Journal: bioRxiv

Article Title: Vitamin C is an efficient natural product for prevention of SARS-CoV-2 infection by targeting ACE2 in both cell and in vivo mouse models

doi: 10.1101/2022.07.14.499651

Figure Lengend Snippet: (A) Western blot analysis of endogenous ACE2 in various types of cell lines, including HEK293T transfected with either control shRNAs (shCtrl) or shRNAs against ACE2 (shACE2), 2fTGH, Caco-2 and A549 cells. (B) HEK293T cells were transfected with shCtrl or increasing amounts of shACE2. Then cells were subjected to RT-qPCR analysis of Ace2 mRNA levels (right), or were infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) for 2 hrs. RT-qPCR was used to analyze SARS-CoV-2 RNA levels (left). (C) Western blot analysis of ACE2 in Caco-2 cells treated with vitamin (Vit) compounds (VitB1, 500 µM; VitB6, 500 µM; VitB12, 50 nM; VitC, 5 mM; VitD3, 25 µM; VitK1, 0.5 µM) for 24 hrs. (D) Western blot analysis of ACE2 in HEK293T, 2fTGH, Caco-2 and A549 cells treated with VitC at indicated concentrations for 24 hrs. (E) Western blot analysis of ACE2 in HEK293T cells treated with 5 mM or 0.2 mM of VitC for different durations. (F) Immunofluorescence analysis of ACE2 proteins in HeLa cells treated with VitC (5 mM) for 24 hrs. DAPI was used for the nucleus. Scale bars, 1 μm. (G) Western blot analysis of ACE2, IRF3 and STAT1 in A549 cells treated with VitC at indicated concentrations for 24 hrs. (H) Fluorescence microscopy of the SARS-CoV-2 GFP/ΔN or VSV-GFP viruses in Caco-2-N cells pretreated with VitC (5 mM and 10 mM) for 24 hrs, and then infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) or VSV-GFP (MOI = 0.1) viruses for 24 hrs. Scale bar: 100 µm. (I) RT-qPCR analysis of SARS-CoV-2 GFP/ΔN or VSV RNA levels in Caco-2 cells pretreated with VitC as (H), and then infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) or VSV (MOI = 0.1) for 2 hrs. Data are representative of three independent experiments (A, C-F), or are shown as mean and s.d. of three biological replicates (B, I). N.S, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S1.

Article Snippet: The antibodies with the indicated dilutions were as follows: anti-human ACE2 (Cell Signaling Technology, #74512, 1:1000), anti-ACE2 (Affinity, AF5165, 1:1000), anti-USP50 (Affinity, AF9225, 1:1000), anti-USP50 (Proteintech, 20374-1-AP, 1:1000), anti-Flag (Sigma, F7425, 1:5000), anti-HA (Abcam, ab9110, 1:3000), anti-Myc (Abmart, M20002H, 1:3000), anti-IRF3 (Santa Cruz, sc-33641, 1:1000), anti-STAT1 (Cell Signaling Technology, 9172, 1:1000), anti-Ubiquitin (Ub) (Santa Cruz, 12987-1-AP, 1:1000), anti-K48 Ub (Cell Signaling Technology, 4289S, 1:1000), anti-VSV-G (Santa Cruz, sc-66180, 1:2000) and anti-Tubulin (Proteintech, 66031-1-Ig, 1:3000).

Techniques: Western Blot, Transfection, Quantitative RT-PCR, Infection, Immunofluorescence, Fluorescence, Microscopy, Two Tailed Test

Journal: bioRxiv

Article Title: Vitamin C is an efficient natural product for prevention of SARS-CoV-2 infection by targeting ACE2 in both cell and in vivo mouse models

doi: 10.1101/2022.07.14.499651

Figure Lengend Snippet: (A) RT-qPCR analysis of Ace2 mRNA in 2fTGH cells treated with VitC (5 mM) as indicated. (B) Western blot analysis of ACE2 in 2fTGH cells pretreated with ddH 2 O (Ctrl) or VitC (5 mM) for 12 hrs and then treated with CHX (50 μM) for 6 and 12 hrs. (C) Western blot analysis of Myc-ACE2 levels in HEK293T cells transfected with Myc-ACE2 and then treated with VitC at indicated concentrations for 12 hrs. (D) Western blot analysis of ACE2 in HEK293T cells pretreated with MG132 (10 µM) or MA (10 µM) for 2 hrs, followed by VitC treatment (5 mM) for 6 hrs. (E) Immunoprecipitation (IP)-immunoblotting (IB) analysis of ubiquitination (Ub) of endogenous ACE2 in 2fTGH cells treated with VitC at indicated concentrations for 12 hrs. (F) IP-IB analysis of ubiquitination types of Myc-ACE2 in HEK293T cells cotransfected with Myc-ACE2 and different types of HA-Ub, and then treated with VitC (5 mM) for 12 hrs. (G) IP-IB analysis of K48-linked polyubiquitination (K48-Ub) of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 and then treated with VitC (2.5 mM and 5 mM) for 12 hrs, using a specific anti-K48-Ub antibody. (H) IP-IB analysis of K48-Ub of endogenous ACE2 in 2fTGH cells treated with VitC (2.5 mM and 5 mM) for 12 hrs. Data are representative of three independent experiments (B-H), or are shown as mean and s.d. of three biological replicates (A, B). N.S, not significant, ** p < 0.01 (two-tailed unpaired Student’s t -test). See also Figure S2.

