Journal: Environmental Science and Pollution Research International

Article Title: Rutin protects against gamma-irradiation and malathion-induced oxidative stress and inflammation through regulation of mir-129-3p, mir-200C-3p, and mir-210 gene expressions in rats’ kidney

doi: 10.1007/s11356-023-27166-z

Figure Lengend Snippet: The activity of acetylcholinesterase, angiotensin I converting enzyme, and angiotensin II converting enzyme in the kidney tissues. C, control; rutin, rutin-treated animals; IRR, gamma-irradiated animals; MT, malathion-treated animals; IRR/MT, gamma-irradiation/malathion-treated animals; IRR/rutin, gamma-irradiation/rutin-treated animals; MT/rutin, malathion/rutin-treated animals; IRR/MT/rutin, gamma-irradiation/malathion/rutin-treated animals; AchE, acetylcholinesterase; ACE I, angiotensin I converting enzyme. The statistical significance to control, IRR, MT, and IRR/MT are denoted by a, b, c, and d, respectively, at p < 0.05. Statistical significance was analyzed by one-way ANOVA with Tukey post hoc multiple comparisons. These enzymes are assessed by ELISA technique

Article Snippet: Elabscience ELISA commercial kits for rats were used to assess the activity of angiotensin-converting enzymes ACE I (Cat. No: E-EL-R2401) and ACE II (Cat. No: E-EL-R2453), in the rats’ kidneys tissues, according to the catalogs’ instructions.

Techniques: Activity Assay, Control, Irradiation, Enzyme-linked Immunosorbent Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: A biosafe mouse model for SARS-CoV-2 infection that more realistically simulates COVID-19 symptoms

doi: 10.1038/s41392-026-02640-5

Figure Lengend Snippet: Generation of the SARS-CoV-2 N conditional knock-in mouse model. a Schematic diagrams illustrating the knock-in strategy, in which the CAG-loxP-Stop-loxP-SARS-CoV-2 N-WPRE-PolyA sequence was inserted into the Tigre locus on chromosome 9, using the K18-hACE2 KI mouse background (N-hACE2 mouse). The stop sequence was flanked by two loxP sites, and upon expression of Cre recombinase, the stop sequence located between these loxP sites was excised. N-hACE2 mice were crossed with Rosa26 SA-CreERT2 or Sftpc-IRES-iCre mice, resulting in the generation of SA-N-hACE2 (TAM-inducible systemic Cre expression) and Sftpc-N-hACE2 (lung-specific Cre expression) mice. b N-hACE2 mouse genotyping by PCR showed the presence of N gene and hACE2 gene fragments. M: GimiRun DM5000 DNA Marker. c PCR confirmed excision of the stop sequence between loxP sites in SA-N-hACE2 mice after 5 days of TAM injection. Western blot analysis of SARS-CoV-2 N protein expression in multiple tissues from SA-N-hACE2 mice after TAM induction ( d ) and from Sftpc-N-hACE2 mice ( e ). f Immunofluorescence staining of lung sections was conducted using 4,6-diamidino-2-phenylindole (DAPI, blue), an anti-SARS-CoV-2 N antibody (red), and an anti-hACE2 antibody (green) to evaluate the expression of SARS-CoV-2 N and hACE2 in lung cells

Article Snippet: The antibodies used for IFAs were as follows: anti-hACE2 antibody (Sino Biological, 10108-RP01), anti-SARS-CoV-2 N antibody (Sino Biological, 40143-MM05), anti-GFP antibody (Sino Biological, 13105-R208), anti-IBA1 antibody (Abways, CY7217), anti-Ly6G antibody (Servicebio, GB11229), anti-CD68 antibody (Starter, SOB2064), anti-CD19 antibody (Servicebio, GB11061), anti-CD3 antibody (AiFang Biological, AFRM0030), anti-CD4 antibody (Abcam, ab183685), and anti-CD8 antibody (Abcam, ab217344).

