Journal: bioRxiv

Article Title: Delivery of loaded MR1 Monomer Results in Efficient Ligand Exchange to Host MR1 and Subsequent MR1T cell activation

doi: 10.1101/2022.07.11.499573

Figure Lengend Snippet: A . Biotinylated MR1 monomer, recombinant TCR, or HLA-A2 were immobilized on a traptavidin-coated plate before incubation with PBS at the indicated pH overnight at 4C. Binding of either anti-MR1 (clone 26.5) or anti-b2m (clone B2M-01) antibody was assess with an HRP-conjugated secondary antibody and TMB substrate. Error bars denote standard deviation. B . MR1T clone (5e3) IFN-γ response to BEAS-2B (1e4) pre-treated with Bafilomycin A1 for 2 hours, followed by addition of the MR1/5-OP-RU monomer or 5-OP-RU prodrug. Data in B are representative of three independent experiments.

Article Snippet: After removal of the acidic buffer and washing with PBS pH 7.2, either anti-human MR1 (unlabeled, clone 26.5, BioLegend 361102) or anti-human B2m (unlabeled, clone B2M-01, Novus Biologicals NB500-317) antibody was applied to the plate, followed by a rabbit anti-mouse IgG H+L, HRP-conjugated secondary antibody (Abcam ab6728).

Techniques: Recombinant, Incubation, Binding Assay, Standard Deviation

Journal: Viruses

Article Title: Garlic Organosulfur Compounds Reduce Inflammation and Oxidative Stress during Dengue Virus Infection

doi: 10.3390/v9070159

Figure Lengend Snippet: Garlic organosulfur compounds do not significantly affect dengue virus infection. Cells were treated with indicated doses of garlic organosulfur compounds and immediately infected with dengue virus 2 New Guinea C (DENV 2 NGC) at multiplicity of infection (MOI) of 1.0. At 24 h post-infection, RNA was isolated from cell lysates and used in quantitative reverse transcription (qRT)-PCR analysis to detect dengue virus infection levels. Viral gene expression was normalized to human β-2 microglobulin expression. ( A ). Huh-7 cells; ( B ). U937 cells. Data is expressed as mean ± SD. Technical and biological replicates were done in triplicate. DAS: diallyl sulfide; DADS: diallyl disulfide.

Article Snippet: Dengue virus primers were: F: 5′-CATTCCAAGTGAGAATCTCTTTGTCA-3′ and R: 5′-CAGATCTCTGATGAATAACCAACG-3′; Viral gene expression was normalized to human β-2 microglobulin using a purchased primer set (#HP207421 Origene, Rockville, MD, USA).

Techniques: Virus, Infection, Isolation, Reverse Transcription, Quantitative RT-PCR, Gene Expression, Expressing

Journal: Scientific reports

Article Title: Targeted clearance of senescent cells using an antibody-drug conjugate against a specific membrane marker.

doi: 10.1038/s41598-021-99852-2

Figure Lengend Snippet: Figure 1. B2M expression increases with age in mice. (A) Representative images of immunohistochemical staining (left) of skin and brain (frontal lobe) samples from mice of different ages (3–87 weeks), showing B2M expression, as compared to p16 (senescence marker) Magnification: × 20. Graphs show quantitation of expression as %DAB (right). Bars represent the average of three independent samples for skin and five for brain, error bars represent standard deviation (SD). Fold change quantifications of B2M protein (B) and mRNA (C) expression levels, as measured by Western blot and quantitative real-time PCR, respectively, in EJp53, EJp21, EJp16 and HT1080-p21-9 cells induced to senesce for 4 days, normalized to the expression of their proliferating counterparts (represented by the dotted line at y = 1). Bars represent the mean ± SD of 3 independent experiments. ** p < 0.005 (compared to proliferating controls).

Article Snippet: In order to express B2M, a B2M cDNA (OriGene) was transfected into EJ cells using Lipofectamine 2000 reagent (Invitrogen), following manufacturer’s instructions, in a 1 μg: 2.5 μl ratio.

Techniques: Expressing, Immunohistochemical staining, Staining, Marker, Quantitation Assay, Standard Deviation, Western Blot, Real-time Polymerase Chain Reaction

Journal: Scientific reports

Article Title: Targeted clearance of senescent cells using an antibody-drug conjugate against a specific membrane marker.

doi: 10.1038/s41598-021-99852-2

Figure Lengend Snippet: Figure 2. Characteristics of B2M-ADCs. (A) Internalization rate of the unconjugated B2M antibody in Control EJp16 cells as measured by CypHer5E fluorescence in a FACS analysis. Results show mean ± SD of three independent experiments. Results show mean ± SD of three independent experiments. (B) Size-Exclusion Chromatography characterization of the purity of the final ADC mix, compared to pure antibody and PBS. (C) SDS page comparing 1 µg of the unconjugated B2M antibody (UC) to an aliquot of the B2M ADC. (D) Quantitation of Drug-Antibody Ratios (DAR) of the B2M conjugated antibodies. Numbers indicate percentages of each DAR population in the mixture. Average DAR: 2.02.

Article Snippet: In order to express B2M, a B2M cDNA (OriGene) was transfected into EJ cells using Lipofectamine 2000 reagent (Invitrogen), following manufacturer’s instructions, in a 1 μg: 2.5 μl ratio.

Techniques: Control, Fluorescence, Size-exclusion Chromatography, SDS Page, Quantitation Assay

Journal: Scientific reports

Article Title: Targeted clearance of senescent cells using an antibody-drug conjugate against a specific membrane marker.

doi: 10.1038/s41598-021-99852-2

Figure Lengend Snippet: Figure 3. A B2M-duocarmycin ADC selectively kills senescent cells. (A) Representative Western Blot of B2M expression in EJp53, EJp21, EJp16 and HT1080-p21-9 cells 4 days after induction of p53 or p16 respectively, compared to EJp53 cells transfected with B2M cDNA for 4 days (+ B) . (B) Western blot showing B2M expression for different periods of time (0–6 days) in EJp53 cells transfected with B2M cDNA. (C–D) Cell viability, as measured by Cell Titre Glo, of proliferating and senescent (4 days after induction) EJp53 and EJp16 cells, incubated with different concentrations (C) or only 0.05 μM (bottom) of the B2M ADC or an isotype control for 72 h. An EJp53 cell transfected with B2M for 4 days was used as a positive control (+ B). Line and bar charts show the mean of 4 independent experiments. Colour codes match all panels. Bar represent the mean ± SD. * p < 0.05, ** p < 0.005. (E) Cell death, as measured by PI staining, in the same cells as above. Graphs show percentage of PI positive cells (dead cells). Bars represent the mean ± SD. * p < 0.05, ** p < 0.005.

Article Snippet: In order to express B2M, a B2M cDNA (OriGene) was transfected into EJ cells using Lipofectamine 2000 reagent (Invitrogen), following manufacturer’s instructions, in a 1 μg: 2.5 μl ratio.

Techniques: Western Blot, Expressing, Transfection, Incubation, Control, Positive Control, Staining