Journal: Oncogene

Article Title: Inhibitor of DNA binding 2 (ID2) regulates the expression of developmental genes and tumorigenesis in ewing sarcoma.

doi: 10.1038/s41388-022-02310-0

Figure Lengend Snippet: Fig. 5 HHT reduces the levels of the ID1, ID2, and ID3 proteins in sarcoma cell lines. A Schematic of a doxycycline-inducible, lentiviral expression vector that expresses Flag-ID1, Myc-ID2, and V5-ID3 from a single mRNA transcript using P2A/T2A elements. B Immunoblot of 293 T cells with doxycycline-inducible expression of Flag-ID1, Myc-ID2, and V5-ID3 (293T-ID1-3) treated with HHT 50 nM for 24 h. C 293T-ID1-3 cells were treated with different doses of HHT for 24 h and then lysates were immunoblotted for the epitope tags. D 293T-ID1-3 cells were treated with HHT 50 nM in the presence or absence of the proteasome inhibitor MG132. Lysates were then collected and immunoblotted for the epitope tags for ID1-3. Immunoblot of Ewing sarcoma (E) and osteosarcoma (F) cell lines expressing Flag-ID1, Myc-ID2, and V5-ID3 treated with HHT (50 nM) for 24 h. G The osteosarcoma cell line U2OS was treated with HHT for 24 h and lysates were then immunoblotted for ID1. H U2OS osteosarcoma cells were treated with HHT 50 nM for different amounts of time and lysates were then immunoblotted for ID1. I The MPNST cell line S462 was treated with HHT for 24 h and lysates were then immunoblotted for ID1. J S462 cells were treated with HHT 50 nM for different amounts of time and lysates were then immunoblotted for ID1. K Dose-response curves for osteosarcoma (U2OS, SAOS), rhabdomyosarcoma (RD, RH30), fibrosarcoma (HT1080), and MPNST (S462) cell lines treated with different concentrations of HHT for 72 h. Cell viability was assessed using the AlamarBlue Fluorescence Assay. The results are representative of two independent experiments. Error bars represent mean ± SD of three technical replicates. L Colony formation assay for sarcoma cell lines treated with HHT 25 nM or vehicle. Error bars represent the mean ± SD of three technical replicates. Pancreatic cancer (M) and leukemia (N) cell lines were treated with HHT (50 nM) for 24 h and then immunoblotted for ID1.

Article Snippet: The HEK293T, HT1080, and U2OS cell lines were obtained from ATCC.

Techniques: Expressing, Plasmid Preparation, Western Blot, Fluorescence, Colony Assay

Journal: Virology

Article Title: Intracellular-activated Notch1 can reactivate Kaposi's sarcoma-associated herpesvirus from latency.

doi: 10.1016/j.virol.2006.03.047

Figure Lengend Snippet: Fig. 2. ICN activates RTA promoter in a dose-dependent manner. (A) Scheme to show the Notch expression constructs used for luciferase assay. Delta E is encoded by a cDNA consisting of codons 1–22 fused to codons 1673–2555 and is predicted to result in the synthesis of a mature polypeptide with an amino terminus lying 61 amino acids external to the transmembrane domain. Delta EA has an additional deletion of ankyrin repeats. ICN is encoded by a cDNA consisting of the first two codons of NOTCH1 fused to codon 1770, which lies 24 amino acids internal to the transmembrane domain (Aster et al., 1997). In the diagram, proteolytic cleavage by furin at site S1 produces two subunits, ECN and NTM, which remain non-covalently associated at the cell surface. Sites S2 and S3 identify the sites of proteolytic cleavage mediated by metalloproteases and presenilins induced upon activation by ligand. Cleavage at S3 yields activated form of Notch ICN (Schroeter et al., 1998). (B) Luciferase assay: 1 million of U2OS cells were transfected with 0.5-μg RTA promoter reporter plasmid along with increasing amount of full-length Notch, Delta E, Delta EA or ICN expression vector (0 ng, 10 ng, 20 ng, 50 ng, 100 ng, 200 ng) by lipofectamine 2000. Twenty four hours post-transfection, cells were harvested and lysed for reporter assay.

Article Snippet: A human osteosarcoma cell line U2OS was obtained from the American Type Culture Collection.

