human u2os dr gfp cells  (ATCC)

Journal: Cell Reports

Article Title: The interplay of TARG1 and PARG protects against genomic instability

doi: 10.1016/j.celrep.2023.113113

Figure Lengend Snippet: The loss of TARG1 sensitizes cells to topoisomerase II and ATR inhibition and induces homologous recombination defects (A–D) Representative images (left) and quantification (right) of colony-formation assays with U2OS WT and TARG1-KO cells treated with DMSO or as indicated. (E) Schematic representation of the DR-GFP HR reporter assay, as described. The SceGFP gene is a GFP gene mutated to contain a recognition site for the I- Sce I endonuclease and two in-frame stop codons. The iGFP gene is a truncated internal WT GFP fragment. Following I-Sce I expression, SceGFP is cleaved, yielding a DSB. A functional GFP gene is restored upon repair via HR using iGFP as a donor sequence. (F) U2OS DR-GFP cells were transfected with non-targeting siRNA control (siCTRL), two different siTARG1, or siCtIP and 24 h later were cotransfected with I-SceI and mCherry for 48 h prior to analysis by flow cytometry. The proportion of GFP-positive cells among the mCherry-positive population was used as a readout for I-SceI-induced HR events. CtIP knockdown acts as a positive control here. Data are shown as mean ± SD, n = 3 (A–D), or mean ± SEM, n = 3 (F); ns, not significant, ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 (two-tailed Student’s t test). See also Figure S1 .

Article Snippet: Human: U2OS DR-GFP cells , ATCC , Cat# CRL-3455.

Techniques: Inhibition, Homologous Recombination, Reporter Assay, Expressing, Functional Assay, Sequencing, Transfection, Control, Flow Cytometry, Knockdown, Positive Control, Two Tailed Test

Journal: Cell Reports

Article Title: The interplay of TARG1 and PARG protects against genomic instability

doi: 10.1016/j.celrep.2023.113113

Figure Lengend Snippet: TARG1 deficiency is synthetically lethal with PARG suppression in a PARP1-dependent manner (A–D) Representative images (top) and quantification (bottom) of colony-formation assays with U2OS WT and TARG1-KO cells (A, C, and D), PEO1 WT and TARG1-KO cells (B), and U2OS TARG1-KO cells complemented with TARG1 WT or catalytically inactive K84A mutant (D) treated with DMSO or as indicated. (C) P1i, PARP1 inhibitor; P2i, PARP2 inhibitor. Data are shown as mean ± SD, n = 3; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 (two-tailed Student’s t test). See also Figure S2 .

Article Snippet: Human: U2OS DR-GFP cells , ATCC , Cat# CRL-3455.

Techniques: Mutagenesis, Two Tailed Test

Journal: Cell Reports

Article Title: The interplay of TARG1 and PARG protects against genomic instability

doi: 10.1016/j.celrep.2023.113113

Figure Lengend Snippet: The joint loss of TARG1 and PARG activity leads to excessive ADPr and induces replication stress (A) U2OS cells were treated with DMSO, 10 μM PARGi and 0.1 μM veliparib, and 10 μM PARGi for 6 days. ADPr and DNA damage marker levels were analyzed using western blotting. (B) Representative images of ADPr staining in detergent pre-extracted cells treated with DMSO, 10 μM PARGi, or 10 μM PARGi and 0.1 μM veliparib for 4 days. Scale bars, 10 μm. A representative image from n = 3 is shown. (C) Quantification of (B). Each point represents the mean ADPr intensity of an individual nucleus. The black line represents the mean ADPr intensity of each condition; at least 220 cells were analyzed per condition. (D) Representative images of RPA32 p-T21 and γH2AX staining in cells treated with DMSO, 10 μM PARGi, 10 μM PARGi and 0.1 μM veliparib, or 0.1 μM veliparib for 4 days. Scale bars, 10 μm. A representative image from n = 3 is shown. (E and F) Quantification of (D). Each point represents the percentage of cells with >5 RPA32 p-T21 foci per image (E) or the percentage of cells with >10 γH2AX foci per image (F). The black line represents the mean percentage of cells per image with >5 RPA32 p-T21 (E) or >10 γH2AX (F) foci for each condition. ∼250 images and a total of ∼20,000 cells were analyzed per condition. (G) Quantification of γH2AX-positive cells by flow cytometry after 5 days of exposure to DMSO or indicated treatment. (H) U2OS cells were treated with DMSO or 10 μM PARGi for 4 days. ADPr and DNA damage marker levels were analyzed using western blotting. (I) Quantification of cell-cycle analysis by flow cytometry of EdU- and DAPI-stained U2OS cells after 5 days of exposure to DMSO or indicated treatment and 1 h EdU pulse. Data are shown as mean ± SEM of four independent experiments (G and I). ∗∗∗ p < 0.001 and ∗∗∗∗ p < 0.0001 (two-tailed Student’s t test). See also Figure S3 .

Article Snippet: Human: U2OS DR-GFP cells , ATCC , Cat# CRL-3455.

Techniques: Activity Assay, Marker, Western Blot, Staining, Flow Cytometry, Cell Cycle Assay, Two Tailed Test

Journal: Cell Reports

Article Title: The interplay of TARG1 and PARG protects against genomic instability

doi: 10.1016/j.celrep.2023.113113

Figure Lengend Snippet: TARG1 and HPF1 both protect cells from toxic PARP1-mediated ADPr (A) Quantification of colony-formation assay with U2OS WT and TARG1-KO cells transfected with siCTRL or siHPF1 and treated with DMSO or as indicated. Data are shown as mean ± SD, n = 3; ∗∗ p < 0.01 and ∗∗∗ p < 0.001 (two-tailed Student’s t test). (B) U2OS cells transfected with siCTRL or siHPF1 were treated with DMSO, 10 μM PARGi, or 10 μM PARGi and 0.1 μM veliparib for 4 days. ADPr and DNA damage marker levels were analyzed using western blotting.

Article Snippet: Human: U2OS DR-GFP cells , ATCC , Cat# CRL-3455.

Techniques: Colony Assay, Transfection, Two Tailed Test, Marker, Western Blot

Journal: Cell Reports

Article Title: The interplay of TARG1 and PARG protects against genomic instability

doi: 10.1016/j.celrep.2023.113113

Figure Lengend Snippet:

Article Snippet: Human: U2OS DR-GFP cells , ATCC , Cat# CRL-3455.

Techniques: Recombinant, Transfection, Protease Inhibitor, Mutagenesis, Flow Cytometry, Negative Control, Plasmid Preparation, Software