Journal: EBioMedicine

Article Title: Primate-specific BTN3A2 protects against SARS-CoV-2 infection by interacting with and reducing ACE2.

doi: 10.1016/j.ebiom.2024.105281

Figure Lengend Snippet: Fig. 4: BTN3A2 competed with ACE2 to bind to the Spike protein. a: BTN3A2 isoform (BTN3A2-L-His or BTN3A2-S-His) interacted with ACE2. b: ACE2 interacted with BTN3A2-L. c: Endogenous BTN3A2 interacted with the ACE2. Procedures were similar to Fig. 3e d: Biolayer interferometry analysis of BTN3A2-L binding to immobilized ACE2-Fc. Different concentrations of BTN3A2-L (2000, 1000, 250, and 62.5 nM; corresponding to kinetic curves from top to bottom) were used. e: Biolayer interferometry analysis of BTN3A2-S binding to immobilized ACE2-Fc. Different concentrations of BTN3A2-S (1000 and 500 nM; corresponding to kinetic curves from top to bottom) were used. f-g: Competition ELISA analysis of competitive binding of BTN3A2-L with Spike RBD. f: Schematic of competition assays. g: Competition between ACE2 (ACE2-His-tagged) and BTN3A2-L (BTN3A2) for immobilized Spike S1 subunit (S1-Fc). ACE2 and S1-Fc protein or human IgG1-Fc and S1-Fc protein were incubated for 12 h, followed by the addition of dilution series of BTN3A2-L-His. Amount of S1 protein remaining in the presence of the competitor was determined by antihuman HRP through a colorimetric readout. h: BTN3A2-L competed with ACE2 to bind to the Spike S1. Procedures for IP and IB in (a-b) and (h) were similar to those in Fig. 3c.

Article Snippet: The membranes were then blocked with 5% non-fat dry milk in Tris-buffered saline (#9997, Cell Signaling Technology, USA) with 0.1% Tween 20 (P1379, Sigma, USA) (TBST) at room temperature for 2 h. Membranes were incubated with primary antibodies against His (1:5000, M20001M, Abmart, China), HA (1:5000, M20003M, Abmart, China), Flag (1:5000, M20008M, Abmart, China), ACE2 (1:1000, 66699-1-Ig, Proteintech, China), BTN3A2 (1:1000, TA500730, Origene, USA), Spike (1:1000, 28869-1-AP, Proteintech, www.thelancet.com Vol 107 September, 2024 China), and Tubulin (1:10,000, OTI3H2, ZSGB-Bio, China) overnight at 4 ◦C, respectively.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation

Journal: EBioMedicine

Article Title: Primate-specific BTN3A2 protects against SARS-CoV-2 infection by interacting with and reducing ACE2.

doi: 10.1016/j.ebiom.2024.105281

Figure Lengend Snippet: Fig. 5: BTN3A2 inhibited ACE2 expression. a: Differential expression of BTN3A2 and ACE2 in scRNA-seq dataset of lung tissues from COVID-19 patients and uninfected individuals. Original dataset was reported in Melms et al.43 Significance was assessed by two-sided Wilcox rank sum test. b: Stable expression of BTN3A2 decreased ACE2 mRNA level in Huh7 cells. RNA was extracted from Huh7 cells overexpressing BTN3A2-L and BTN3A2-S, or control (Vector Huh7) cells (each group 1 × 105 cells). BTN3A2 and ACE2 mRNA levels were measured by qRT-PCR, with normalization to GAPDH. c-d: Overexpression of BTN3A2-L (c) and BTN3A2-S (d) suppressed ACE2 protein expression in HEK293T cells. Cells (5 × 105) were transfected with indicated expression vector or empty vector (each 1.25 μg) for 48 h, then harvested for western blotting. e: Dose-dependent inhibitory effect of BTN3A2-L on ACE2 protein expression. HEK293T cells (5 × 105) were transfected with indicated expression

Article Snippet: The membranes were then blocked with 5% non-fat dry milk in Tris-buffered saline (#9997, Cell Signaling Technology, USA) with 0.1% Tween 20 (P1379, Sigma, USA) (TBST) at room temperature for 2 h. Membranes were incubated with primary antibodies against His (1:5000, M20001M, Abmart, China), HA (1:5000, M20003M, Abmart, China), Flag (1:5000, M20008M, Abmart, China), ACE2 (1:1000, 66699-1-Ig, Proteintech, China), BTN3A2 (1:1000, TA500730, Origene, USA), Spike (1:1000, 28869-1-AP, Proteintech, www.thelancet.com Vol 107 September, 2024 China), and Tubulin (1:10,000, OTI3H2, ZSGB-Bio, China) overnight at 4 ◦C, respectively.

Techniques: Expressing, Quantitative Proteomics, Control, Plasmid Preparation, Quantitative RT-PCR, Over Expression, Transfection, Western Blot

Journal: EBioMedicine

Article Title: Primate-specific BTN3A2 protects against SARS-CoV-2 infection by interacting with and reducing ACE2.

doi: 10.1016/j.ebiom.2024.105281

Figure Lengend Snippet: Fig. 6: BTN3A2 suppressed ACE-mediated SARS-CoV-2 entry. a: Schematic of animal experiments with SARS-CoV-2 inoculation. BTN3A2-tg (n = 6) and WT mice (C57BL/6J, n = 7) were intranasally infected with AAV9-hACE2 (2 × 1012 viral genome copies) for 14 days, with all animals then infected with BA.2 (5 × 104 TCID50). All mice were euthanized at 3 dpi for tissue collection. b: Viral RNA copies in lung tissues of SARS- CoV-2-infected mice at 3 dpi. Each dot indicates log copies of viral genomic N gene, E gene, and subgenomic E (sgE) of a mixed sample of five lung lobes from an individual mouse. Viral copies are presented as mean ± SD. ***, P < 0.001; ANOVA with Dunnett’s multiple comparisons. c: Representative image of immunohistochemical staining of SARS-CoV-2 N protein in lung tissues of WT and BTN3A2-tg mice at 3 dpi. Scale bars: 500 μm for entire lung lobe section (left) and 100 μm for enlarged view of boxed areas labeled by numbers in entire section (right). d: Representative of image of H&E staining of lung sections in SARS-CoV-2-infected WT and BTN3A2-tg mice. e: AAV-mediated hACE2 expression in lungs tissues of WT (C57BL/6J, n = 7) and BTN3A2-tg mice (n = 6) at 3 dpi. f: Representative image of immunohistochemical staining for ACE2 and BTN3A2 in lung tissues of WT and BTN3A2-tg mice at 3 dpi. Scale bars were same as those in (c).

Article Snippet: The membranes were then blocked with 5% non-fat dry milk in Tris-buffered saline (#9997, Cell Signaling Technology, USA) with 0.1% Tween 20 (P1379, Sigma, USA) (TBST) at room temperature for 2 h. Membranes were incubated with primary antibodies against His (1:5000, M20001M, Abmart, China), HA (1:5000, M20003M, Abmart, China), Flag (1:5000, M20008M, Abmart, China), ACE2 (1:1000, 66699-1-Ig, Proteintech, China), BTN3A2 (1:1000, TA500730, Origene, USA), Spike (1:1000, 28869-1-AP, Proteintech, www.thelancet.com Vol 107 September, 2024 China), and Tubulin (1:10,000, OTI3H2, ZSGB-Bio, China) overnight at 4 ◦C, respectively.

Techniques: Infection, Immunohistochemical staining, Staining, Labeling, Expressing

Journal: PLoS Pathogens

Article Title: SARS-CoV-2 variants with reduced infectivity and varied sensitivity to the BNT162b2 vaccine are developed during the course of infection

doi: 10.1371/journal.ppat.1010242

Figure Lengend Snippet: (A) Phylogenetic trees depicting N genes isolated from nose-throat swabs by SGS. IDs of individuals from whom swabs were isolated are indicated in the figure. Black rectangles depict the dominant N gene isolated from each swab sample. Red rectangles depict N genes with missense mutations, grey rectangles depict N genes with silent mutations and blue rectangles depict N genes with nonsense mutations. Mutations that are not documented in the GSAID are marked with an asterisk. For each N-gene variant, the nucleotide substitution is indicated (based on Wuhan-Hu-1 numbering). The segment with the number below each phylogenetic tree shows the length of branch that represents an amount genetic change. The amount of genetic change is the number of nucleotide substitutions divided by the length of the N-gene sequence. (B) Pie charts showing the proportion of all N-gene sequences isolated from each swab sample. The White pie slice depicts unmutated sequences. The Colored pie slices depict mutated N genes. The different mutations are indicated in the figure. The number in the middle of the pie chart depicts the total number of N-gene variants that were sequenced. (C) Relative p24 value as measured by adding 20 μl of supernatant containing the pseudovirus to Lenti-X GoStix Plus. The x-axis depicts the SARS-CoV-2 pseudoviruses that were produced, and the y-axis depicts the relative p24 protein (GoStix values) in the supernatant of each pseudovirus. (D) 293T-ACE2 infection with SARS-CoV-2 pseudovirses expressing mutated N proteins and unmutated (Wuhan-Hu-1) N protein (WT). The x-axis depicts the mutation in the N proteins of the pseudovirus that was used for infection. The y-axis depicts the luminescence levels that were measured 48 hours post infection. Experiments were done in triplicates and repeated three times. One representative experiment is shown. Mean values and standard errors are shown. Statistically significant differences in comparison to the WT SARS-CoV-2 pseudovirus are indicated (student’s t test, p < 0.005). Figure was generated using biorender.com .

Article Snippet: 293T-ACE2 (5x10 4 ) cells were seeded in a 96-well plate, washed, and were incubated with polyclonal anti-ACE2 antibody (Bioss, BS-23028R) or RBD-Ig [ ] for 1 hour at 4°C at 1:20 dilution, followed by incubation with PE-conjugated goat anti-human IgG (Jackson, Cat #109-116-088, 1:200) for 45 minutes.

Techniques: Isolation, Variant Assay, Sequencing, Produced, Infection, Expressing, Mutagenesis, Generated

Journal: PLoS Pathogens

Article Title: SARS-CoV-2 variants with reduced infectivity and varied sensitivity to the BNT162b2 vaccine are developed during the course of infection

doi: 10.1371/journal.ppat.1010242

Figure Lengend Snippet: (A) Schematic representation of the ten spike variants that were tested for infectivity. Amino acids substitution in each spike mutants is shown in the figure. NTD = N-terminal domain, RBD = receptor binding domain, FP = fusion peptide, HR1 = heptad repeat 1, HR2 = heptad repeat 2, TM = transmembrane domain, CT = cytoplasmic domain. (B) Relative p24 value as measured by adding 20 μl of supernatant containing the pseudovirus to Lenti-X GoStix Plus. The y-axis depicts the SARS-CoV-2 pseudoviruses that were produced, and the x-axis depicts the relative p24 protein (GoStix values) in the supernatant of each pseudovirus. WT = pseudovirus that expresses an unmutated (Wuhan-Hu-1) spike. (C) FACS staining of 293T-ACE2 cells. The gray histogram shows the staining of the 293T-ACE2 cells with secondary antibody only. The empty black histograms depict the staining anti-ACE2 antibody or RBD-Ig. Shown is one representative experiment out of three preformed. (D) 293T-ACE2 infection with SARS-CoV-2 pseudovirses expressing mutated spikes, unmutated (Wuhan-Hu-1) spike protein and bald pseudovirus. The x-axis depicts the mutation in the spike of the pseudovirus that was used for infection. The y-axis depicts the luminescence levels that were measured 48 hours post infection. Experiments were done in triplicates and repeated three times. One representative experiment is shown. Mean values and standard errors are shown. WT = pseudovirus that expresses an unmutated (Wuhan-Hu-1) spike. Statistically significant differences in comparison to the WT SARS-CoV-2 pseudovirus are indicated (student’s t test, *p < 0.05, **p < 0.005). Figure was generated using biorender.com .

Article Snippet: 293T-ACE2 (5x10 4 ) cells were seeded in a 96-well plate, washed, and were incubated with polyclonal anti-ACE2 antibody (Bioss, BS-23028R) or RBD-Ig [ ] for 1 hour at 4°C at 1:20 dilution, followed by incubation with PE-conjugated goat anti-human IgG (Jackson, Cat #109-116-088, 1:200) for 45 minutes.

