Journal: medRxiv

Article Title: ARF1 prevents aberrant type I IFN induction by regulating STING activation and recycling

doi: 10.1101/2023.04.28.23289152

Figure Lengend Snippet: a , Induction of the ISRE promoter in THP-1-Dual WT (grey) and THP-1-Dual KO-cGAS (pink) cells transduced with lentiviral vectors expressing ARF1 WT or R99C as indicated, quantified by Lucia luciferase (LLuc) activity 72 h post transduction. IFN-β (1000 U/mL, 16 h) and cGAMP (10 µg/ml, 16 h) served as positive controls. Bars represent mean of n=3 ± SEM (biological replicates). Lower panel: Corresponding immunoblots of WCLs stained by anti-FLAG, anti-STING, anti-cGAS and anti-GAPDH. b , Exemplary electron microscopy analysis of HEK293T cells transiently transfected with ARF1 WT or ARF1 R99C as indicated. Mitochondria (m) are highlighted in insets in bottom panels. Electron-dense granules and inflated cristae are highlighted by black and white arrows, respectively. Annotations: cp, cytoplasm; er, endoplasmic reticulum; g , Golgi apparatus; lv, large vesicle; ly, lysosome; m, mitochondria; nc, nucleus. c, Exemplary immunoblots showing fractionation of ARF1 WT, R99C, Q71L and vector transfected HEK293T cells as indicated. WCLs and fraction blots stained by anti-FLAG, anti-TFAM (mitochondria), anti-LAMIN B1 (nucleus) and anti-GAPDH (cytosol). d, qPCR of mtDNA (MT-D-Loop) in the cytosolic fraction of ( c ) relative to total normalized cellular mtDNA (mtDNA/nuclear DNA) using the ΔΔCT method. n = 3 ± SEM. e , qPCR of mtDNA (MT-D-Loop) in the cytosolic fraction of primary fibroblasts from healthy donors (n2, f1, I7) or a patient (Patient 1) relative to total normalized cellular mtDNA (mtDNA/nuclear DNA) using the ΔΔCT method. n = 5 ± SEM. f, qPCR of representative ISG OAS1 in U2OS cells stably expressing STING and depleted of mtDNA by ddC, or untreated (NT), upon transfection with empty vector, ARF1 WT or R99C n=3 ± SEM. g , Exemplary immunoblot of WCLs of HEK293T cells transiently expressing ARF1 WT, R99C or vector. Blots were stained with anti-MFN1, anti-RHOT1, anti-FLAG and anti-GAPDH. Quantification of the band intensities for MFN1 normalized to the band intensities of GAPDH. Bars represent mean of n = 6 ± SEM (biological replicates). h , Exemplary immunoblots showing fractionation of HEK293T cells expressing ARF1 WT, R99C or vector control as well as VCP. WCLs and fraction blots stained by anti-FLAG, anti-HA, anti-TFAM (mitochondria), anti LAMIN B1 (nucleus) and anti-GAPDH (cytosol). i , qPCR of mtDNA (MT-D-Loop) in the cytosolic fraction of (h) relative to total normalized cellular mtDNA (mtDNA/nuclear DNA) using 725 the ΔΔCT method. n = 5 ± SEM.

Article Snippet: U2OS cells (ATCC) were maintained in McCoy medium (Invitrogen) supplemented with 10% (v/v) fetal bovine serum.

