Journal: Veterinary Pathology

Article Title: Techniques to study chimerism at the tissue level in humanized mice

doi: 10.1177/03009858251386916

Figure Lengend Snippet: (a) Liver from a NOG-EXL mouse with chimeric myeloid cells hyperactivation syndrome. CD11b (white) is expressed in the infiltrating human multinucleated giant cells (inset) and reactive histiocytes that are identified by labeling with human CD45 LCA labeling (red). Similarly, CD11b (white) is expressed in the mouse Kupffer cells and macrophages that are identified by mouse CD45 LCA labeling (green). Multiplex immunofluorescence with mouse-specific CD45 LCA (green), human-specific CD45 LCA (red), human and mouse cross-binding CD11b (white), and DAPI counterstain (blue). (b) Lungs from an NSG mouse treated with human CAR T-cells. CAR expression in the perivascular pulmonary infiltrate of mononuclear cells is labeled with in situ hybridization (ISH) probes targeting the UTRs of the lentiviral vector used for transduction. Inset: Pulmonary infiltrate of untransduced human T-cells serve as a negative control. Chromogenic ISH for lentiviral UTRs. (c) Human tumor xenograft in an NSG mouse treated with human CAR T-cells. Both human T-cells for CAR T-cell manufacturing and the tumor cell line have been manipulated in vitro with lentiviral vectors. ISH for UTRs is positive in both tumor-infiltrating CAR T-cells and engrafted tumor cells, making it impossible to distinguish between the 2 populations. Inset: An example of UTRs expression in nontransduced CAR T-cells infiltrating a tumor xenograft. Chromogenic ISH for lentiviral UTRs. (d) Lungs from an NSG mouse treated with human armored CAR T-cells producing IL-18. Activated CAR T-cells in the perivascular pulmonary infiltrate of mononuclear cells are highlighted using immunohistochemistry for human IL-18. Inset: Perivascular infiltrate of nonarmored CAR T-cells that do not express IL-18. (e) Human tumor xenograft in an NSG mouse treated with human CAR T-cells. Multiplex fluorescent ISH assay allows the visualization of lentiviral UTRs in CAR T-cells (red) and the transcripts of the CAR-specific target in tumor cells (green). Multiplex fluorescent ISH with DAPI counterstain.

Article Snippet: For the multiplex fluorescent RNAscope assay, the specific ISH detection kit (322800; ACD) and the Automation Multiplex Detection Kit (NEL830001KT; Akoya Biosciences) were implemented with a Leica BOND Research Detection System (DS9455).

Techniques: Labeling, Multiplex Assay, Immunofluorescence, Binding Assay, Expressing, In Situ Hybridization, Plasmid Preparation, Transduction, Negative Control, Xenograft Assay, In Vitro, Immunohistochemistry

Journal: Molecular Cancer Research

Article Title: Gut Microbiota–Mediated hsa_circ_0126925 Targets BCAA Metabolic Enzyme BCAT2 to Exacerbate Colorectal Cancer Progression

doi: 10.1158/1541-7786.MCR-24-0434

Figure Lengend Snippet: HFD-modulated gut microbiota regulates the expression of circRNAs. A, The circRNA expression profiles of intestinal tumors from CTL, HFD, and HFD+Abx AOM/DSS model mice were compared via high-throughput circRNA microarray sequencing. B, Mmu_circ_017841, mmu_circ_006689, mmu_circ_32011, circ_38945, mmu_circ_19191, mmu_circ_41893, and mmu_circ_45525, which appeared simultaneously in the upregulated circRNAs of the HFD vs. CTL and HFD+Abx groups. C–E , RT-qPCR was used to verify the differential expression of target mmu_circRNAs (mmu_circ_38945, mmu_circ_32011, mmu_circ_19191, and mmu_circ_45525) in groups of mice generated via three different modeling methods. F, Sanger sequencing results for circ_38945 and circ_0126925. G, Divergent and convergent primers were used to amplify circ_0126925, gDNA, and cDNA with or without RNase treatment before RT-qPCR. H, FISH analysis of the subcellular localization of circ_0126925. I, The proportions of cells in which circ_0126925 was expressed in the nucleus and cytoplasm. J and K, RT-qPCR and ISH were used to detect the expression of circ_0126925 in cancer and adjacent tissues. L, KM curve of the overall survival rate of patients with different expression levels of circ_0126925. In C–E , J , and K , unpaired and paired t tests were used for statistical analyses. In K , the differences between the KM curves were tested for significance by the log-rank test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. ns indicates no significant difference between the two indicated groups.

Article Snippet: The expression level of circ_0126925 in adjacent and cancerous tissues was detected using a Boster ISH Detection Kit (Boster Bio).

Techniques: Expressing, High Throughput Screening Assay, Microarray, Sequencing, Quantitative RT-PCR, Quantitative Proteomics, Generated