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