3 Δ Env variant (defective in viral envelope) pseudotyped with the SARS-CoV-2 spike (S) envelope (S-GFP-LV) or convalescent COVID-19 serum neutralized S-GFP-LV (E-H) , and analyzed for GFP fluorescence by flow cytometry (A-F) or for intracellular HIV-1 CAp24 capsid and β-actin expression (G, H) . Representative flow cytometry analysis (A-E) and percentages of GFP + cells (F) are shown. I-P ACE2-TMPRSS2-A549 (I-O) or ACE2-A549 (P) cells were incubated during 4 hours with control (I, J) , 100 µM Tranilast (K) or 100 µM YVAD (L) and infected for 48 hours (J-L) or 4 hours (O, P) with S-GFP-LV (J-L, N-P) or with convalescent COVID-19 serum neutralized S-GFP-LV (M, N) . Then, cells were analyzed for GFP fluorescence by flow cytometry (I-N) or for intracellular HIV-1 CAp24 capsid and β-actin expression (O, P) . Representative flow cytometry analysis (I-M) and percentages of GFP + cells (N) are shown. In (P) , the asterisk (*) is indicating a non-specific band. The data are presented as means ± SEM from at least 3 independent experiments. p values (**** p < 0.0001) were determined using one-way ANOVA Tukey’s multiple comparisons test (F, N) .

Article Snippet: Primary antibodies against ACE2 (#AF933) and Spike (S) (#GTX632604) were from R&D Systems and Genetex, respectively.

Techniques: Control, Incubation, Infection, Variant Assay, Fluorescence, Flow Cytometry, Expressing

Journal: Cells

Article Title: Effects of Chronic Inflammatory Activation of Murine and Human Arterial Endothelial Cells at Normal Lipoprotein and Cholesterol Levels In Vivo and In Vitro

doi: 10.3390/cells13090773

Figure Lengend Snippet: ACE2 expression in human arterial endothelial cells upon inflammatory activation. Human arterial endothelial cells (HAECs) were treated for different periods of time with TNFα (50 ng/mL) or IL-1β (10 ng/mL), followed by RNA extraction. ( A ) RNA was reversed transcribed, and ACE2 mRNA was measured via quantitative PCR (mean ± SEM, n = 3, ANOVA was performed with GraphPad Prism 6.0 with Fisher’s LSD test between control and treated samples; p -values as indicated). ( B ) ACE2 protein levels were determined in HAECs treated as indicated by immunofluorescence staining and normalization of the staining intensity to the DNA staining (mean values ± SEM, n = 3; ANOVA and Fisher’s LSD test between treated samples and untreated control; * p < 0.05, ** p < 0.01, *** p < 0.0001).

Article Snippet: The cells were stained with primary rabbit monoclonal antibody against ACE2 (1:100, #MA5-32307, Invitrogen) and goat anti-rabbit Alexa Fluor 647 (1:500, #A21245, Invitrogen) antibody.

Techniques: Expressing, Activation Assay, RNA Extraction, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining