Journal: Scientific Reports

Article Title: Insights into Ag-NPs-mediated pathophysiology and ultrastructural aberrations in ovarian tissues of darkling beetles

doi: 10.1038/s41598-022-17712-z

Figure Lengend Snippet: Evaluation of DNA damage following comet analysis in the ovarian tissues of B. polychresta , ( a ) a slide for cells from untreated beetles displays normal DNA (class 0), and ( b – c ) slides for cells from Ag-NPs-treated beetles show damaged DNA (class 1, class (2), and class 3), while ( a' – c' ) reveal the slides after analysis using Imagej Fiji software ( https://imagej.net/software/fiji/downloads ), demonstrating the tails of DNA. Fig. ( d , e ) illustrate the analysis of 300 cells taken for the comet assay, showing the different classes in (d) and the comet cells in ( e ). ( f , g ) exhibit the flow cytometry analyses of annexin-V-FITC and propidium iodide staining of the Ag-NPs-untreated group and the Ag-NPs-treated group 3, respectively, and ( h ) demonstrates the analysis of the flow cytometry results, showing the significant lessening in viable cells and a significant rise in apoptotic cells for the Ag-NPs-treated group 3 compared to the control group. The (UL) represents (PI + /Annexin V −), indicating necrotic cells, the (LL) represents (PI − /Annexin V −), which refers to normal cells, the (UR) is (PI + /Annexin V +), pointing to early apoptotic cells (EAC), and the (LR) alludes to (PI − /Annexin V +), showing late apoptotic cells (LAC). The data was analyzed using CellQuest Pro software version 5.2.1, 2005 (BD Biosciences, San Jose, CA). For comet analysis, five ovarian tissues from different beetles of Ag-NPs-treated group 3 and the control group were used for developing five slides and 100 comets per slide were investigated. Flow cytometric analyses were also performed using five ovarian tissues from different beetles of Ag-NPs-treated group 3 and untreated beetles. The results are presented as mean ± SEM (**** P ≤ 0.0001, and *** P ≤ 0.001), indicating the significant differences between the Ag-NPs-treated group 3 and the control beetles in relation to genotoxicity and cellular toxicity.

Article Snippet: Annexin V-FITC/PI stained cells were instantly examined using a flow cytometry analyzer (Becton Dickinson, Franklin Lakes, NJ, USA) as previously proposed .

Techniques: Software, Single Cell Gel Electrophoresis, Flow Cytometry, Staining

Journal: Molecular Cancer Therapeutics

Article Title: IMiDs Augment CD3-Bispecific Antibody–Induced CD8 + T-Cell Cytotoxicity and Expansion by Enhancing IL2 Production

doi: 10.1158/1535-7163.MCT-22-0498

Figure Lengend Snippet: IMiDs enhance TDB-induced IL2 release, CD8 + T-cell proliferation and expansion. A, IL2 RNA sequencing (RNA-seq) analysis of purified CD8 + T and CD4 + T cells induced by anti-HER2/CD3-TDB ( B ). Luciferase readout from JURKAT-IL2 Luciferase reporter cell line co-cultured with KPL-4 and treated 16 hours with 100 ng/mL anti-HER2/CD3-TDB and lenalidomide, pomalidomide, iberomide, or CC-92480 as indicated. C, Ikaros Western blot of FACS-sorted CD4 + T and CD8 + T cells from SKBR-3 co-cultures treated with anti-HER2/CD3-TDB (15 ng/mL), pomalidomide (1 μmol/L), anti-HER2/CD3-TDB-pomalidomide combination, or CD3/CD28 beads. D, Flow-cytometry analysis of CFSE-labeled CD4 + T and CD8 + T cells after 7-day treatment with 500 ng/mL cevostamab in the absence (red) or presence (blue) of 1 μmol/L lenalidomide. E, The cell number change of CD4 + T cells and CD8 + T-cell over 14 days in the co-culture of PBMC and MOLP-2 cells with the treatments of either single-agent cevostamab (500 ng/mL), lenalidomide (1 μmol/L), or the combination. F, CD4 + T cells and CD8 + T-cell number change over 14 days in a co-culture of PBMC and MOLP-2 cells upon the treatment of a combination of 500 ng/mL of cevostamab and 1 μmol/L of different IMiDs. D–F, Representative data from the results of PBMC from three healthy blood donors. Data are shown as means ± SD ( A , B , E, and F ). B and E, The t test was used for statistical analysis (*, P < 0.01).

Article Snippet: All antibodies for flow cytometry cell staining were purchased from BD Biosciences or BioLegend.

Techniques: RNA Sequencing Assay, Purification, Luciferase, Cell Culture, Western Blot, Flow Cytometry, Labeling, Co-Culture Assay

Journal: Molecular Cancer Therapeutics

Article Title: IMiDs Augment CD3-Bispecific Antibody–Induced CD8 + T-Cell Cytotoxicity and Expansion by Enhancing IL2 Production

doi: 10.1158/1535-7163.MCT-22-0498

Figure Lengend Snippet: IL2 is a critical mediator of the effect of IMiDs on TDB-induced T-cell expansion and killing activity. A, CD8 + T-cell number change over 14 days in the co-culture of PBMC and MOLP-2 cells upon the treatments of 500 ng/mL cevostamab, 1 μmol/L pomalidomide, and 2 μg/mL anti-IL2–blocking antibody as indicated. B, CD8 + T-cell number change in PBMCs from three different healthy donors at 14 days in the co-culture of PBMC and MOLP-2 cells. C, CD8 + T-cell number change over 14 days in the coculture of PBMC and KPL-4 cells treated with 15 ng/mL anti-HER2/CD3-TDB, 1 μmol/L pomalidomide and 2 μg/mL anti-IL2–blocking antibody. D, CD8 + T-cell number change in PBMC from three different healthy donors at 14 days in the co-culture of PBMC and KPL-4 cells. E, The percentage of granzyme B–positive cells and the geometric mean fluorescence intensity (gMFI) of the granzyme B–positive cells in CD8 + T cells were detected by intracellular staining followed by flow-cytometry analysis in PBMCs treated with 50 ng/mL anti-HER2/CD3-TDB, 1 μmol/L pomalidomide, and 2 μg/mL anti-IL2 in the time points as indicated. F, The percentage of IFNγ–positive cells in CD8 + T cells was detected by intracellular staining followed by flow-cytometry analysis after the second dose of anti-HER2/CD3-TDB. G, Apoptosis of SKBR3 cells was detected using IncuCyte Caspase 3/7 assay after the second dose of anti-HER2/CD3-TDB. Number of CD8 + T cells pretreated with 50 ng/mL anti-HER2/CD3-TDB, ±1 μmol/L pomalidomide, and ±2 μg/mL anti-IL2 T cells were normalized for the caspase assay with the single agent TDB and freshly plated target cells in an E:T ratio of 0.5:1. Data are shown as means ± SD ( A , C , E , F, and G ), means ± SEM ( B and D ). A , C, and G, Representative data of the results of PBMCs from three healthy blood donors. B , D – F, A paired t test was used for statistical analysis of PBMCs from three to five health donors co-cultured with target cells and the treatments as indicated. A and C, The t test was used for statistical analysis.

Article Snippet: All antibodies for flow cytometry cell staining were purchased from BD Biosciences or BioLegend.

Techniques: Activity Assay, Co-Culture Assay, Blocking Assay, Fluorescence, Staining, Flow Cytometry, Caspase Assay, Cell Culture