Journal: Signal Transduction and Targeted Therapy

Article Title: In vivo evaluation of guide-free Cas9-induced safety risks in a pig model

doi: 10.1038/s41392-024-01905-1

Figure Lengend Snippet: Analysis of guide-free Cas9-induced genomic damages in pig model. a Comet assay detection of the PBMCs of WT and DIC pigs on Day 0, Day 3, Day 9 and Day 14 of Dox administration. b Quantification of the tail length of the PBMCs of the WT and DIC pigs at four time points. *** p = 0.0001, **** p < 0.0001, n = 6, unpaired t test. c Fluorescence microscopy images of pH2AX (green), tdTomato (red) and DAPI (blue) in WT and DIC pig PBMCs. Genome damaged cells are marked by the white dashed circle, scale bar = 10 μm. Negative indicates a normal cell, and positive indicates a cell with DNA DSBs, scale bar = 2 μm. d Percentage of pH2AX-positive cells in the PBMCs of WT pigs and DIC pigs, *** p = 0.0005, n = 3, unpaired t test. e Fluorescence microscope images of pH2AX (green), tdTomato (red), and DAPI (blue) in the stomach, intestine, lungs, kidneys, and heart of the WT and DIC pigs; white arrows indicate genome-damaged cells, scale bar = 20 μm, area bar = 5 μm. f Quantification of the fluorescence density of pH2AX in the stomach, intestine, lungs, kidneys, and heart of the WT and DIC pigs from three independent repeats. * p < 0.0332, ** p < 0.0021, n = 3, unpaired t test. Scale bar = 10 μm. g Fluorescence microscope images of CASP3 (green), Cas9 (red), and DAPI (blue) in the stomach, intestine, lungs, kidneys, and heart of the WT and DIC pigs; white arrows indicate apoptotic cells. Data in b , d , and f are presented as mean ± SEMs

Article Snippet: After red blood cell lysis, PBMCs were seeded in poly-L-lysine-coated cover glasses, fixed with 4% paraformaldehyde for 15 min and permeabilized with 9% Triton-X for 1 h. IF staining was performed using primary antibodies to detect pH2AX (CST, 9718).

Techniques: Single Cell Gel Electrophoresis, Fluorescence, Microscopy

Journal: Oncogene

Article Title: Spermine oxidase promotes Helicobacter pylori-mediated gastric carcinogenesis through acrolein production.

doi: 10.1038/s41388-024-03218-7

Figure Lengend Snippet: Fig. 5 Effect of 2-HOBA on DNA damage. AGS cells pretreated or not with 2-HOBA (100 μM) for 2 h and then treated or not with acrolein (10 μM) for 24 h; cells were stained by the AcroleinRED assay (A), and the staining was quantified (B). C, D AGS cells were pretreated or not with 2-HOBA (100 μM) for 2 h and then exposed to acrolein (10 μM) and/or infected with H. pylori. After 24 h, cells were immunostained for pH2AX (green; C) and the fluorescence was quantified (D) from 3 independent experiments. E Representative immunofluorescence images of acrolein adducts in the gastric tissues of INS-GAS mice infected or not with H. pylori ± 2-HOBA; data are representative of n = 2 uninfected mice and n = 3 infected animals per group. Scale bars, 50 μm (C and E, bottom row) and 100 μm (all other images). Data represent the mean ± SEM. Statistical analyses, where shown, **P < 0.01, ****P < 0.0001 by one-way ANOVA and Tukey test (B), and two-way ANOVA and Tukey test (D).

Article Snippet: Tissues were incubated with prediluted rabbit polyclonal anti-Ki-67 [28] (Biocare, Cat # PRM 325 AA), rabbit polyclonal anti-phospho-gamma H2AX [p Ser 139] polyclonal antibody [22] (pH2AX, 1:200, Novus, Cat # NB100-2280), or prediluted rabbit monoclonal anti-myeloperoxidase [28] (MPO, Biocare Cat # PP 023 AA) overnight at 4 °C.

Techniques: Staining, Infection