Techniques: Transduction, Expressing, Luciferase, Activity Assay, Western Blot, Staining, Electron Microscopy, Transfection, Fractionation, Plasmid Preparation, Stable Transfection, 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) 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: 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: ACS Applied Nano Materials

Article Title: Cell–Substrate Interactions Lead to Internalization and Localization of Layered MoS2 Nanosheets

doi: 10.1021/acsanm.0c03338

Figure Lengend Snippet: Figure 2. Cell−substrate interactions and uptake. (a) Cell count data for U2OS cells grown on MoS2 substrates (blue) and pristine glass controls (orange); error bars calculated as standard error in the mean from three repeat experiments (two samples per type for each experiment). (b) Optical micrograph of U2OS cells (7 days of growth) on a split substrate (left half of the substrate pristine glass and the right half coated in a MoS2 Langmuir thin film of typical density shown in the inset); MoS2 nanosheets have been accreted by the cells (dark regions). The inset scale bar is also 200 μm. (c, d) AFM height images of U2OS cells (3 days of growth) on MoS2 nanosheets, with cytoskeletal extensions and adhesion sites directed toward the MoS2 nanosheets on the substrate. (e, f) Optical micrographs showing the internalization of the MoS2 nanosheets and the localization around the nucleus in the ER (taken of the same sample as shown in (b)). The inset scale bar is also 20 μm.

Article Snippet: The cell lines used for the cell studies were U2OS (obtained from the American Type Culture Collection) and 1BR primary fibroblasts (obtained from the GDSC Research Tissue Bank, University of Sussex).

Techniques: Cell Counting

Journal: ACS Applied Nano Materials

Article Title: Cell–Substrate Interactions Lead to Internalization and Localization of Layered MoS2 Nanosheets

doi: 10.1021/acsanm.0c03338

Figure Lengend Snippet: Figure 3. Localization and lifetime of MoS2 nanosheets. (a) 2D Raman map of the intensity of the ∼405 cm−1 A1g peak of MoS2 (green) overlaid on the optical microscope image of cells. (b, c) Metrics37 indicate the same layer number (8 layers) but reduced length (<200 nm). (d) Top-down optical view of the location of (e) volumetric Raman mapping of the intensity of the A1g peak (green), with some nanosheets inside the cell and some still on the substrate. (f) Cell count data for U2OS cells grown on MoS2 substrates and glass controls for 7 days and then removed from the substrate, split, and reseeded such that cells initially from MoS2 substrates (M) are then grown on further fresh MoS2 substrates (MM) or glass controls (MC), and cells from control substrates (C) are seeded onto MoS2 substrates (CM) or further control substrates (CC); the error bars are calculated from standard error in the mean from two repeat experiments (two samples per type in each experiment). (g) Raman mapping of cells taken from MoS2 substrates and reseeded onto pristine glass controls, indicating that MoS2 nanosheets are still localized within the ER and present in daughter cells, with (h) minimal modification of innate Raman spectra indicating limited degradation.

Article Snippet: The cell lines used for the cell studies were U2OS (obtained from the American Type Culture Collection) and 1BR primary fibroblasts (obtained from the GDSC Research Tissue Bank, University of Sussex).

Techniques: Microscopy, Cell Counting, Control

Journal: ACS Applied Nano Materials

Article Title: Cell–Substrate Interactions Lead to Internalization and Localization of Layered MoS2 Nanosheets

doi: 10.1021/acsanm.0c03338

Figure Lengend Snippet: Figure 4. Generalization of the methodology and applications. (a, b) Optical images of U2OS cells grown on (a) MoS2 and (b) control substrates; a substantial increase in the size of the ER is observed for the cells grown on MoS2 nanosheets. One endoplasmic reticulum from each image is highlighted with blue dotted circles to give a guide to its size and location within the cell. (c) A cell with internalized MoS2 after irradiation with a 100 mW 660 nm laser showing severe local damage. (d) Cell count data for primary fibroblast cells, showing no toxicity after 7 days; error bars calculated from the experimental error of single experiment. (e, f) Optical micrographs of fibroblast cells grown on MoS2 substrates with nanosheets localized in the ER, but less accretion under the cells compared to those on U2OS.

Article Snippet: The cell lines used for the cell studies were U2OS (obtained from the American Type Culture Collection) and 1BR primary fibroblasts (obtained from the GDSC Research Tissue Bank, University of Sussex).

Techniques: Control, Irradiation, Cell Counting

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