Techniques: Knock-In, Sequencing, Expressing, Marker, Injection, Western Blot, Immunofluorescence, Staining

Journal: Signal Transduction and Targeted Therapy

Article Title: A biosafe mouse model for SARS-CoV-2 infection that more realistically simulates COVID-19 symptoms

doi: 10.1038/s41392-026-02640-5

Figure Lengend Snippet: SA-N-hACE2 mice are susceptible to SARS-CoV-2 ΔN/GFP-HiBiT infection. a Experimental design for intranasal infection. After TAM treatment, SA-N-hACE2 mice were infected either with 5 × 10 4 or 1 × 10 6 TCID 50 of SARS-CoV-2 ΔN/GFP-HiBiT. Tissue samples were collected at the indicated dpi. b Changes in the weights of the mice are shown ( n = 4 per group). c qRT‒PCR was used to quantify viral loads in tissues at 7 dpi ( n = 4 per group). The viral loads ( d ) and luminescence ( e ) were measured in the lungs collected at 2, 4, and 7 dpi ( n = 4 per group). f Immunofluorescence staining of lung sections at 2, 4, and 7 dpi was conducted using DAPI (blue) and an anti-GFP antibody (green) specific to GFP in SARS-CoV-2 ΔN/GFP-HiBiT. g – j Pathological changes observed using H&E staining in lung ( g ) and brain ( i ) tissues from SA-N-hACE2 mice challenged with 1 × 10 6 TCID 50 at 0, 2, 4, and 7 dpi. Pathology scores for the lungs ( h ) and brain ( j ) were calculated ( n = 4 per group). Data are presented as means ± SD. Statistical significance was determined using one-way ANOVA with Dunnett’s multiple comparisons test ( h ). ns not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. ND not detected

Article Snippet: The antibodies used for IFAs were as follows: anti-hACE2 antibody (Sino Biological, 10108-RP01), anti-SARS-CoV-2 N antibody (Sino Biological, 40143-MM05), anti-GFP antibody (Sino Biological, 13105-R208), anti-IBA1 antibody (Abways, CY7217), anti-Ly6G antibody (Servicebio, GB11229), anti-CD68 antibody (Starter, SOB2064), anti-CD19 antibody (Servicebio, GB11061), anti-CD3 antibody (AiFang Biological, AFRM0030), anti-CD4 antibody (Abcam, ab183685), and anti-CD8 antibody (Abcam, ab217344).

Techniques: Infection, Immunofluorescence, Staining

Journal: Signal Transduction and Targeted Therapy

Article Title: A biosafe mouse model for SARS-CoV-2 infection that more realistically simulates COVID-19 symptoms

doi: 10.1038/s41392-026-02640-5

Figure Lengend Snippet: Sftpc-N-hACE2 mice are susceptible to SARS-CoV-2 ΔN/GFP-HiBiT infection. a Illustration of the protocol for intranasal infection with tissue samples collected at the indicated dpi. Sftpc-N-hACE2 mice were infected with 5 × 10 4 or 1 × 10 6 TCID 50 of SARS-CoV-2 ΔN/GFP-HiBiT. b Changes in the body weights of the mice are shown ( n = 4 per group). c Viral loads in the tissues obtained at 7 dpi were quantified via qRT‒PCR ( n = 4 per group). The viral loads ( d ) and luminescence ( e ) were measured in the lungs collected at 2, 4, and 7 dpi ( n = 4 per group). f Immunofluorescence staining of lung sections at 2, 4, and 7 dpi was conducted using DAPI (blue) and an anti-GFP antibody (green) specific to GFP in SARS-CoV-2 ΔN/GFP-HiBiT. g – j Pathological changes in the lungs ( g ) and brains ( i ) of Sftpc-N-hACE2 mice challenged with 1 × 10 6 TCID 50 at 0, 2, 4, and 7 dpi were assessed using H&E staining. Pathology scores for the lungs ( h ) and brains ( j ) are shown ( n = 4 per group). Data are presented as means ± SD. Statistical significance was determined using one-way ANOVA with Dunnett’s multiple comparisons test ( h ). ns not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. ND not detected

Article Snippet: The antibodies used for IFAs were as follows: anti-hACE2 antibody (Sino Biological, 10108-RP01), anti-SARS-CoV-2 N antibody (Sino Biological, 40143-MM05), anti-GFP antibody (Sino Biological, 13105-R208), anti-IBA1 antibody (Abways, CY7217), anti-Ly6G antibody (Servicebio, GB11229), anti-CD68 antibody (Starter, SOB2064), anti-CD19 antibody (Servicebio, GB11061), anti-CD3 antibody (AiFang Biological, AFRM0030), anti-CD4 antibody (Abcam, ab183685), and anti-CD8 antibody (Abcam, ab217344).