Article Snippet: The antibodies with the indicated dilutions were as follows: anti-human ACE2 (Cell Signaling Technology, #74512, 1:1000), anti-ACE2 (Affinity, AF5165, 1:1000), anti-USP50 (Affinity, AF9225, 1:1000), anti-USP50 (Proteintech, 20374-1-AP, 1:1000), anti-Flag (Sigma, F7425, 1:5000), anti-HA (Abcam, ab9110, 1:3000), anti-Myc (Abmart, M20002H, 1:3000), anti-IRF3 (Santa Cruz, sc-33641, 1:1000), anti-STAT1 (Cell Signaling Technology, 9172, 1:1000), anti-Ubiquitin (Ub) (Santa Cruz, 12987-1-AP, 1:1000), anti-K48 Ub (Cell Signaling Technology, 4289S, 1:1000), anti-VSV-G (Santa Cruz, sc-66180, 1:2000) and anti-Tubulin (Proteintech, 66031-1-Ig, 1:3000).

Techniques: Quantitative RT-PCR, Western Blot, Transfection, Immunoprecipitation, Two Tailed Test

Journal: bioRxiv

Article Title: Vitamin C is an efficient natural product for prevention of SARS-CoV-2 infection by targeting ACE2 in both cell and in vivo mouse models

doi: 10.1101/2022.07.14.499651

Figure Lengend Snippet: (A) Western blot analysis of ACE2 in HEK293T cells pretreated with PR619 (50 µM, 2 hrs) and then treated with VitC (5 mM) for 12 hrs. (B) HEK293T cells were individually transfected with the plasmids from the human DUBs expression library. Western blot was used to identify the key deubiquitinase that significantly increases ACE2 levels. (C) IP-IB analysis of the interaction between Flag-USP50 and Myc-ACE2 in HEK293T cells cotransfected with these two constructs. (D) Immunoprecipitation analysis of the interaction between endogenous USP50 and ACE2 in 2fTGH cells. (E) Western blot analysis of ACE2 in HEK293T cells transfected with increasing amounts of Flag-USP50. (F) Western blot analysis of ACE2 in HEK293T cells transfected with shCtrl or shUSP50 (#1 or #2). (G) Western blot analysis of ACE2 in HEK293T cells transfected with Flag-USP50 and then treated with CHX (50 μM) as indicated. (H) Western blot analysis of ACE2 in Usp50 +/+ and Usp50 -/- HEK293T cells. (I) Western blot analysis of ACE2 in Usp50 +/+ and Usp50 -/- HEK293T cells treated with VitC (5 mM) for 12 hrs. (J) RT-qPCR analysis of SARS-CoV-2 GFP/ΔN RNA levels in HEK293T cells transfected with Flag-USP50 and then infected with the SARS-CoV-2 GFP/ΔN virus (MOI = 0.1) for 24 hrs. (K) Fluorescence microscopy of the SARS-CoV-2-S pseudovirus in Usp50 +/+ and Usp50 -/- HEK293T cells pretreated with or without VitC (5 mM) for 12 hrs, followed by infection with SARS-CoV-2-S pseudovirus (MOI = 0.1) for 24 hrs. Scale bar: 100 µm. Data are representative of three independent experiments (A-I), or are shown as mean and s.d. of three biological replicates (J, K). N.S, not significant, * p < 0.05, *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S3.

Article Snippet: The antibodies with the indicated dilutions were as follows: anti-human ACE2 (Cell Signaling Technology, #74512, 1:1000), anti-ACE2 (Affinity, AF5165, 1:1000), anti-USP50 (Affinity, AF9225, 1:1000), anti-USP50 (Proteintech, 20374-1-AP, 1:1000), anti-Flag (Sigma, F7425, 1:5000), anti-HA (Abcam, ab9110, 1:3000), anti-Myc (Abmart, M20002H, 1:3000), anti-IRF3 (Santa Cruz, sc-33641, 1:1000), anti-STAT1 (Cell Signaling Technology, 9172, 1:1000), anti-Ubiquitin (Ub) (Santa Cruz, 12987-1-AP, 1:1000), anti-K48 Ub (Cell Signaling Technology, 4289S, 1:1000), anti-VSV-G (Santa Cruz, sc-66180, 1:2000) and anti-Tubulin (Proteintech, 66031-1-Ig, 1:3000).

Techniques: Western Blot, Transfection, Expressing, Construct, Immunoprecipitation, Quantitative RT-PCR, Infection, Fluorescence, Microscopy, Two Tailed Test

Journal: bioRxiv

Article Title: Vitamin C is an efficient natural product for prevention of SARS-CoV-2 infection by targeting ACE2 in both cell and in vivo mouse models

doi: 10.1101/2022.07.14.499651

Figure Lengend Snippet: (A) IP-IB analysis of ubiquitination of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2, together with Flag-USP50 (WT) or its deubiquitinase inactive mutant (C53S). (B) IP-IB analysis of ubiquitination of endogenous ACE2 in HEK293T cells transfected with either shCtrl (-) or shUSP50 (#1, #2). (C) IP-IB analysis of ubiquitination types of Myc-ACE2 in HEK293T cells cotransfected with Myc-ACE2, Flag-USP50 and different types of HA-Ub. (D) IP-IB analysis of K48-Ub of endogenous ACE2 in HEK293T cells transfected with shCtrl (-) or shUSP50 (#1, #2). (E) Putative ubiquitination sites of ACE2 in the PhosphoSitePlus database (Upper). Myc-ACE2 K48-linked ubiquitination was analyzed by IP-IB in HEK293T cells cotransfected with Myc-ACE2 (WT or its mutants) and HA-K48-Ub (Lower). (F) IP-IB analysis of Myc-ACE2 K48-linked ubiquitination in HEK293T cells cotransfected with Myc-ACE2 (WT or K788R) and HA-K48, together with shCtrl or shUSP50. (G) Western blot analysis of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 (WT or K788R) and then treated with CHX (50 μM) as indicated. (H) IP-IB analysis of K48-Ub of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 (WT or K788R) and then treated with VitC (5 mM) for 12 hrs, by a specific anti-K48-Ub antibody. (I) Western blot analysis of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 (WT or K788R) and then treated with VitC (5 mM) as indicated. Data are representative of three independent experiments (A-I). See also Figure S3 and S4.