Techniques: Expressing, Construct, Luciferase, Activation Assay, Transfection, Plasmid Preparation, Reporter Assay

Journal: Virology

Article Title: Intracellular-activated Notch1 can reactivate Kaposi's sarcoma-associated herpesvirus from latency.

doi: 10.1016/j.virol.2006.03.047

Figure Lengend Snippet: Fig. 5. Transcriptional activity of ICN on RTA promoters. The reporter plasmid pRpluc contains a 3-kb sequence upstream of the translational initiation site of the RTA gene that drives the expression of firefly luciferase. A series of truncation promoters named as pRplucΔ2570, pRplucΔ1490 and pRplucΔ1327 were made for deletion of RBP–Jκ binding sites (bottom panel). A fixed amount (0.5 μg) of the reporter plasmids was transfected or co-transfected into U2OS cell with 50 ng of pflu-ICN. The promoter activity was expressed as the fold activation relative to the reporters alone control. The means and standard deviations from three independent transfections are shown.

Article Snippet: A human osteosarcoma cell line U2OS was obtained from the American Type Culture Collection.

Techniques: Activity Assay, Plasmid Preparation, Sequencing, Expressing, Luciferase, Binding Assay, Transfection, Activation Assay, Control

Journal: Virology

Article Title: Intracellular-activated Notch1 can reactivate Kaposi's sarcoma-associated herpesvirus from latency.

doi: 10.1016/j.virol.2006.03.047

Figure Lengend Snippet: Fig. 7. (A) LANA represses ICN transactivation on RTA promoter. 1 million of U2OS cells were transfected with 0.5 μg RTA promoter reporter plasmid and 50 ng ICN expression vector along with increasing amount of LANA expression vector (0 ng, 10 ng, 20 ng, 50 ng, 100 ng, 200 ng) by lipofectamine 2000. Twenty four hours post-transfection, cells were harvested and lysed for reporter assay. (B) For each transfection, 15 million BCBL1 cells were transfected with mock, pA3M empty vector, and ICN with or without LANA. Twenty four hours post-transfection, total RNAwas collected from the cells. A total of 5 μg of RNA was used with the Superscript First Strand Synthesis system to construct cDNA. Real-time PCR was performed using the DyNAmo SYBR Green qPCR kit with β-actin as the standard. The PCR data are expressed as the Ct values for RTA transcription. Each sample was tested in triplicate for the calculation of the mean and standard deviation. The relative transcript abundance (Log molecules) based on the Ct values for RTA. (C) Twenty million of Vero cells stably infected with Bac36 were mock-transfected or transfected with empty vector pA3M, ICN, or ICN plus LANA. Four days post-transfection, supernatant from each transfection was harvested and concentrated for infection of 293 cells. Twenty four hours post-infection, cells were harvested for FACS analysis to detect GFP expression marking the virus existence.

Article Snippet: A human osteosarcoma cell line U2OS was obtained from the American Type Culture Collection.

Techniques: Transfection, Plasmid Preparation, Expressing, Reporter Assay, Construct, Real-time Polymerase Chain Reaction, SYBR Green Assay, Standard Deviation, Stable Transfection, Infection, Virus

Journal: Cell Cycle

Article Title: A dual role for A-type lamins in DNA double-strand break repair

doi: 10.4161/cc.10.15.16531

Figure Lengend Snippet: Defects in NHEJ of dysfunctional telomeres are rescued by reconstitution of 53BP1. (A) Levels of 53BP1 and A-type lamins in U2OS cells after ectopic expression of 53BP1 (EV, empty vector control), followed by lentiviral transduction with shLmna or shCtrl. β-tubulin was used as loading control. Note how ectopic expression of 53BP1 prevents the decrease in protein levels upon depletion of A-type lamins. (B) Key of the different categories of metaphases based on the extent of chromosome end-to-end fusions induced by expression of TRF2ΔBΔM. Representative images of the different categories are shown. (C) Histograms showing the percentage of metaphases belonging to each category from the different cell lines. Top part: cells transduced with EV, shCtrl and TRF2ΔBΔM. Middle part: cells transduced with EV, shLmna and TRF2ΔBΔM. Bottom part: cells transduced with 53BP1, shLmna and TRF2ΔBΔM. 79–88 metaphases were analyzed per condition.

Article Snippet: MCF-7 and U2OS cells were obtained from ATCC (Manassas, VA).

Techniques: Expressing, Plasmid Preparation, Control, Transduction

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