Techniques: Infection, Immunofluorescence, Staining

Journal: Signal Transduction and Targeted Therapy

Article Title: A biosafe mouse model for SARS-CoV-2 infection that more realistically simulates COVID-19 symptoms

doi: 10.1038/s41392-026-02640-5

Figure Lengend Snippet: SARS-CoV-2 ΔN/GFP-HiBiT infection in Sftpc-N-hACE2 mice can lead to lethal disease. a The schematic outlines the procedure for infecting the mice with 5 × 10 6 TCID 50 of SARS-CoV-2 ΔN/GFP-HiBiT. At 0, 7, 10, 14, and 21 dpi, four mice were euthanized at each time point for sample collection. Mice that experienced a loss of more than 20% of their initial body weight were euthanized as a humane endpoint. Mice were monitored for body weight changes ( b ) and survival ( c ) ( n = 16). E gene copies in lung ( d ) and brain ( e ) tissues were quantified via qRT‒PCR. f – i Pathological changes were assessed using H&E staining in the lungs ( f ) and brains ( g ). Pathology scores for the lungs ( h ) and brains ( i ) were recorded ( n = 4 per group). j Immunofluorescence staining of brain sections was performed using DAPI (blue) and an anti-GFP antibody (green) specific to GFP in SARS-CoV-2 ΔN/GFP-HiBiT. k Immunofluorescence analysis was performed on brain sections from mock-infected mice, K18-hACE2 KI mice challenged with live SARS-CoV-2, and both SA-N-hACE2 and Sftpc-N-hACE2 mice exposed to SARS-CoV-2 ΔN/GFP-HiBiT. The sections were stained with anti-IBA1 (red) and anti-CD68 (green) antibodies. l Light-sheet imaging of cleared lung tissues from Sftpc-N-hACE2 mice at 7 dpi confirmed the distribution of SARS-CoV-2 N in lung epithelial cells and SARS-CoV-2 ΔN/GFP-HiBiT infection. Data are presented as means ± SD. Statistical significance was determined using one-way ANOVA with Dunnett’s multiple comparisons test ( h ). ns not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. ND not detected

Article Snippet: The antibodies used for IFAs were as follows: anti-hACE2 antibody (Sino Biological, 10108-RP01), anti-SARS-CoV-2 N antibody (Sino Biological, 40143-MM05), anti-GFP antibody (Sino Biological, 13105-R208), anti-IBA1 antibody (Abways, CY7217), anti-Ly6G antibody (Servicebio, GB11229), anti-CD68 antibody (Starter, SOB2064), anti-CD19 antibody (Servicebio, GB11061), anti-CD3 antibody (AiFang Biological, AFRM0030), anti-CD4 antibody (Abcam, ab183685), and anti-CD8 antibody (Abcam, ab217344).

Techniques: Infection, Staining, Immunofluorescence, Imaging

Journal: Signal Transduction and Targeted Therapy

Article Title: A biosafe mouse model for SARS-CoV-2 infection that more realistically simulates COVID-19 symptoms

doi: 10.1038/s41392-026-02640-5

Figure Lengend Snippet: Transcriptomic profiling of the lungs of SA-N-hACE2 and Sftpc-N-hACE2 mice revealed distinct molecular features. a Scheme illustrating the transcriptome sequencing. Gene expression heatmaps of IFN-I-related genes ( b ) and cytokines and chemokines ( c ) in the lungs of the mice. Mock: PBS-treated mice; the SA-N-hACE2 and Sftpc-N-hACE2 mice were infected with 1 × 10 6 TCID 50 of SARS-CoV-2 ΔN/GFP-HiBiT. d Bubble plot showing gene set enrichment analysis (GSEA) results for pathways enriched in the lungs of the infected mice. The color scale indicates normalized enrichment scores (NES), while the size of the bubbles corresponds to −log 10 ( p values)

Article Snippet: The antibodies used for IFAs were as follows: anti-hACE2 antibody (Sino Biological, 10108-RP01), anti-SARS-CoV-2 N antibody (Sino Biological, 40143-MM05), anti-GFP antibody (Sino Biological, 13105-R208), anti-IBA1 antibody (Abways, CY7217), anti-Ly6G antibody (Servicebio, GB11229), anti-CD68 antibody (Starter, SOB2064), anti-CD19 antibody (Servicebio, GB11061), anti-CD3 antibody (AiFang Biological, AFRM0030), anti-CD4 antibody (Abcam, ab183685), and anti-CD8 antibody (Abcam, ab217344).