Article Snippet: The antibodies with the indicated dilutions were as follows: anti-human ACE2 (Cell Signaling Technology, #74512, 1:1000), anti-ACE2 (Affinity, AF5165, 1:1000), anti-USP50 (Affinity, AF9225, 1:1000), anti-USP50 (Proteintech, 20374-1-AP, 1:1000), anti-Flag (Sigma, F7425, 1:5000), anti-HA (Abcam, ab9110, 1:3000), anti-Myc (Abmart, M20002H, 1:3000), anti-IRF3 (Santa Cruz, sc-33641, 1:1000), anti-STAT1 (Cell Signaling Technology, 9172, 1:1000), anti-Ubiquitin (Ub) (Santa Cruz, 12987-1-AP, 1:1000), anti-K48 Ub (Cell Signaling Technology, 4289S, 1:1000), anti-VSV-G (Santa Cruz, sc-66180, 1:2000) and anti-Tubulin (Proteintech, 66031-1-Ig, 1:3000).

Techniques: Transfection, Mutagenesis, Western Blot

Journal: bioRxiv

Article Title: Vitamin C is an efficient natural product for prevention of SARS-CoV-2 infection by targeting ACE2 in both cell and in vivo mouse models

doi: 10.1101/2022.07.14.499651

Figure Lengend Snippet: (A) The procedure for analysis of the binding of VitC to Flag-USP50 or Myc-ACE2 (left) can be seen detailedly in the Methods. The concentrations of VitC that binds with either Flag-USP50 (middle) or Myc-ACE2 (right) proteins were measured by the Vitamin-C Detection Kit. (B) Recombinant human ACE2-IgG-Fc proteins (r-hACE2-Fc) or anti-Tyk2 IgG proteins were incubated with protein-G beads for 2 hrs, and then VitC was added for binding. After washing and centrifuging, the concentrations of VitC that binds to anti-Tyk2 proteins or r-hACE2-Fc proteins were detected as (A). (C) The concentrations of VitC that binds to Myc-ACE2 (WT) or its deletion mutants were detected as (A). (D) VitC docking to the binding pocket of ACE2 by standard precision (SP) using the Glide docking module in the Schrödinger molecular simulation software. (E) The docking diagram between amino acids of ACE2 and VitC based on the SP docking scoring function. (F) IP-IB analysis of the interaction between USP50 and Myc-ACE2 (WT) or its deletion mutants (Δ19-200, Δ201-400, Δ401-600 and Δ601-805) in HEK293T. (G) IP-IB analysis of the interaction between Flag-USP50 and Myc-ACE2 in HEK293T cells transfected with these two constructs and then treated with VitC (2.5 mM and 5 mM) for 12 hrs. (H) IP-IB analysis of the in vivo interaction between endogenous USP50 and ACE2 in 2fTGH cells treated with VitC (5 mM) for 12 hrs. (I) Myc-ACE2 and Flag-USP50 proteins were immunoprecipitated from HEK293T cells transfected with either Myc-ACE2 or Flag-USP50. Flag-USP50 proteins were eluted by the Flag (M2) agarose. After washing, Flag-USP50 proteins were mixed with the Myc beads with Myc-ACE2, together with or without VitC (5 mM) for 2 hrs. After centrifuging, Flag-USP50 proteins interacting with Myc-ACE2 were analyzed by immunoblotting. (J) Flag-USP50 proteins were obtained as (I). Flag-USP50 and r-hACE2 proteins were mixed, together with increasing amounts of VitC or with VitC-Na + (20 mM). HCl is a pH control (pH = 4). After 2 hrs incubation, ACE2 proteins were analyzed by immunoblotting by a specific anti-ACE2 antibody. Data are representative of three independent experiments (F-J), or are shown as mean and s.d. of three biological replicates (A-C). N.S, not significant. *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S5.

Article Snippet: The antibodies with the indicated dilutions were as follows: anti-human ACE2 (Cell Signaling Technology, #74512, 1:1000), anti-ACE2 (Affinity, AF5165, 1:1000), anti-USP50 (Affinity, AF9225, 1:1000), anti-USP50 (Proteintech, 20374-1-AP, 1:1000), anti-Flag (Sigma, F7425, 1:5000), anti-HA (Abcam, ab9110, 1:3000), anti-Myc (Abmart, M20002H, 1:3000), anti-IRF3 (Santa Cruz, sc-33641, 1:1000), anti-STAT1 (Cell Signaling Technology, 9172, 1:1000), anti-Ubiquitin (Ub) (Santa Cruz, 12987-1-AP, 1:1000), anti-K48 Ub (Cell Signaling Technology, 4289S, 1:1000), anti-VSV-G (Santa Cruz, sc-66180, 1:2000) and anti-Tubulin (Proteintech, 66031-1-Ig, 1:3000).

