Journal: Nucleic Acids Research

Article Title: Transposon DNA sequences facilitate the tissue-specific gene transfer of circulating tumor DNA between human cells

doi: 10.1093/nar/gkae427

Figure Lengend Snippet: Integration of ctDNA is mediated by non-homologous end joining (NHEJ) repair. ( A ) Immunofluorescence index images of ctDNA (red) integration into chromatids (blue) in MM (MM1S), PC (ASPC-1) and CC (HCT116) cell lines. Circles define zoomed regions of interest. White arrows identify an area of ctDNA integration. ( B ) Scatter plot displaying the number of chromatids with ctDNA integrations in different MM (MM1s, RPMI, OPM1), PC (MIA, PANC1, ASPC-1) and CC (HCT 116, HT29, RKO) cell lines. Error bars indicate standard deviations of triplicate experiments ( n = 20 metaphases per experiment). Color and symbol shapes signify different cell types. ( C ) Incorporation of rhodamine-ctDNA fragments into chromosomes of MM (MM1s), PC (ASPC-1) and CC (HCT 116) cell lines after treatment with NHEJ (DNAPKcs and ATM) and PARP ( n = 10) inhibitors. Cells were pretreated for 2 h with inhibitors of DNA-PK (inhibitor II, 30 nM), ATM (KU-55933, 100 nM) and PARP (NU1025, 200 nM) before the addition of rhodamine-ctDNA to the cell culture. The number of integrated sites was measured in 10–20 metaphases per condition. ( D ) Gain of nucleotide variants in cells cocultured with ctDNA. Comparative SNV analysis between cell genome, ctDNA, and coculture. The Venn diagram displays exclusive and shared SNVs for each experimental condition. The area marked in red highlights SNVs commonly shared between ctDNA and coculture condition ctDNA/cell. ( E ) Stacked bar diagram demonstrating the changes in allele depth of the variant (red) and reference (Blue) allele in MM ctDNA, cell line genome, and coculture condition. Cells under coculture conditions have more depth in the variant allele in several locations compared to the control cell genome. ( F ) Index IVG variant calls images and their allele frequency in MM and PC experimental conditions. Green horizontal bars (Adenine) and purple (Cytosine) are, in these cases, the alternate nucleotide, and gray bars are the reference nucleotide. ( G ) Blast images demonstrate the transition point of insertion between cell genome contigs (red boxes) and ctDNA contigs (green boxes). (*) The cumulative of all reads, including the insertion transition site between cell genome contigs (red boxes) and ctDNA contigs (green boxes), is displayed at the bottom. Results were obtained after comparing the contigs carrying insertions in the coculture condition with the reference Coculture (ctDNA + cell) genome. MM: multiple myeloma; PC: pancreatic cancer; CC: colon Cancer.

Article Snippet: DNA-PK inhibitor II and Raltegravir were purchased from Santa Cruz Biotechnology, KU-55933 (ATM kinase inhibitor) from Selleckchem, and NU1025 (PARP inhibitor VI) from Calbiochem.

Techniques: Non-Homologous End Joining, Immunofluorescence, Cell Culture, Variant Assay, Control

Journal: Journal of the National Cancer Center

Article Title: DCS, a novel classifier system based on disulfidptosis reveals tumor microenvironment heterogeneity and guides frontline therapy for clear cell renal carcinoma

doi: 10.1016/j.jncc.2024.06.003

Figure Lengend Snippet: Addition of DU1025 could inhibit ccRCC progression. (A) Nearest template prediction analysis of the expression matrix of ccRCC cell lines from CCLE based on biomarkers from four subtypes. (B, C) Different proliferation ability of ccRCC cell in CCK8 kit and (C) clone formation. (D) Wound healing experiments showed difference migration ability of different groups. (E) Migration and invasion assays of 786p and A704 treated or not treated with NU1025. NC, negative control. ⁎⁎ , P < 0.01; ⁎⁎⁎ , P < 0.001.

Article Snippet: NU1025 (id: S773001) was purchased from Selleckchem.

Techniques: Expressing, Migration, Negative Control

Journal: bioRxiv

Article Title: Retrotransposons facilitates tissue specific horizontal transfer of circulating tumor DNA between human cells

doi: 10.1101/2022.08.10.501131

Figure Lengend Snippet: A. Immunofluorescence index images of ctDNA (red) integration into chromatids (blue) in MM (MM1S), PC (ASPC-1), and CC (HCT116) cell lines. Circles define zoomed regions of interest. White arrows identify an area of ctDNA integration. B. Scatter plot displaying the number of chromatids with ctDNA integrations in different MM (MM1s, RPMI, OPM1), PC (MIA, PANC1, ASPC-1), and CC (HCT 116, HT29, RKO) cell lines. Error bars indicate standard deviations of triplicate experiments (n=20 metaphases per experiment). Color and symbol shapes signify different cell types. C. Incorporation of rhodamine-ctDNA fragments into chromosomes of multiple myeloma (MM1s), pancreatic cancer (ASPC-1), and colon cancer (HCT 116) cell lines after treatment with NHEJ (DNAPKcs and ATM), and PARP (n=10). Cells were pretreated for 2 hours with inhibitors of DNA-PK (inhibitor II), ATM (KU-55933), and PARP (NU1025) before the addition of rhodamine-ctDNA to the cell culture. The number of integrated sites was measured in 10-20 metaphases per condition. D. Gain of nucleotide variants in cells cocultured with ctDNA. Comparative SNV analysis between cell genome, ctDNA, and coculture. Venn diagram displays exclusive and shared SNVs between each experimental condition. The area marked in red highlights SNVs commonly shared between ctDNA and coculture condition ctDNA/cell. E. Stacked bar diagram demonstrating the changes in allele depth of the variant (red) and reference (Blue) allele in multiple myeloma ctDNA, cell line genome, and coculture condition. Cells under coculture conditions have more depth in the varian allele in several locations compared to the control cell genome. F. Figures demonstrating two index IVG variant calls images and their allele frequency in multiple myeloma and pancreatic cancer experimental conditions. Green horizontal bars (Adenine) and blue (guanine) are, in these cases, the alternate nucleotide, and gray bars are the reference nucleotide. G. Blast images demonstrate the transition point of insertion between cell genome contigs (red boxes) and ctDNA contigs (green boxes). Results were obtained after comparing the contigs carrying insertions in the coculture condition with the reference cell genome. Coculture contigs carrying a ctDNA insertion were identified using NucDiff analysis. MM: Multiple myeloma, PC: Pancreatic cancer, and CC: Colon Cancer.

Article Snippet: In addition, we used inhibitors for alternative NHEJ repair pathways, also known as microhomology-end joining, such as the poly ADP ribose polymerase (PARP) inhibitor NU1025 (200 μM, Sigma-Aldrich, MO) and the transposase inhibitor raltegravir (100nM, Sigma-Aldrich, MO).

Techniques: Immunofluorescence, Cell Culture, Variant Assay