Techniques: Sequencing, Gene Expression, Infection

Journal: Signal Transduction and Targeted Therapy

Article Title: A biosafe mouse model for SARS-CoV-2 infection that more realistically simulates COVID-19 symptoms

doi: 10.1038/s41392-026-02640-5

Figure Lengend Snippet: Assessment of anti-SARS-CoV-2 therapeutics using SA-N-hACE2 and Sftpc-N-hACE2 mouse models. a This schematic diagram illustrates the procedure for evaluating the neutralizing antibody 7B3 and NMV. SA-N-hACE2, Sftpc-N-hACE2, and K18-hACE2 KI mice received 10 mg/kg of 7B3 at 1 dpi or 300 mg/kg NMV via oral gavage from 0–3 dpi. SA-N-hACE2 and Sftpc-N-hACE2 mice were challenged with 1 × 10 6 TCID 50 of SARS-CoV-2 ΔN/GFP-HiBiT, whereas K18-hACE2 KI mice were infected with 3.57 × 10 2 TCID 50 of SARS-CoV-2 WT at 0 dpi. At 4 dpi, all mice were sacrificed, and lung samples were collected. The expression of the viral E gene in lung tissues from mock-, NMV- and 7B3-treated SA-N-hACE2 ( b ), Sftpc-N-hACE2 ( c ) and K18-hACE2 KI mice ( d ) was quantified via qRT‒PCR ( n = 4 per group). e – j Lung tissues from mock-, NMV-, and 7B3-treated SA-N-hACE2 ( e ), Sftpc-N-hACE2 ( f ), and K18-hACE2 KI ( g ) mice were analyzed using H&E staining to assess pathological changes, with histological scores documented for each group ( n = 4 per group) ( h – j ). Data are presented as means ± SD. Statistical significance was determined using one-way ANOVA with Dunnett’s multiple comparisons test ( b – d , h – j ). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: The antibodies used for IFAs were as follows: anti-hACE2 antibody (Sino Biological, 10108-RP01), anti-SARS-CoV-2 N antibody (Sino Biological, 40143-MM05), anti-GFP antibody (Sino Biological, 13105-R208), anti-IBA1 antibody (Abways, CY7217), anti-Ly6G antibody (Servicebio, GB11229), anti-CD68 antibody (Starter, SOB2064), anti-CD19 antibody (Servicebio, GB11061), anti-CD3 antibody (AiFang Biological, AFRM0030), anti-CD4 antibody (Abcam, ab183685), and anti-CD8 antibody (Abcam, ab217344).

Techniques: Infection, Expressing, Staining

Journal: EBioMedicine

Article Title: Monoclonal antibodies targeting two immunodominant epitopes on the Spike protein neutralize emerging SARS-CoV-2 variants of concern

doi: 10.1016/j.ebiom.2022.103818

Figure Lengend Snippet: Binding characteristics and inhibition activities of monoclonal antibodies specific to SARS-CoV-2 S/RBD. (a) Immunoreactivity of hybridoma clones culture supernatants derived from mice immunized with the spike (S) protein of SARS-CoV-2 and with RBD of Spike (b) in ELISA against the pre-fusion stabilized and trimerized S protein ectodomain (“S protein”) or RBD (“RBD”). Asterisks mark non-binders. (c) Blocking of interaction between RBD and ACE2 in a competition assay based on ELISA (RBD-ACE2), inhibition of the S protein internalization by cells expressing ACE2 (S-ACE2), inhibition of cell infection by MLV pseudotyped with SARS-CoV-2 spike protein (Pseudovirus) and plaque reduction neutralization test performed with an authentic SARS-CoV-2 virus isolate Slovakia/SK-BMC5/2020 (PRNT). Experiments were done with hybridoma culture supernatants adjusted with fresh cell culture medium to the same mAb concentration and then diluted for the assays as follows: RBD-ACE2 competitive ELISA 1:6 in PBS-T; S-ACE2 cell assay 1:50 in DMEM; pseudoviral assay 1:25 in DMEM; live virus PRNT 1:50 in EMEM. Positive control: serum of a mouse immunized with the S protein, diluted 1:200; Negative control: irrelevant mAb. All experiments are an average of at least two measurements, pseudoviral tests were all done in tetraplicates. (d) The purified monoclonal antibodies were tested for blocking the RBD-ACE2 interaction in competitive ELISA, the inhibition of ACE2-mediated S protein internalization by HEK293/17-hACE2 cells (e), and plaque reduction neutralization test (f) performed with strain Slovakia/SK-BMC5/2020. The curves are calculated from two replicates using Prism 6 for Windows (GraphPad Software). (g) Summary of assays from d-f. (h, i) Kinetic characteristics of the interactions of RBD with selected neutralizing antibodies. On-rate (k a ), off-rate (k d ) constants and equilibrium dissociation constant (K D ) of individual antibody-RBD complexes (±SD). Green dots mark antibodies AX290 and AX677 selected for further development.

Article Snippet: Cells were fixed with 4% paraformaldehyde and labelled with anti-human ACE2 antibody at 10 μg/ml (cat. # 600-401-X59; Rockland Immunochemicals), followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (Alexa Fluor 488; #A-11008; Invitrogen).