Techniques: Binding Assay, Recombinant, Incubation, Software, Transfection, Construct, In Vivo, Immunoprecipitation, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Vitamin C is an efficient natural product for prevention of SARS-CoV-2 infection by targeting ACE2 in both cell and in vivo mouse models

doi: 10.1101/2022.07.14.499651

Figure Lengend Snippet: (A) IP-IB analysis of the interaction between endogenous hACE2 and USP50 in the lung and liver tissues of hACE2 mice. (B) The hACE2 mice were intraperitoneally administrated with VitC (300 mg/day/kg body weight) for two days. The interaction between USP50 and hACE2 in mouse lung tissues was analyzed by IP-IB. (C) IP-IB analysis of K48-Ub of hACE2 in mouse lung tissues from (B). (D) Western blot analysis of hACE2 levels in lung tissues of hACE2 mice administrated with VitC as (B). (E) Immunohistochemical staining of hACE2 protein in the lung, kidney and liver tissues from (B). (F) The hACE2 mice were administrated with VitC as (B). Mice were then given intraperitoneal injections of SARS-CoV-2-S pseudoviruses (1×10 6 PFU per gram body). After 24 hrs, immunohistochemical staining was performed to analyze the SARS-CoV-2 Spike proteins in mouse lung and kidney tissues. Scale bar: 100 µm. (G) RT-qPCR analysis of the SARS-CoV-2 Spike mRNA levels in lung, kidney, liver and spleen tissues of hACE2 mice treated with VitC and SARS-CoV-2-S pseudoviruses as (F). Data are representative of three independent experiments (A-D). All graphs show the mean ± SEM for five individual mice (G). *** p < 0.001 (two-tailed unpaired Student’s t -test). See also Figure S6 and S7.

Article Snippet: The antibodies with the indicated dilutions were as follows: anti-human ACE2 (Cell Signaling Technology, #74512, 1:1000), anti-ACE2 (Affinity, AF5165, 1:1000), anti-USP50 (Affinity, AF9225, 1:1000), anti-USP50 (Proteintech, 20374-1-AP, 1:1000), anti-Flag (Sigma, F7425, 1:5000), anti-HA (Abcam, ab9110, 1:3000), anti-Myc (Abmart, M20002H, 1:3000), anti-IRF3 (Santa Cruz, sc-33641, 1:1000), anti-STAT1 (Cell Signaling Technology, 9172, 1:1000), anti-Ubiquitin (Ub) (Santa Cruz, 12987-1-AP, 1:1000), anti-K48 Ub (Cell Signaling Technology, 4289S, 1:1000), anti-VSV-G (Santa Cruz, sc-66180, 1:2000) and anti-Tubulin (Proteintech, 66031-1-Ig, 1:3000).

Techniques: Western Blot, Immunohistochemical staining, Staining, Quantitative RT-PCR, Two Tailed Test

Journal: Acta Pharmacologica Sinica

Article Title: Transcriptional regulation and small compound targeting of ACE2 in lung epithelial cells

doi: 10.1038/s41401-022-00906-6

Figure Lengend Snippet: a Putative transcription factors of ACE2 predicted by Cistrome Data Browser. Each dot represents a ChIP-seq sample and the top 24 factors that ranked by the regulatory potential score over all ChIP-seq samples are shown. b , c Association between ACE2 and the representative transcription factors ( b ) including STAT3 ( c ) was analyzed using the RNA-Seq data of non-cancer samples in the GEPIA dataset.

Article Snippet: The antibodies used in Western blotting were as follows: rabbit monoclonal anti-human ACE2 (#ab108252, Abcam, Cambridge, MA, USA; 1:1000 for Western blot); rabbit polyclonal anti-human ACE2 (#4355, 1:1000), mouse anti-STAT3 (#9139 S, 1:1000), rabbit anti-phospho-STAT3 (#9145 S, 1:2000), anti-GAPDH (#5174, 1:1000) were obtained from Cell Signaling Technology, Danvers, MA, USA; anti-Actin (#A5441, 1:5000) was purchased from Sigma, St. Louis, MO, USA; rabbit monoclonal anti-human ACE2 (#ab108252, Abcam; 1:200) and mouse anti-phospho-STAT3 (#9145S, Cell Signaling Technology, 1:200) were used in IHC assay.

Techniques: ChIP-sequencing, RNA Sequencing Assay

Journal: Acta Pharmacologica Sinica

Article Title: Transcriptional regulation and small compound targeting of ACE2 in lung epithelial cells

doi: 10.1038/s41401-022-00906-6

Figure Lengend Snippet: a , b Immunohistochemical staining experiments were performed in 12 patients with benign disease using anti-ACE2 and anti-phospho-Stat3 antibodies ( a ), and the immunoreactivity score was evaluated ( b ). Scale bars, 50 μm. * P < 0.05, two-tailed Student’s t -test. c Western blot assays were performed in 49 normal lung tissues isolated from patients with lung cancer at surgery. d The relative ACE2 and p-Stat3 levels were determined by densitometry analysis of immunoblot bands and normalized to Actin, and the potential association between ACE2 and p-Stat3 levels was analyzed.

Article Snippet: The antibodies used in Western blotting were as follows: rabbit monoclonal anti-human ACE2 (#ab108252, Abcam, Cambridge, MA, USA; 1:1000 for Western blot); rabbit polyclonal anti-human ACE2 (#4355, 1:1000), mouse anti-STAT3 (#9139 S, 1:1000), rabbit anti-phospho-STAT3 (#9145 S, 1:2000), anti-GAPDH (#5174, 1:1000) were obtained from Cell Signaling Technology, Danvers, MA, USA; anti-Actin (#A5441, 1:5000) was purchased from Sigma, St. Louis, MO, USA; rabbit monoclonal anti-human ACE2 (#ab108252, Abcam; 1:200) and mouse anti-phospho-STAT3 (#9145S, Cell Signaling Technology, 1:200) were used in IHC assay.

