u2os dr gfp cells  (Addgene inc)

Journal: bioRxiv

Article Title: SFPQ Promotes Homologous Recombination via mRNA Stabilization of RAD51 and Its Paralogs

doi: 10.1101/2025.09.08.674956

Figure Lengend Snippet: (A) Representative images show DAPI (blue), EdU (green), pATM (magenta), SFPQ (cyan), and merged (right) staining in siNTC-treated DIvA U2OS cells under break and no break conditions. Breaks were induced with 4-hydroxytamoxifen for 24 hours, and images were captured using a 10X magnification. Cells (∼20,000 per well) were imaged across three wells per condition (16 fields per well; 48 images total) and quantified in Cell Profiler for nuclear intensity, foci count, and cell-cycle stage based on EdU/DAPI. Data are representative of n=48 images. (B) Quantification of SFPQ–pATM and pATM–γH2AX co-localization in G2-phase cells. Violin plots show correlation coefficients of SFPQ and pATM (left) and pATM and γH2AX (right) in G2 cells with or without DNA breaks. (C) ChIP-seq data representing SFPQ-bound chromatin at 122 defined AsiSI sites under uncut (noDSB) and cut (+4OHT, 4 hours) conditions (left). ChIP-seq data representing SFPQ bound to RNU sites (right). Immunoprecipitation was performed using SFPQ polyclonal antibody. Normalized ChIP-seq signal was plotted for ±1.5 kb around AsiSI sites. SFPQ occupancy profiles are shown for two independent replicates with DSB induction (dark blue and light blue) and for the noDSB control (yellow).

Article Snippet: U2OS DR-GFP cells (female; provided by Jeremy Stark’s laboratory, City of Hope, Duarte, California, USA) and wild-type U2OS cells (female; ATCC) were both grown in DMEM supplemented with 10% FBS and 1% P/S.

Techniques: Staining, ChIP-sequencing, Immunoprecipitation, Control

Journal: bioRxiv

Article Title: SFPQ Promotes Homologous Recombination via mRNA Stabilization of RAD51 and Its Paralogs

doi: 10.1101/2025.09.08.674956

Figure Lengend Snippet: (A) (Top) SFPQ mean intensity: Violin plots (with embedded boxplots) show the single-cell distribution of nuclear SFPQ mean fluorescence intensity in DIvA U2OS cells under no break (untreated) and break (4-hydroxytamoxifen, 4-OHT) conditions. Each dot is one nucleus; boxplots denote median and interquartile range. Cell-cycle phase (G1, S, G2) was assigned per cell using EdU incorporation (green) and DAPI DNA content (blue). (Bottom) SFPQ foci per cell: Violin plots (with embedded boxplots) show the number of SFPQ nuclear foci per cell under the same conditions and cell-cycle stratification. Quantification: Cells were left untreated or treated with 4-OHT to induce AsiSI-mediated DSBs, then stained for SFPQ (cyan), EdU, and DAPI. Images were analyzed in Cell Profiler to segment nuclei, call SFPQ foci, compute per-nucleus mean intensity and foci counts, and assigned cell-cycle stage from EdU/DAPI features. (B) Non–pre-extracted immunofluorescence staining of pATM and SFPQ in DIvA U2OS cells with or without DSB induction. Cells were left untreated or treated with 4-hydroxytamoxifen (4-OHT) to induce AsiSI-mediated DSBs and stained for DNA (DAPI, blue), EdU incorporation (green), phosphorylated ATM (pATM, magenta), and SFPQ (cyan). Images were acquired without cytoskeletal (CSK) pre-extraction to visualize total nuclear staining patterns. Merged images show nuclear co-localization of pATM and SFPQ signals in the presence and absence of DNA damage.

Article Snippet: U2OS DR-GFP cells (female; provided by Jeremy Stark’s laboratory, City of Hope, Duarte, California, USA) and wild-type U2OS cells (female; ATCC) were both grown in DMEM supplemented with 10% FBS and 1% P/S.