Techniques: Binding Assay, Inhibition, Bioprocessing, Clone Assay, Derivative Assay, Enzyme-linked Immunosorbent Assay, Blocking Assay, Competitive Binding Assay, Expressing, Infection, Plaque Reduction Neutralization Test, Virus, Cell Culture, Concentration Assay, Competitive ELISA, Positive Control, Negative Control, Purification, Software

Journal: EBioMedicine

Article Title: Monoclonal antibodies targeting two immunodominant epitopes on the Spike protein neutralize emerging SARS-CoV-2 variants of concern

doi: 10.1016/j.ebiom.2022.103818

Figure Lengend Snippet: The panel of selected neutralization antibodies target three non-overlapping epitopes on RBD. (a) Competitive ELISA was used for determination of the epitopes on RBD by NAbs. Signal reduction for more than 30 % was considered a positive competition. The experiments were performed in duplicates. (b) Difference plots depicting the changes in deuterium uptake in the RBD peptides upon binding to AX677 (left panels) and AX290 (right panels). The HDX reaction times are indicated in seconds. Numbers on x-axis represent amino acid positions of the recombinant RBD protein (adding 316 aligns them with the S protein numbering). Each plot represents an average of three technical replicates. (c, d) The positions of the peptides bound by the antibodies and identified in the HDX experiments are highlighted in the structure of SARS-CoV-2 spike receptor-binding domain bound to ACE2 reproduced from PDB 6M0J . The AX290 binding site is represented by shades of blue, AX677 by orange and yellow. Darker colours represent peptides that exhibit reduction in deuterium exchange (%D) ≥20%, light colours represent peptides with reduction in %D between 5-20%. ACE2 is shown in a green cartoon model, RBD as a grey surface model, mutations N439K (within AX677 binding site) and E484K (within AX290 binding site) are shown in red. The model was rendered using PyMOL Molecular Graphics System, Version 2.5.0a0 Open-Source, Schrödinger, LLC.

Article Snippet: Cells were fixed with 4% paraformaldehyde and labelled with anti-human ACE2 antibody at 10 μg/ml (cat. # 600-401-X59; Rockland Immunochemicals), followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (Alexa Fluor 488; #A-11008; Invitrogen).

Techniques: Neutralization, Competitive ELISA, Binding Assay, Recombinant

Journal: EBioMedicine

Article Title: Monoclonal antibodies targeting two immunodominant epitopes on the Spike protein neutralize emerging SARS-CoV-2 variants of concern

doi: 10.1016/j.ebiom.2022.103818

Figure Lengend Snippet: A combination of AX290 and AX677 prevents rapid mutational escape of authentic live SARS-CoV-2 virus. (a) A schematic diagram of microplate well allocations in the SARS-CoV-2 escape mutants experiment. MAbs were serially five-fold diluted starting with 50 µg/mL (left panels), pre-incubated with live SARS-CoV-2 (Slovakia/SK-BMC5/2020) virus (diluted to MOI=0·5) and added to Vero E6 cells (1 st passage, middle panels). Culture medium from the first wells of the mAb dilution series where CPE appeared (indicated with the yellow circles) were used for the 2 nd passage (right panels). White squares highlight wells with CPE where virus genomes were sequenced. C - control wells with virus without mAbs were sequenced to monitor possible tissue culture adaptations. (b) The table shows amino acid changes resulting from nonsynonymous mutations in the virus genome that appeared under selection pressure of mAbs. Combination of the two antibodies prevented appearance of escape mutations in the Spike protein. (c) The positions of the amino acids that were mutated in the viruses that escaped from the neutralization by the antibodies are highlighted in pink in the structure of SARS-CoV-2 Spike receptor-binding domain bound to ACE2 (reproduced from PDB 6M0J , see also Figure 2 c, d). T345 is located close to the AX677 binding site (orange and yellow, the structure on the left), S477 is located in the binding site of AX290 (blue, structure on the right). (d) Changes in the proportion of the mutant viruses in the mixture with the parental virus before and after three days of co-cultivation with Vero E6 cells in the absence of the monoclonal antibodies determined by nanopore sequencing. The mutant viruses were arbitrarily named A1, B11 and C25 for the escape viruses from AX290, AX677 and AX290+AX677 mix, respectively. Mean and SD are shown from three separate passages of the viral mixtures.

Article Snippet: Cells were fixed with 4% paraformaldehyde and labelled with anti-human ACE2 antibody at 10 μg/ml (cat. # 600-401-X59; Rockland Immunochemicals), followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (Alexa Fluor 488; #A-11008; Invitrogen).