Techniques: Immunohistochemical staining, Staining, Two Tailed Test, Western Blot, Isolation

Journal: Acta Pharmacologica Sinica

Article Title: Transcriptional regulation and small compound targeting of ACE2 in lung epithelial cells

doi: 10.1038/s41401-022-00906-6

Figure Lengend Snippet: a The Stat3 binding site of ACE2 promoter. TSS, transcription start site. b EMSA using Stat3 protein and biotin-labeled Stat3-binding (BD) DNA in the presence or absence of IL-6. Biotin-labeled GATA1-BD DNA or unlabeled Stat3-BD DNA was used as controls. IL-6, interleukin-6. c , d Relative mRNA levels of Stat3 and ACE2 in 16HBE cells transfected with si Stat3 ( c ) or Flag- Stat3 ( d ), measured by qRT-PCR. Data are expressed as mean ± SEM, n = 3 for biological independent replicates. * P < 0.05, two-tailed Student’s t -test. e , f Western blot analyses of ACE2 in 16HBE cells transfected with si Stat3 ( e ) or Flag -Stat3 ( f ). g 16HBE cells were treated with IL-6 at 50 ng/mL for 30 min, lysed, and subjected to Western blot using indicated antibodies. h , i Western blot analyses of ACE2 in 293 T cells transfected with si Stat3 ( h ) or Flag -Stat3 ( i ).

Article Snippet: The antibodies used in Western blotting were as follows: rabbit monoclonal anti-human ACE2 (#ab108252, Abcam, Cambridge, MA, USA; 1:1000 for Western blot); rabbit polyclonal anti-human ACE2 (#4355, 1:1000), mouse anti-STAT3 (#9139 S, 1:1000), rabbit anti-phospho-STAT3 (#9145 S, 1:2000), anti-GAPDH (#5174, 1:1000) were obtained from Cell Signaling Technology, Danvers, MA, USA; anti-Actin (#A5441, 1:5000) was purchased from Sigma, St. Louis, MO, USA; rabbit monoclonal anti-human ACE2 (#ab108252, Abcam; 1:200) and mouse anti-phospho-STAT3 (#9145S, Cell Signaling Technology, 1:200) were used in IHC assay.

Techniques: Binding Assay, Labeling, Transfection, Quantitative RT-PCR, Two Tailed Test, Western Blot

Journal: Acta Pharmacologica Sinica

Article Title: Transcriptional regulation and small compound targeting of ACE2 in lung epithelial cells

doi: 10.1038/s41401-022-00906-6

Figure Lengend Snippet: a HLF cells were transfected with ACE2 promoter-luciferase constructs and treated with 64 compounds and negative controls, and the luciferase activity was measured. b Luciferase activity was detected in ACE2 promoter-luciferase-expressing HLF cells that were treated with 6-OAP at 2.5 µM for 24–48 h. Data are expressed as mean ± SEM, n = 3 for biological independent replicates. * P < 0.05, ** P < 0.01, two-tailed Student’s t -test. c A chromatin immunoprecipitation (ChIP) assay was performed in 6-OAP treated or untreated 16HBE cells, and STAT3-bound ACE2 expression level was detected by quantitative PCR. Data are expressed as mean ± SEM, n = 3 for biological independent replicates. ** P < 0.01, two-tailed Student’s t -test. d EMSA using Stat3 protein and biotin-labeled Stat3-BD DNA in the presence of IL-6 and/or 6-OAP. e , f The ACE2 promoter-luciferase in 16HBE cells transfected with si Stat3 ( e ) or Flag- Stat3 ( f ), in the presence of absence of 6-OAP. * P < 0.05, one-way ANOVA. g The luciferase activity of HLF cells driven by wild-type or mutant ACE2 promoter in the presence or absence of 6-OAP. * P < 0.05, one-way ANOVA.

Article Snippet: The antibodies used in Western blotting were as follows: rabbit monoclonal anti-human ACE2 (#ab108252, Abcam, Cambridge, MA, USA; 1:1000 for Western blot); rabbit polyclonal anti-human ACE2 (#4355, 1:1000), mouse anti-STAT3 (#9139 S, 1:1000), rabbit anti-phospho-STAT3 (#9145 S, 1:2000), anti-GAPDH (#5174, 1:1000) were obtained from Cell Signaling Technology, Danvers, MA, USA; anti-Actin (#A5441, 1:5000) was purchased from Sigma, St. Louis, MO, USA; rabbit monoclonal anti-human ACE2 (#ab108252, Abcam; 1:200) and mouse anti-phospho-STAT3 (#9145S, Cell Signaling Technology, 1:200) were used in IHC assay.

Techniques: Transfection, Luciferase, Construct, Activity Assay, Expressing, Two Tailed Test, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Labeling, Mutagenesis

Journal: Acta Pharmacologica Sinica

Article Title: Transcriptional regulation and small compound targeting of ACE2 in lung epithelial cells

doi: 10.1038/s41401-022-00906-6

Figure Lengend Snippet: a , b Western blot assays using lysates of 16HBE cells treated with 6-OAP at indicated concentrations and time points. c Western blot assays using lysates of Beas-2B cells upon 6-OAP treatment. d 16HBE cells were treated with 6-OAP and/or IL-6, lysed, and subjected to Western blot. e , f 16HBE cells were treated with CM at indicated concentrations and indicated time points, lysed, and subjected to Western blot assays using indicated antibodies. CM, Centipeda minima . g Beas-2B cells were treated with CM, lysed, and subjected to Western blot. h C57BL/6 mice were treated with 6-OAP or CM for one month, sacrificed, and lung tissues were isolated and lysed for Western blot analyses using anti-ACE2 and anti-p-Stat3 antibodies. i The relative ACE2 and p-Stat3 expression levels were determined by densitometry analysis of immunoblot bands and normalized to Actin. * P < 0.05, two-tailed Student’s t -test. j C57BL/6 mice were treated with CM for two months, sacrificed, and lung tissues were isolated and lysed for Western blot analyses. k The relative ACE2 and p-Stat3 expression levels were determined by densitometry analysis of immunoblot bands and normalized to Actin. ** P < 0.01, two-tailed Student’s t -test.