Techniques: Infection, Binding Assay, Produced, Staining, Expressing, Mutagenesis, Generated

Journal: PLoS Pathogens

Article Title: SARS-CoV-2 variants with reduced infectivity and varied sensitivity to the BNT162b2 vaccine are developed during the course of infection

doi: 10.1371/journal.ppat.1010242

Figure Lengend Snippet: (A, C) OD values of ELISA against spike S1 subunit of plasma from vaccinated individuals (A) and of convalescent plasma (C). Experiments were done in triplicates and repeated three times. One representative experiment is shown. Mean values and standard errors are shown. OD values of control plasma (unvaccinated healthy individual) are shown in the red dashed graph. (B) The luminescence values derived from 293T-ACE2 cells 48 hours after infection with nanoluc-expressing SARS-CoV-2 pseudovirus and following incubation with increasing concentrations of plasma from vaccinated individuals. The plasma dilution is shown in the x-axis. Luminescence values after incubation with control (unvaccinated healthy individual) is shown in the red-dashed graph. Experiments were done in triplicates and repeated two times. Mean values and standard errors are shown; representative of two independent experiments is shown. (D) The luminescence values derived from 293T-ACE2 cells 48 hours post infection with nanoluc-expressing SARS-CoV-2 pseudovirus and following incubation with convalescent plasma at 10 −3 dilution. The study ID of the plasma samples is shown in the x-axis. Experiments were done in triplicates and repeated two times. Mean values and standard errors are shown; representative of two independent experiments is shown.

Article Snippet: 293T-ACE2 (5x10 4 ) cells were seeded in a 96-well plate, washed, and were incubated with polyclonal anti-ACE2 antibody (Bioss, BS-23028R) or RBD-Ig [ ] for 1 hour at 4°C at 1:20 dilution, followed by incubation with PE-conjugated goat anti-human IgG (Jackson, Cat #109-116-088, 1:200) for 45 minutes.

Techniques: Enzyme-linked Immunosorbent Assay, Derivative Assay, Infection, Expressing, Incubation

Journal: PLoS ONE

Article Title: Endoplasmic Reticulum Stress-Sensing Mechanism Is Activated in Entamoeba histolytica upon Treatment with Nitric Oxide

doi: 10.1371/journal.pone.0031777

Figure Lengend Snippet: Genes upregulated encoding metabolism enzymes.

Article Snippet: The primary antibody used were a rabbit anti-PFOR polyclonal antibody (1∶400 dilution, a kind gift from Dr Ester Orozco, CINVESTAV, Mexico), a mouse anti-actin monoclonal antibody (1∶20,000 dilution; #69100, MP Biomedicals).

Techniques:

Journal: PLoS ONE

Article Title: Endoplasmic Reticulum Stress-Sensing Mechanism Is Activated in Entamoeba histolytica upon Treatment with Nitric Oxide

doi: 10.1371/journal.pone.0031777

Figure Lengend Snippet: PFOR of 120 kDa was revealed upon SDS-PAGE and immunobloting of crude extracts from SNP-treated and control trophozoites (4, 2 and 1×10 4 cells). The loaded amount of proteins for each condition is evidenced by actin (43 kDa) immunodetection on the same western blot. Quantification of signal emission did not reveal differences between the tested conditions in 3 independent experiments.

Article Snippet: The primary antibody used were a rabbit anti-PFOR polyclonal antibody (1∶400 dilution, a kind gift from Dr Ester Orozco, CINVESTAV, Mexico), a mouse anti-actin monoclonal antibody (1∶20,000 dilution; #69100, MP Biomedicals).

Techniques: SDS Page, Western Blot, Control, Immunodetection

Journal: PLoS ONE

Article Title: Endoplasmic Reticulum Stress-Sensing Mechanism Is Activated in Entamoeba histolytica upon Treatment with Nitric Oxide

doi: 10.1371/journal.pone.0031777

Figure Lengend Snippet: PFOR activities upon no treatment.

Article Snippet: The primary antibody used were a rabbit anti-PFOR polyclonal antibody (1∶400 dilution, a kind gift from Dr Ester Orozco, CINVESTAV, Mexico), a mouse anti-actin monoclonal antibody (1∶20,000 dilution; #69100, MP Biomedicals).

Techniques: Activity Assay

Journal: Biomedicines

Article Title: Morphological and Immunocytochemical Characterization of Paclitaxel-Induced Microcells in Sk-Mel-28 Melanoma Cells

doi: 10.3390/biomedicines12071576

Figure Lengend Snippet: Primary and secondary antibodies.

Article Snippet: Caspase-2 , Primary antibody: rabbit anti-human, rabbit polyclonal , FITC Conjugated; Ex.494 nm/Em.518 nm , 1:100 , BS-5802R-FITC, Bioss antibodies/Nordic BioSite, Täby, Sweden.

Techniques: Concentration Assay, Recombinant