Techniques: Virus, Incubation, Control, Selection, Neutralization, Binding Assay, Mutagenesis, Bioprocessing, Nanopore Sequencing

Journal: EBioMedicine

Article Title: Monoclonal antibodies targeting two immunodominant epitopes on the Spike protein neutralize emerging SARS-CoV-2 variants of concern

doi: 10.1016/j.ebiom.2022.103818

Figure Lengend Snippet: Antibody prophylaxis against SARS-CoV-2 infection in a mouse model. (a) Schematic of the in vivo experimental procedures. Wild-type mice were transduced with AAV-hACE2 by forced inhalation. After 11 days, the mice were inoculated subcutaneously (s.c.) with 1·25 mg of the indicated mAbs antibodies one day (-24 h, n=10 animals per group) before being infected intranasally (i.n.) with SARS-CoV-2 (1 × 10 4 pfu). Health characteristics and body weight were monitored daily for the duration of the experiment. 5 animals per group were sacrificed 3 and 7 days after infection and lung tissue analyzed for viral load (on day 3 only), viral RNA and histopathological changes. (b) Body weight was monitored daily for 7 days (n=5 mice per group). Mean with s.d. is shown. (c) Viral burden in the lungs of mice infected with SARS-CoV-2 (n = 5 per group) as measured 3 dpi by plaque assays. Mean with s.d. is shown. Dashed line indicates the limit of detection (2·14 log pfu/g of tissue). All three treated groups were compared to the Isotype Ctrl group, p values were calculated using non-parametric Mann-Whitney test, in all comparisons p=0·0079 (exact p values were calculated). (d) The analysis of viral RNA copy numbers in the lungs shows clear reduction in all animals treated with anti-Spike antibodies compared to isotype control on day 3 (p=0·0079 for AX290, p=0·0159 for AX677 and p=0·0079 for AX290+AX677; evaluated by non-parametric Mann-Whitney test). On day 7 the copy numbers were greatly reduced in all animals, probably due to the elimination of the replicating viruses. The bars represent mean with s.d. Histological analysis of hematoxylin/eosin-stained lung sections collected 7 days after infection showed accumulation of the interstitial inflammatory infiltrates (e) and reduction of normal tissue (f) in animals treated with isotype antibody control. These histopathological changes were reduced by the anti-Spike antibodies AX290 and AX677 applied either alone or in combination. Data were evaluated by Mann-Whitney test (** p=0·0079, * p=0·0159).

Article Snippet: Cells were fixed with 4% paraformaldehyde and labelled with anti-human ACE2 antibody at 10 μg/ml (cat. # 600-401-X59; Rockland Immunochemicals), followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (Alexa Fluor 488; #A-11008; Invitrogen).

Techniques: Infection, In Vivo, Transduction, MANN-WHITNEY, Control, Staining

Journal: Frontiers in oncology

Article Title: The Angiotensin-Converting Enzyme Inhibitory State Promotes the Transformation of Non-Small Cell Lung Cancer Blood Supply Pattern Toward Vasculogenic Mimicry Formation.

doi: 10.3389/fonc.2021.663671

Figure Lengend Snippet: FIGURE 1 | High ACE2 expression was linked to increased VM and better prognosis in NSCLC. (A) Typical image of ACE2, VE-cadherin, EphA2 protein expression and CD34/PAS double staining in TMA tissues. Case B1 had massive CD34−/PAS+ VM (yellow arrows) lined by ACE2, VE-cadherin and EphA2 high expressing tumor cells. Case G1 had abundant CD34+/PAS−MVs (black arrows) with ACE2, VE-cadherin and EphA2 low expressing tumor cells. (B) Kaplan-Meier analysis of OS in NSCLC patients with ACE2 low or high expression. P = 0.044.

Article Snippet: The sections were dewaxed, rehydrated, treated with antigen retrieval, hatched with primary antibodies of ACE2 (1:200), VE-cadherin (1:200), EphA2 (1:100), and CD34 (1:200, ab81289, Abcam) overnight at 4°C, individually exposed to secondary antibody (SA1020, BOSTER, USA) 1 h at RT, and colored with DAB peroxidase substrate.

Techniques: Expressing, Double Staining

Journal: Frontiers in oncology

Article Title: The Angiotensin-Converting Enzyme Inhibitory State Promotes the Transformation of Non-Small Cell Lung Cancer Blood Supply Pattern Toward Vasculogenic Mimicry Formation.

doi: 10.3389/fonc.2021.663671

Figure Lengend Snippet: FIGURE 2 | ACE2-induced better outcome in NSCLC patients might be attributed to less vessels and more VM formation. (A, C, E, G) Typical tissue images of each group stained with ACE2 or CD34/PAS. Yellow arrows: CD34−/PAS+ VMs; black arrows: CD34+/PAS−MV. (B, D, F, H) Kaplan–Meier analysis of OS in each group.