Article Snippet: The antibodies used in Western blotting were as follows: rabbit monoclonal anti-human ACE2 (#ab108252, Abcam, Cambridge, MA, USA; 1:1000 for Western blot); rabbit polyclonal anti-human ACE2 (#4355, 1:1000), mouse anti-STAT3 (#9139 S, 1:1000), rabbit anti-phospho-STAT3 (#9145 S, 1:2000), anti-GAPDH (#5174, 1:1000) were obtained from Cell Signaling Technology, Danvers, MA, USA; anti-Actin (#A5441, 1:5000) was purchased from Sigma, St. Louis, MO, USA; rabbit monoclonal anti-human ACE2 (#ab108252, Abcam; 1:200) and mouse anti-phospho-STAT3 (#9145S, Cell Signaling Technology, 1:200) were used in IHC assay.

Techniques: Western Blot, Isolation, Expressing, Two Tailed Test

Journal: Acta Pharmacologica Sinica

Article Title: Transcriptional regulation and small compound targeting of ACE2 in lung epithelial cells

doi: 10.1038/s41401-022-00906-6

Figure Lengend Snippet: a , b Relative mRNA levels of indicated genes in 16HBE cells that were treated with CM ( a ) or 6-OAP ( b ) for 48 h. Data are expressed as mean ± SEM, n = 3 for biological independent replicates. * P < 0.05, ** P < 0.01, *** P < 0.001, two-tailed Student’s t -test. c IL-6 levels in 16HBE cell supernatants were measured by ELISA. Data are represented as means ± SD of three independent experiments. * P < 0.05, two-tailed Student’s t -test. d , e The effects of 6-OAP (5 µM)/CM (0.5%) on SARS-CoV-2 spike (S) protein pseudovirus entry into 16HBE ( d ) and 293T- ACE2 ( e ) cells, revealed by relative luciferase activity. * P < 0.05, ** P < 0.01, two-tailed Student’s t -test. f Schematic representation of the mechanisms of the action of 6-OAP/CM in modulation of Stat3 and ACE2.

Article Snippet: The antibodies used in Western blotting were as follows: rabbit monoclonal anti-human ACE2 (#ab108252, Abcam, Cambridge, MA, USA; 1:1000 for Western blot); rabbit polyclonal anti-human ACE2 (#4355, 1:1000), mouse anti-STAT3 (#9139 S, 1:1000), rabbit anti-phospho-STAT3 (#9145 S, 1:2000), anti-GAPDH (#5174, 1:1000) were obtained from Cell Signaling Technology, Danvers, MA, USA; anti-Actin (#A5441, 1:5000) was purchased from Sigma, St. Louis, MO, USA; rabbit monoclonal anti-human ACE2 (#ab108252, Abcam; 1:200) and mouse anti-phospho-STAT3 (#9145S, Cell Signaling Technology, 1:200) were used in IHC assay.

Techniques: Two Tailed Test, Enzyme-linked Immunosorbent Assay, Luciferase, Activity Assay

Journal: bioRxiv

Article Title: Lentiviral vector-based SARS-CoV-2 pseudovirus enables analysis of neutralizing activity in COVID-19 convalescent plasma

doi: 10.1101/2020.12.28.424590

Figure Lengend Snippet: Upper panels in (A) and (B) represent the maps for the empty LeGO-iT2puro vector backbone and LeGO-hACE2-iT2puro vector used to genetically modify 293FT cells. Lower panels show representative flow cytometry analysis of RBD-GFP fusion protein staining on tdTomato or hACE2-tdTomato expressing 293FT cells. (C) Representative confocal microscopy images generated from RBD-GFP fusion protein stained tdTomato and hACE2-tdTomato expressing HEK293FT cells.

Article Snippet: The membrane was blocked with 5% skimmed milk in TBS-T and incubated overnight at 4°C with polyclonal rabbit anti human-ACE2 antibody (#4355), (Cell Signaling Technology (CST), USA).

Techniques: Plasmid Preparation, Flow Cytometry, Staining, Expressing, Confocal Microscopy, Generated

Journal: bioRxiv

Article Title: Lentiviral vector-based SARS-CoV-2 pseudovirus enables analysis of neutralizing activity in COVID-19 convalescent plasma

doi: 10.1101/2020.12.28.424590

Figure Lengend Snippet: (A) Representative protocol for the production of Spike-pseudotyped lentiviral vectors encoding GFP. Lego-G2 lentiviral plasmid was co-transfected into 293FT cells along with packaging constructs and the plasmid for Spike expression. Collected supernatants containing pseudovirus particles were used to infect 293FT-hACE2 cells. (B) Percentage of GFP+ 293FT-hACE2 cells after transduction with pseudovirus containing supernatants prepared with varying levels of the plasmid pCMV-SpikeΔ19 during transfection. (Data from one representative experiment runt in triplicates, Mean +/− SEM plotted) (C) Percentage of GFP+ 293FT-hACE2 cells after transduction with pseudovirus containing supernatants collected in cull growth medium containing different concentrations of Sodium Butyrate (Data from one representative experiment runt in triplicates. **p<0.005, n.s. not significant, One-way ANOVA, Tukey’s test) (D) Percentage of GFP+ 293FT-hACE2 cells after transduction with pseudovirus containing supernatants collected under different concentrations of FBS and temperatures. (Data from one representative experiment runt in triplicates. n.s. not significant) (E) Representative flow cytometry analysis of GFP expression 3 days after pseudovirus treatment of 293FT-hACE2 cells. Effects of (F) spinoculation and (G) exposure time to pseudovirus during transduction of 293FT-hACE2 cells. (Data from two experiments with two different batches of pseudovirus run in triplicates, **** p<0.001, paired t-test, two-tailed)

Article Snippet: The membrane was blocked with 5% skimmed milk in TBS-T and incubated overnight at 4°C with polyclonal rabbit anti human-ACE2 antibody (#4355), (Cell Signaling Technology (CST), USA).