Techniques: Fluorescence, Staining, Immunofluorescence, Extraction

Journal: bioRxiv

Article Title: SFPQ Promotes Homologous Recombination via mRNA Stabilization of RAD51 and Its Paralogs

doi: 10.1101/2025.09.08.674956

Figure Lengend Snippet: (A) mRNA-seq log₂ fold changes of RAD51 paralogs and pooled transcripts in the indicated Gene Ontology (GO) categories in DIvA U2OS cells treated with siSFPQ compared to siNTC control for 72 hours in the absence of DSBs. Data represent the mean of three biological replicates. Individual p-values were adjusted for multiple comparisons. Aggregate p-values were combined by Fisher’s method. (B) Differential transcript utilization analysis for RAD51 paralogs. mRNA-seq data from siNTC versus siSFPQ DIvA U2OS cells were analyzed for transcript isoform usage. Bars represent the likelihood ratio statistic for each gene, with blue bars indicating genes showing significant shifts in transcript utilization (RAD51B, RAD51C) upon SFPQ depletion. Grey bars indicate genes without significant changes. (C) Western blot analysis of SFPQ and RAD51 protein levels of the three biological replicates used for mRNA-seq following siNTC or siSFPQ treatment. Total protein staining is shown as a loading control. (D) Representative images show DAPI (blue), EdU (green), RAD51 (magenta), SFPQ (cyan), and merged (right) staining in siNTC-treated DIvA U2OS cells under break and no break conditions, pre-extracted with CSK. Breaks were induced with 4-OHT for 4 hours. Cells (∼20,000 per well) were imaged across four wells per condition (16 fields per well; 64 images total) and quantified in Cell Profiler for nuclear intensity, foci count, and cell-cycle stage based on EdU/DAPI. (E) Quantification of SFPQ and RAD51 foci per cell in DIvA U2OS cells following siRNA treatment and DNA damage induction. Violin plots show the distribution of foci counts across conditions with or without 4-hydroxytamoxifen (4-OHT) treatment and following transfection with non-targeting control (NTC), RAD51-targeting, or SFPQ-targeting siRNAs. Data are representative of n=64 images. (F) Violin plots showing correlation coefficients of SFPQ and RAD51 in G2 cells with or without DNA breaks. Quantification of SFPQ-RAD51 foci co-localization in G2-phase cells was performed using Cell Profiler analysis of single-cell fluorescence signals.

Article Snippet: U2OS DR-GFP cells (female; provided by Jeremy Stark’s laboratory, City of Hope, Duarte, California, USA) and wild-type U2OS cells (female; ATCC) were both grown in DMEM supplemented with 10% FBS and 1% P/S.

Techniques: Control, Western Blot, Staining, Transfection, Fluorescence

Journal: bioRxiv

Article Title: SFPQ Promotes Homologous Recombination via mRNA Stabilization of RAD51 and Its Paralogs

doi: 10.1101/2025.09.08.674956

Figure Lengend Snippet: (A) Differential expression analysis of mRNA-seq data comparing DSB versus no-DSB conditions in siNTC-treated DIvA U2OS cells (n=3 biological replicates). Mean log₂ fold change for the same targets is shown as . No significant expression differences were detected for these targets upon DSB induction in control cells. (B) ChIP-seq data showing SFPQ abundance at sites upstream and downstream of RAD51-paralog genes both without (noDSB) or with (+DSB) 4 hours of DSB induction. Data displayed is the average signal across all 6 RAD51 paralogs. (C) mRNA-seq log₂ fold changes of transcript expression of the indicated gene or GO category in DIvA U2OS cells treated with siSFPQ compared to siNTC control for 72 hours in the absence of DSBs. Data represent the mean of three biological replicates. Individual p-values were adjusted for multiple comparisons. Aggregate p-values were combined by Fisher’s method. (D) Western blot of DIvA U2OS cells treated with siSFPQ with or without p53 inhibition by PFT-α (30 µM) for 24 hours. Lysates were blotted for SFPQ, HSP70, MDM2 and RAD51. (E) (Left) Western blot of p53-null K562 cells treated with siSFPQ. Total protein staining is shown as a loading control. (Right) Quantification of SFPQ and RAD51 normalized band intensities relative to total protein is graphed.

