drgfp reporter  (Addgene inc)

Journal: bioRxiv

Article Title: Proximity labeling reveals cell cycle–specific NEK2 interactions and a regulatory axis controlling NUSAP1 stability

doi: 10.64898/2026.01.25.701545

Figure Lengend Snippet: (A) Schematic overview of the experimental design. (B) Intracellular localization of TurboID, TurboID–NEK2-WT, and TurboID–NEK2-KD proteins upon doxycycline induction. Blue: DAPI (DNA); Green: FLAG tag (TurboID-fusion proteins); Red: γ-tubulin (centrosomes). (C) Western blot analysis of FLAG and NEK2 after biotin labeling and streptavidin pull-down. (D) Localization of biotinylated proteins in U2OS–TurboID cell groups. Top: dox–/biotin–; Middle: dox+/biotin–; Bottom: dox+/biotin+. Blue: DAPI (DNA); Green: Streptavidin-488 (biotinylation); Red: γ-tubulin (centrosomes). EV: Empty vector. Scale bars show 10 μm distance.

Article Snippet: U2OS (ATCC, CRL3455), MDA-MB-231 (ATCC, HTB-26) and HEK293-T (ATCC, CRL-3216) cells were maintained in DMEM (Sigma, D6429) supplemented with 10% FBS (Biowest, S1520) and 1% penicillin-streptomycin at 37°C in 5% CO 2 and were obtained from ATCC.

Techniques: FLAG-tag, Western Blot, Labeling, Plasmid Preparation

Journal: bioRxiv

Article Title: Proximity labeling reveals cell cycle–specific NEK2 interactions and a regulatory axis controlling NUSAP1 stability

doi: 10.64898/2026.01.25.701545

Figure Lengend Snippet: (A) Western blotting analysis of KIF2C, MAPRE3, NUSAP1, and MPG in unsynchronized samples. (B) Western blotting analysis of NUSAP1, KIF2C, KIF2A, MPG, MAPRE3, and SMURF2 in synchronized samples. (C) Immunofluorescence images showing co-localization of NEK2 with MAPRE3, NUSAP1, SMURF2, and KIFC1. (D–F) Co-immunoprecipitation (co-IP) validation of NEK2 interaction partners in (D) U2OS, (E) MDA-MB-231, and (F) HEK293T cells. CEP68 was used as a positive control for NEK2 interaction. EV: Empty vector. IgG: Immunoglobulin G. Scale bars show 5 μm distance.

Article Snippet: U2OS (ATCC, CRL3455), MDA-MB-231 (ATCC, HTB-26) and HEK293-T (ATCC, CRL-3216) cells were maintained in DMEM (Sigma, D6429) supplemented with 10% FBS (Biowest, S1520) and 1% penicillin-streptomycin at 37°C in 5% CO 2 and were obtained from ATCC.

Techniques: Western Blot, Immunofluorescence, Immunoprecipitation, Co-Immunoprecipitation Assay, Biomarker Discovery, Positive Control, Plasmid Preparation

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: Nar Molecular Medicine

Article Title: Rare SNP in the HELB gene interferes with RPA interaction and cellular function of HELB

doi: 10.1093/narmme/ugaf019

Figure Lengend Snippet: D506G substitution in HELB interferes with localization of HELB to chromatin in response to replication stress. ( A ) HELB KO U2OS cells expressing SFB-HELB variants (WT, D506G, and 3xA) were treated with 10 μM CPT for 1 h followed by pre-extraction to remove nonchromatin-bound proteins before fixing and staining with an anti-FLAG antibody to detect SFB-HELB. ( B ) The intensity of HELB in the nuclei of >130 cells for each condition was quantified using CellProfiler from biological triplicate experiments. Scale bar indicates 1 μm. Significance was assessed using a one-way ANOVA; n.s. is not significant. ( C ) Lysates of HELB KO HEK293T cells expressing SFB-HELB variants (WT, D506G, and 3xA) with or without CPT treatment were separated into soluble and chromatin fractions and visualized by western blotting. Additional replicates are shown in . ( D ) Quantification of western blots in panel (C) and in .

Article Snippet: HEK293T (ATCC CRL-3216), U2OS (ATCC HTB-96), U2OS-265 DSB reporter [ ], and U2OS DR-GFP (ATCC CRL-3455) cells were grown in Dulbecco's Modified Eagle Medium (DMEM) (Gibco) supplemented with 10% FBS (R&D solutions S11150 ) at 37°C with 5% CO 2 .

Techniques: Expressing, Extraction, Staining, Western Blot

Journal: Nar Molecular Medicine

Article Title: Rare SNP in the HELB gene interferes with RPA interaction and cellular function of HELB

doi: 10.1093/narmme/ugaf019

Figure Lengend Snippet: D506G HELB has reduced co-localization with DSBs relative to WT HELB. ( A ) A single array of mCherry labeled DSBs is produced upon treatment of U2OS DSB reporter cells with 4-OHT and Shield1. ( B ) DSB reporter cells expressing GFP-HELB variants (WT, D506G, and 3xA) were treated with 1 μM 4-OHT and 1 μM Shield1 for 4 h. Cells were fixed and stained with an antibody to mCherry before imaging. ( C ) Localization of GFP-HELB relative to Cherry-FokI foci was quantified blind for >150 cells per condition from biological triplicate experiments. Significance was assessed using a two-way ANOVA; n.s. is not significant.

Article Snippet: HEK293T (ATCC CRL-3216), U2OS (ATCC HTB-96), U2OS-265 DSB reporter [ ], and U2OS DR-GFP (ATCC CRL-3455) cells were grown in Dulbecco's Modified Eagle Medium (DMEM) (Gibco) supplemented with 10% FBS (R&D solutions S11150 ) at 37°C with 5% CO 2 .

Techniques: Labeling, Produced, Expressing, Staining, Imaging

Journal: Nar Molecular Medicine

Article Title: Rare SNP in the HELB gene interferes with RPA interaction and cellular function of HELB

doi: 10.1093/narmme/ugaf019

Figure Lengend Snippet: Expression of D506G HELB increases HR levels relative to expression of WT HELB. ( A ) Expression of I-SceI in DR-GFP-U2OS cells produces a DSB in the I-SceI GFP gene. Cells that repair the DSB by HR are identified as GFP-positive cells by the flow cytometry. ( B ) The percentage of GFP-positive cells in each sample are plotted. Significance was assessed using a one-way ANOVA; n.s. is not significant.

Article Snippet: HEK293T (ATCC CRL-3216), U2OS (ATCC HTB-96), U2OS-265 DSB reporter [ ], and U2OS DR-GFP (ATCC CRL-3455) cells were grown in Dulbecco's Modified Eagle Medium (DMEM) (Gibco) supplemented with 10% FBS (R&D solutions S11150 ) at 37°C with 5% CO 2 .

Techniques: Expressing, Flow Cytometry