Techniques: Plasmid Preparation, Transfection, Construct, Expressing, Transduction, Flow Cytometry, Two Tailed Test

Journal: bioRxiv

Article Title: Lentiviral vector-based SARS-CoV-2 pseudovirus enables analysis of neutralizing activity in COVID-19 convalescent plasma

doi: 10.1101/2020.12.28.424590

Figure Lengend Snippet: (A) Percentage of GFP+ 293FT-hACE2 cells after transduction with pseudovirus containing supernatants using the WT or D614G SpikeΔ19 plasmids with or without the M protein plasmid. Transductions are done with either fresh or frozen and thawed viruses. (Results from three different batches of pseudovirus each run in triplicates, ** p<0.005, two-way ANOVA, Sidak’s test) (B) Stability of SpikeΔ19 and SpikeΔ19(D614G) pseudoviruses with or without the M protein. Pseudovirus supernatants were incubated at 37°C for up to 48 hours followed by transduction to tdTomato-hACE2 expressing HEK293FT cells. Data normalized to transduction results of freshly thawed supernatant. (Analyzed with one-phase decay model with least squares fit, R2>0.9 for all curves) (C) ACE2-IgG mediated neutralization of SpikeΔ19 and SpikeΔ19(D614G) lentiviruses with or without M protein. Spike-pseudotype and VSV-g enveloped lentiviruses were pre-incubated with various concentrations (20μg/ml – 39ng/ml) of ACE2-IgG for 1 hour at 37°C followed by transduction of tdTomato-hACE2 expressing 293FT cells. VSV-G pseudotyped particles incubated with hACE2-IgG were used as control. Graph shows the percentage of GFP expressing cells as normalized to samples transduced without any ACE2-IgG pre-incubation. (Curve fitting was done by 4-parameter non-linear regression using variable slope, R2 values for all curves except VSV-G were above 0.9) (D) Results of flow based analysis of neutralization by ACE2-IgG from one representative experiment.

Article Snippet: The membrane was blocked with 5% skimmed milk in TBS-T and incubated overnight at 4°C with polyclonal rabbit anti human-ACE2 antibody (#4355), (Cell Signaling Technology (CST), USA).

Techniques: Transduction, Plasmid Preparation, Incubation, Expressing, Neutralization

Journal: bioRxiv

Article Title: Lentiviral vector-based SARS-CoV-2 pseudovirus enables analysis of neutralizing activity in COVID-19 convalescent plasma

doi: 10.1101/2020.12.28.424590

Figure Lengend Snippet: Spike or VSV-G pseudotyped particles were incubated with serial dilutions (1:20 – 1:20480) of CP samples for 1 hour at 37°C followed by transduction to 293FT-hACE2 cells. VSV-G enveloped lentiviruses incubated with CP samples were used as negative controls. (A) Representative flow cytometry analysis of CP dependent neutralization assay from one donor. Plasma samples from healthy donors and 16 patients that recovered from COVID-19 were analyzed (B) Healthy donors are used as a control group and their plasma showed no neutralization effect. (Results from one representative healthy donor) (C) 9/16 of CP samples showed high neutralization activity. (Results from one representative high-activity CP) (D) 7/16 of CP samples showed no or low neutralization. (Results from one representative low-activity CP) Curve fitting was done by 4-parameter non-linear regression using variable slope.

Article Snippet: The membrane was blocked with 5% skimmed milk in TBS-T and incubated overnight at 4°C with polyclonal rabbit anti human-ACE2 antibody (#4355), (Cell Signaling Technology (CST), USA).

Techniques: Incubation, Transduction, Flow Cytometry, Neutralization, Activity Assay

Journal: Journal of Clinical Immunology

Article Title: N-Glycan Modification in Covid-19 Pathophysiology: In vitro Structural Changes with Limited Functional Effects

doi: 10.1007/s10875-020-00905-4

Figure Lengend Snippet: Expression and enzymatic digestion of SARS-CoV-2 S and human ACE2 in transfected HEK 293T cells. HEK293T cells were transfected with SARS-CoV-2 S (a) or ACE2 (b). On the following day, cell lysates were digested with PNGase F (lane 2) or Endo H (lane 3) and immunoblotted with anti-SARS-CoV-2 S antibody specific for the S2 subunit of the S protein or anti-ACE2 antibody, as indicated. ß-actin was used as a loading control. Data shown are representative of 2 independent experiments

Article Snippet: The membranes were incubated with antibodies to SARS-CoV-2 S glycoprotein (GeneTex, Cat. GTX632604, recognizing an epitope within the S2 subunit), human ACE2 (Cell Signaling Technology, Cat. 4355), anti-FLAG antibody (Cell signaling technology, Cat. 14793), or β-actin (Cell Signaling Technology, Cat. 4970).