Article Snippet: The sections were dewaxed, rehydrated, treated with antigen retrieval, hatched with primary antibodies of ACE2 (1:200), VE-cadherin (1:200), EphA2 (1:100), and CD34 (1:200, ab81289, Abcam) overnight at 4°C, individually exposed to secondary antibody (SA1020, BOSTER, USA) 1 h at RT, and colored with DAB peroxidase substrate.

Techniques: Staining

Journal: Frontiers in oncology

Article Title: The Angiotensin-Converting Enzyme Inhibitory State Promotes the Transformation of Non-Small Cell Lung Cancer Blood Supply Pattern Toward Vasculogenic Mimicry Formation.

doi: 10.3389/fonc.2021.663671

Figure Lengend Snippet: FIGURE 3 | Human ACE2 was stably overexpressed in A549-ACE2-OE cells. (A) Schematic representation of pLenti6.3-MCS/V5 DEST. (B) pLenti6.3-ACE2 expression vector was detected by PCR. (C) Fluorescence of EGFP in A549-ACE2-OE cells (left) and parental cells (right) was determined by fluorescence microscopy. (D) RT-PCR experiment of ACE2 mRNA level in A549-ACE2-OE cells and control cells, Mean ± SD, n = 3, *p < 0.05. (E) Western blot analysis of ACE2 expression level in A549-ACE2-OE cells and parental cells. (F) Quantification of ACE2 expression level in A549-ACE2-OE cells and parental cells. Mean ± SD, n = 3, ***p < 0.001.

Article Snippet: The sections were dewaxed, rehydrated, treated with antigen retrieval, hatched with primary antibodies of ACE2 (1:200), VE-cadherin (1:200), EphA2 (1:100), and CD34 (1:200, ab81289, Abcam) overnight at 4°C, individually exposed to secondary antibody (SA1020, BOSTER, USA) 1 h at RT, and colored with DAB peroxidase substrate.

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Fluorescence, Microscopy, Reverse Transcription Polymerase Chain Reaction, Control, Western Blot

Journal: Frontiers in oncology

Article Title: The Angiotensin-Converting Enzyme Inhibitory State Promotes the Transformation of Non-Small Cell Lung Cancer Blood Supply Pattern Toward Vasculogenic Mimicry Formation.

doi: 10.3389/fonc.2021.663671

Figure Lengend Snippet: FIGURE 4 | Tube formation ability of A549 cells was improved with ACE inhibitory state. (A) Morphologies of a panel of A549-ACE2-OE cells, A549-NC cells, and A549-NC cells treated with ACEI (1, 5, and 10 nM/L) were shown as sheet-like and thread-like cell types in 2D culture, which were outlined partly with yellow lines. Representative images were shown above. Upper: white light; lower: fluorescence. (B) Quantification of sheet-like or thread-like cells and pebble-like cells in three groups, representatively, Mean ± SD, n = 3, **p < 0.01. (C) Images of both above cell lines grown in 3D Matrix gel. A 10 nM/L ACEI dilution was performed. Representative images were shown above. Yellow arrows point out the free cancer cells escaping from tube wall and isolated segments. Upper: white light; lower: fluorescence. (D, E) Images of 3D culture were applied to determine average number of nodes, branches, isolated segments, meshes and mean mesh area in those groups, per field, Mean ± SD, n = 3, **p < 0.01, *p < 0.05. ns, no significance.

Article Snippet: The sections were dewaxed, rehydrated, treated with antigen retrieval, hatched with primary antibodies of ACE2 (1:200), VE-cadherin (1:200), EphA2 (1:100), and CD34 (1:200, ab81289, Abcam) overnight at 4°C, individually exposed to secondary antibody (SA1020, BOSTER, USA) 1 h at RT, and colored with DAB peroxidase substrate.