Article Snippet: U2OS DR-GFP cells (female; provided by Jeremy Stark’s laboratory, City of Hope, Duarte, California, USA) and wild-type U2OS cells (female; ATCC) were both grown in DMEM supplemented with 10% FBS and 1% P/S.

Techniques: Quantitative Proteomics, Expressing, Control, ChIP-sequencing, Western Blot, Inhibition, Staining

Journal: bioRxiv

Article Title: SFPQ Promotes Homologous Recombination via mRNA Stabilization of RAD51 and Its Paralogs

doi: 10.1101/2025.09.08.674956

Figure Lengend Snippet: (A) Cycloheximide (CHX) ± carfilzomib (Carf) protein stability assay in DIvA U2OS cells. Cells were transfected with either non-targeting control (siNTC) or SFPQ-targeting (siSFPQ) siRNAs for 72 h, then treated with CHX alone or CHX + Carf to inhibit protein synthesis and proteasomal degradation, respectively. Lysates were collected at 0-, 2-, and 4-hours post-drug treatment from three independent biological replicates. (B) RAD51 abundance from normalized to total protein and then to 0 hr. condition. Data points represent individual replicates; lines indicate the mean. (C) RIP-seq analysis of SFPQ binding across RAD51 family paralogs in melanoma cells. Read coverage tracks show SFPQ-associated RNA fragments aligned to the genomic loci of RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3. Peaks indicate regions of enriched SFPQ binding, with annotations of exon–intron structure shown below each track. Model for SFPQ-mediated stabilization of RAD51 mRNA and its impact on homologous recombination (HR). In the presence of SFPQ, the protein binds to RAD51 mRNA, promoting transcript stabilization. Stable RAD51 mRNA ensures sufficient RAD51 protein production, enabling efficient RAD51 filament formation on DNA and supporting robust HR (left). Upon SFPQ loss, RAD51 family mRNAs are destabilized, leading to reduced RAD51 protein abundance. This reduction impairs HR efficiency (right).

Article Snippet: U2OS DR-GFP cells (female; provided by Jeremy Stark’s laboratory, City of Hope, Duarte, California, USA) and wild-type U2OS cells (female; ATCC) were both grown in DMEM supplemented with 10% FBS and 1% P/S.

Techniques: Stability Assay, Transfection, Control, Binding Assay, Homologous Recombination, Quantitative Proteomics

Journal: Cell Reports Medicine

Article Title: UBA1 inhibition sensitizes cancer cells to PARP inhibitors

doi: 10.1016/j.xcrm.2024.101834

Figure Lengend Snippet: TAK243 treatment reduces homologous recombination (A) Schematic of the direct repeat (DR)-GFP assay. Stable cells are generated expressing the DR-GFP construct. The first GFP repeat contains an I-SceI endonuclease site, which initially prevents expression, and the second GFP is a truncated fragment overlapping the region with the I-SceI site. Transient transfection with I-SceI induces a double-strand break in the first GFP, which can be repaired by HR using the second truncated GFP fragment, resulting in GFP expression in cells that successfully perform HR. (B) DR-GFP U2OS cells were treated with DMSO, TAK243, or Mirin, were transfected with I-SceI, and GFP-positive cells were analyzed by flow cytometry. Mirin treatment was used as a positive control. At least 5,000 single and viable cells were analyzed for each condition. Graph represents the mean with standard deviation. ANOVA with Holm-Sidak post hoc test. ∗∗∗∗ p < 0.0001. n = 3. See gating strategy in Figure S5 . (C) Representative image of DNA damage-induced Rad51 foci in OVCAR8 and HCC1806 cells. Cells were treated with either DMSO or TAK243 for 48 h prior to irradiation (10 Gy) or mock irradiation, and then processed 4 h after irradiation to stain for cycling cells (EdU, red) and Rad51 foci (green). Scale bar. 10 μm. (D) Quantification of RAD51 foci (at least 50 cells per group) in EdU+ DMSO and TAK243-treated cells. Line represents mean, dots represent individual cells. ANOVA with Holm-Sidak post hoc test. ∗∗∗∗ p < 0.0001. n = 3. (E) OVCAR8 cells were transfected with His-ubiquitin. 48 h post-transfection cells were treated with 2 mM HU and then harvested after 2 h. His-tagged ubiquitinated proteins purified from whole-cell lysates were blotted for endogenous RPA1 and RPA2. Inset values indicate average band intensity from two independent experiments.