Techniques: Expressing, Transfection

Journal: Journal of Clinical Immunology

Article Title: N-Glycan Modification in Covid-19 Pathophysiology: In vitro Structural Changes with Limited Functional Effects

doi: 10.1007/s10875-020-00905-4

Figure Lengend Snippet: Miglustat treatment of SARS-CoV-2 S and human ACE2 expressing HEK293T cells. HEK293T cells were transfected with SARS-CoV-2 S (a) or ACE2 (b). On the next day, iminosugar treatment (miglustat 1 mM or castanospermine 1 mM) or glucosylceramide synthase inhibitor (eliglustat 10 μM) was started. After 24 h, cells were lysed and half of the lysates were digested with PNGase F for 1 h. Protein lysates before and after PNGase F digestion were separated by Western blotting with anti-SARS-CoV-2 S antibody and anti-ACE2 antibody as indicated. Cs = castanospermine. Data shown are representative of 2 independent experiments

Article Snippet: The membranes were incubated with antibodies to SARS-CoV-2 S glycoprotein (GeneTex, Cat. GTX632604, recognizing an epitope within the S2 subunit), human ACE2 (Cell Signaling Technology, Cat. 4355), anti-FLAG antibody (Cell signaling technology, Cat. 14793), or β-actin (Cell Signaling Technology, Cat. 4970).

Techniques: Expressing, Transfection, Western Blot

Journal: Journal of Clinical Immunology

Article Title: N-Glycan Modification in Covid-19 Pathophysiology: In vitro Structural Changes with Limited Functional Effects

doi: 10.1007/s10875-020-00905-4

Figure Lengend Snippet: Effect of miglustat treatment on SARS-CoV-2 S to ACE2 binding. (a) HEK293T cells were transfected with Flag-tagged ACE2 for 24 h, followed by indicated inhibitor treatment for an additional 24 h. Protein extracts were prepared in lysis buffer containing 0.5% Triton X-100 and mixed with 5 μg of SARS-CoV-2 S protein. Same amount of untreated and treated protein lysates was immunoprecipitated with anti-Flag antibodies and probed with anti-SARS-CoV-2 S and anti-Flag antibodies. (b) HEK293T cells were transfected with Flag-tagged ACE2 together with HA-tagged SARS-CoV-2 S for 24 h, followed by indicated inhibitor treatment for an additional 24 h. Protein extracts were prepared in lysis buffer containing 0.5% Triton X-100 and immunoprecipitated with anti-Flag antibodies and probed with anti-SARS-CoV-2 S and anti-Flag antibodies. Two percent of the total volumes of the whole cellular lysates used for IP reactions were loaded as input controls. Cs = castanospermine. Data shown are representative of 3 independent experiments

Article Snippet: The membranes were incubated with antibodies to SARS-CoV-2 S glycoprotein (GeneTex, Cat. GTX632604, recognizing an epitope within the S2 subunit), human ACE2 (Cell Signaling Technology, Cat. 4355), anti-FLAG antibody (Cell signaling technology, Cat. 14793), or β-actin (Cell Signaling Technology, Cat. 4970).

Techniques: Binding Assay, Transfection, Lysis, Immunoprecipitation

Journal: Journal of Clinical Immunology

Article Title: N-Glycan Modification in Covid-19 Pathophysiology: In vitro Structural Changes with Limited Functional Effects

doi: 10.1007/s10875-020-00905-4

Figure Lengend Snippet: Effect of miglustat treatment on SARS-CoV-2 S to human ACE2 binding affinity. HEK293T cells were transfected with Flag-tagged ACE2 together with HA-tagged SARS-CoV-2 S for 24 h, followed by 1 mM miglustat treatment for an additional 24 h. Protein extracts were prepared in lysis buffer containing 0.5% Triton X-100 and 0.5% NP-40. The same amount of protein lysates was divided and indicated percentage of SDS was added in the lysates and performed immunoprecipitation. Data shown are representative of 3 independent experiments

Article Snippet: The membranes were incubated with antibodies to SARS-CoV-2 S glycoprotein (GeneTex, Cat. GTX632604, recognizing an epitope within the S2 subunit), human ACE2 (Cell Signaling Technology, Cat. 4355), anti-FLAG antibody (Cell signaling technology, Cat. 14793), or β-actin (Cell Signaling Technology, Cat. 4970).

Techniques: Binding Assay, Transfection, Lysis, Immunoprecipitation

Journal: Journal of Clinical Immunology

Article Title: N-Glycan Modification in Covid-19 Pathophysiology: In vitro Structural Changes with Limited Functional Effects

doi: 10.1007/s10875-020-00905-4

Figure Lengend Snippet: Effect of ER α-glucosidase inhibition on syncytia formation between SARS-CoV-2 S and human ACE2 transfected cells. (a) NIH/3T3 cells (upper panels) transfected with SARS-CoV-2 S-HA or ACE2-Flag were cultured in the presence or absence of 1 mM miglustat. Primary fibroblasts (bottom panels) from a MOGS-null patient or a normal control were transfected with the same plasmids. SARS-CoV-2 S transfected cells were mixed at 1:1 ratio with ACE2 transfected cells and cocultured for 48 h. Cells were then fixed, permeabilized, and labeled with mouse anti-HA antibody and rabbit anti-Flag antibody followed by Alexa Fluor 488 (green) anti-mouse and Alexa Fluor 594 (red) anti-rabbit secondary antibodies. DAPI was used for nuclei staining. Images are representative of multinucleated giant cells (syncytia) formation from 2 independent experiments (original magnification × 175). (b) Quantitative comparison of syncytia formation between miglustat-treated samples and untreated controls (top row) or normal control (NC) and MOGS-null patient (bottom row). * p < 0.05, student t test. Data are mean ± SEM

Article Snippet: The membranes were incubated with antibodies to SARS-CoV-2 S glycoprotein (GeneTex, Cat. GTX632604, recognizing an epitope within the S2 subunit), human ACE2 (Cell Signaling Technology, Cat. 4355), anti-FLAG antibody (Cell signaling technology, Cat. 14793), or β-actin (Cell Signaling Technology, Cat. 4970).

Techniques: Inhibition, Transfection, Cell Culture, Labeling, Staining