Techniques: Isolation

Journal: Frontiers in oncology

Article Title: The Angiotensin-Converting Enzyme Inhibitory State Promotes the Transformation of Non-Small Cell Lung Cancer Blood Supply Pattern Toward Vasculogenic Mimicry Formation.

doi: 10.3389/fonc.2021.663671

Figure Lengend Snippet: FIGURE 5 | VM formation was increased, and vasculature was lessened due to inhibition of RAS in vivo. (A) Growth curve of allograft tumors of A549-ACE2-OE cells, A549-NC cells with or without ACEI treatment, Mean ± SD, n = 3, *p < 0.05. ns, no significance. (B) Weight of resected tumors, Mean ± SD, n = 3, **p < 0.01, *p < 0.05. (C) Continuous sections of allograft tumor tissues stained with PAS, CD34, VE-cadherin, or EphA2 immunohistochemical stain. Black arrow points out a typical MV (CD34+/PAS−); yellow arrows point out typical VM (CD34−/PAS+). (D) Quantification of MV and VM in different groups, Mean ± SD, n = 3, per field, ***p < 0.001, **p < 0.01. (E) Quantification of VE-cadherin and EphA2 mean optical density in three groups, Mean ± SD, n = 3, ***p < 0.001, **p < 0.01.

Article Snippet: The sections were dewaxed, rehydrated, treated with antigen retrieval, hatched with primary antibodies of ACE2 (1:200), VE-cadherin (1:200), EphA2 (1:100), and CD34 (1:200, ab81289, Abcam) overnight at 4°C, individually exposed to secondary antibody (SA1020, BOSTER, USA) 1 h at RT, and colored with DAB peroxidase substrate.

Techniques: Inhibition, In Vivo, Staining, Immunohistochemical staining

Journal: Frontiers in oncology

Article Title: The Angiotensin-Converting Enzyme Inhibitory State Promotes the Transformation of Non-Small Cell Lung Cancer Blood Supply Pattern Toward Vasculogenic Mimicry Formation.

doi: 10.3389/fonc.2021.663671

Figure Lengend Snippet: FIGURE 6 | VE-cadherin and EphA2 expression was upregulated in A549 cells and NSCLC tissues with impaired local RAS status. (A, B) RT-PCR experiment of VE-cadherin and EphA2 mRNA level in A549-ACE2-OE cells and control cells, Mean ± SD, n = 3, ***p < 0.001. (C–E) Western blot analysis and quantification of VE- cadherin and EphA2 expression level in A549-ACE2-OE cells and control cells, Mean ± SD, n = 3, ***p < 0.001. (F, G) Linear regressions of VM number and VE- cadherin (P < 0.0001) or EphA2 (P = 0.0108) score in TMA. (H) Typical tissue images of both groups stained with VE-cadherin, ACE2 or CD34/PAS. Case F13 with ACE2 low status was provided with rambling VM covered by tumor cells which only expressed VE-cadherin in nuclei; case D6 with ACE2 high status had ordered VM lined by tumor cells expressing VE-cadherin in both nuclei and cytomembranes. Red arrow: VE-cadherin membrane expression.

Article Snippet: The sections were dewaxed, rehydrated, treated with antigen retrieval, hatched with primary antibodies of ACE2 (1:200), VE-cadherin (1:200), EphA2 (1:100), and CD34 (1:200, ab81289, Abcam) overnight at 4°C, individually exposed to secondary antibody (SA1020, BOSTER, USA) 1 h at RT, and colored with DAB peroxidase substrate.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Western Blot, Staining, Membrane

Journal: Frontiers in oncology

Article Title: The Angiotensin-Converting Enzyme Inhibitory State Promotes the Transformation of Non-Small Cell Lung Cancer Blood Supply Pattern Toward Vasculogenic Mimicry Formation.

doi: 10.3389/fonc.2021.663671

Figure Lengend Snippet: FIGURE 7 | PI3K/AKT, p38MAPK, and HIF1-a were inactivated, and Nodal/Notch4 pathway was activated in A549-ACE2-OE cell model. (A–E) Western blot analysis and quantification of AKT, p-AKT, p38, and p-p38 expression level in A549-ACE2-OE cells and negative control, Mean ± SD, n = 3, ***p < 0.001, **p < 0.01. (F, G) Immunoflurescence assay and quantification of HIF1-a mean optical density in A549-ACE2-OE cells and negative control, Mean ± SD, n = 4, *p < 0.05. (H, I) Western blot analysis and quantification of Nodal and Notch4 expression level in A549-ACE2-OE cells and negative control, Mean ± SD, n = 3, ***p < 0.001, **p < 0.01.

Article Snippet: The sections were dewaxed, rehydrated, treated with antigen retrieval, hatched with primary antibodies of ACE2 (1:200), VE-cadherin (1:200), EphA2 (1:100), and CD34 (1:200, ab81289, Abcam) overnight at 4°C, individually exposed to secondary antibody (SA1020, BOSTER, USA) 1 h at RT, and colored with DAB peroxidase substrate.

Techniques: Western Blot, Expressing, Negative Control