Article Snippet: U2OS DR-GFP cells were transfected with I-SceI (Addgene #26477) by using Lipofectamine 3000 as per the manufacturer’s instruction.

Techniques: Homologous Recombination, Generated, Expressing, Construct, Transfection, Flow Cytometry, Positive Control, Standard Deviation, Irradiation, Staining, Ubiquitin Proteomics, Purification

Journal: bioRxiv

Article Title: Cell-cycle dependent inhibition of BRCA1 signaling by the lysine methyltransferase SET8

doi: 10.1101/2023.11.22.567520

Figure Lengend Snippet: ( A ) Representative images of RAD51, BRCA1 and 53BP1 staining in EdU positive nuclei from control (-Tet) and FLAG-Set8 PIPmut (+Tet) expressing cells treated with 5 or 25 nM of camptothecin during 2 hours or with vehicle (DMSO) as indicated. Scale bar = 10 µm. ( B ) Scattered box-plot representing the quantification of RAD51, BRCA1 and 53BP1 foci per EdU-positive nuclei from control (-Tet) and FLAG-Set8 PIPmut expressing cells (+ Tet) treated with 5 nM or 25 nM of camptothecin during 2 hours, or with vehicle (DMSO). Interquartile range and statistical significance are shown as in . n ≥ 3; *** p<0.001 (t test). Number of nuclei per condition and experiment >100. ( C ) Left panel: Bar-plot representing the relative efficiency of DNA repair by homologous recombination of I-SceI-induced DNA breaks as evaluated by flow cytometry using the U2OS DR-GFP cell line after expression of different FLAG-SET8 proteins as indicated. Data = mean ± s.d., n = 3. *** p<0.001 (t-test). Number of events per sample > 10 000. Right panel: immunoblots showing the similar expression of different FLAG-SET8 proteins and of MYC-tagged nuclease I-SceI in U2OS DR-GFP cells. Tubulin was used as loading control.

Article Snippet: The HR assays were performed using the U2OS DR-GFP cell line (ATCC) that allows to monitor homologous recombination efficiency through the detection of GFP positive cells via flow cytometry.

Techniques: Staining, Control, Expressing, Homologous Recombination, Flow Cytometry, Western Blot

Journal: bioRxiv

Article Title: Cell-cycle dependent inhibition of BRCA1 signaling by the lysine methyltransferase SET8

doi: 10.1101/2023.11.22.567520

Figure Lengend Snippet: ( A ). Representative immunoblots showing the localization of BRCA1 and 53BP1 in soluble cytoplasmic (S1) and nuclei (S2) fractions and in chromatin-enriched fractions (P3) at early G1 phase in control and SET8 SiRNA-treated cells that excited from mitosis without SET8 and the conversion of H4K20me0 to H4K20me1 as shown in supplementary figure S7. Cells were harvested 15 hours after G1/S release. n=3 ( B ) FACS analysis of DNA content and BrdU signal after mitotic exit of the control and SET8 SiRNA-treated cells as described above. Cells were harvested 24 hours after G1/S release. n=3 ( C ) Representative images showing EdU incorporation (DNA synthesis) and staining of 53BP1 and BRCA1 in control and siRNA SET8 treated cells progressing from G1 to S-phase after a first mitosis without SET8 and H4K20me1 as described above. Scale bar = 10 µm ( D ). FACS analysis of DNA content and BrdU incorporation levels of control (shLuc/siCtrl), SET8-depleted U2OS cells (shRNA SET8/siRNA Ctrl), BRCA1-depleted U2OS cells (shRNA Luc/siRNA BRCA1) and cells depleted for both BRCA1 and SET8 (shRNA SET8/siRNA BRCA1). n=3. Number of of events per sample > 10 000.

Article Snippet: The HR assays were performed using the U2OS DR-GFP cell line (ATCC) that allows to monitor homologous recombination efficiency through the detection of GFP positive cells via flow cytometry.

Techniques: Western Blot, Control, DNA Synthesis, Staining, BrdU Incorporation Assay, shRNA

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