u2os dr gfp  (ATCC)

Journal: bioRxiv

Article Title: PARP inhibitor synthetic lethality reveals homologous recombination sub-pathway architecture

doi: 10.64898/2026.03.06.709881

Figure Lengend Snippet: (A) Survival of HeLa RAD54B KO cells transfected with the siCTRL, siRAD54L or siBRCA2 upon treatment with 5 µM of olaparib or untreated. Cell viability was measured using growth curve analyses. All data show mean ± SEM (n=3). (B) HeLa (i) EV, (ii) RAD54L KO, (iii) RAD51AP1 KO and (iv) RAD54B KO cells were transfected with the indicated siRNAs, and γH2AX foci were enumerated after treatment with 1 µM olaparib for 24 h in EdU-negative G2 cells. Spontaneous foci were subtracted. All data show mean ± SEM (n = 2-3). Results from individual experiments, each derived from 40 cells, are indicated. (C) HeLa pGC reporter cells carry a GFP cassette that is disrupted by the I-SceI sequence and a second truncated GFP cassette (left). I-SceI-induced HR repair events result in the expression of functional GFP. HeLa pGC cells were transfected with the indicated siRNAs, followed by transfection with the I-SceI expression plasmid to induce site-specific DSBs. The percentage of GFP-positive cells was measured by flow cytometry. All data show mean ± SEM (n=3). All data show mean ± SEM (n = 3). Results from individual experiments are indicated. (D) (Top) U2OS cells were transfected with the indicated siRNAs, and γH2AX foci were enumerated after treatment with 1 µM olaparib for 24 h in EdU-negative G2 cells. Spontaneous foci were subtracted. All data show mean ± SEM (n = 2-5). Results from individual experiments, each derived from 40 cells, are indicated. (Bottom) Knockdown of BRCA2, RAD51AP1 and RAD54L was confirmed by western blotting. (E) Quantitative RT–PCR analysis of RAD54B mRNA expression levels in HeLa and U2OS cells after transfection with siCTRL or siRAD54B. Expression levels were normalized to GAPDH, with siCTRL defined as 100%. All data show mean ± SEM (n = 2). (F) U2OS DR-GFP cells were transfected with the indicated siRNAs, followed by transfection with the I-SceI expression plasmid to induce site-specific DSBs. The percentage of GFP-positive cells, representing HR repair events, was measured by flow cytometry. All data show mean ± SEM (n = 4). Results from individual experiments are indicated. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns: not significant (two-tailed t test).

Article Snippet: U2OS (ATCC), U2OS DR-GFP, HeLa-S3 cells (ATCC), HeLa EV, HeLa RAD54L, HeLa RAD54B KO, HeLa RAD51AP1 KO, HeLa ATRX KO and HeLa pGC cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma), supplemented with 10% fetal bovine serum (FBS; Bio&SELL), 1% non-essential amino acid (NEAA; Sigma-Aldrich) and 1% penicillin/streptomycin (Sigma-Aldrich).

Techniques: Transfection, Derivative Assay, Sequencing, Expressing, Functional Assay, Plasmid Preparation, Flow Cytometry, Knockdown, Western Blot, Quantitative RT-PCR, Two Tailed Test

Journal: bioRxiv

Article Title: PARP inhibitor synthetic lethality reveals homologous recombination sub-pathway architecture

doi: 10.64898/2026.03.06.709881

Figure Lengend Snippet: U2OS cells were transfected with the indicated siRNAs, and γH2AX foci were enumerated 10 h following 2 Gy IR in EdU-negative G2 cells. Spontaneous foci were subtracted. All data show mean ± SEM (n = 3). Results from individual experiments, each derived from 40 cells, are indicated. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns: not significant (two-tailed t test).

Article Snippet: U2OS (ATCC), U2OS DR-GFP, HeLa-S3 cells (ATCC), HeLa EV, HeLa RAD54L, HeLa RAD54B KO, HeLa RAD51AP1 KO, HeLa ATRX KO and HeLa pGC cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma), supplemented with 10% fetal bovine serum (FBS; Bio&SELL), 1% non-essential amino acid (NEAA; Sigma-Aldrich) and 1% penicillin/streptomycin (Sigma-Aldrich).

Techniques: Transfection, Derivative Assay, Two Tailed Test

Journal: bioRxiv

Article Title: PARP inhibitor synthetic lethality reveals homologous recombination sub-pathway architecture

doi: 10.64898/2026.03.06.709881

Figure Lengend Snippet: (A) Representative images of γH2AX foci and ATRX expression in U2OS ATRX G2 cells following transfection with the indicated siRNAs and subsequent irradiation are shown. (B) U2OS ATRX cells, with (blue) and without (green) doxycycline-induced ATRX expression, were transfected with the indicated siRNAs, and γH2AX foci were enumerated at 12 h following 2 Gy IR in EdU-negative G2 cells. Spontaneous foci were subtracted. All data show mean ± SEM (n = 3). Results from individual experiments, each derived from 40 cells, are indicated. Knockdown was confirmed by western blotting. (C) U2OS ATRX cells, with (blue) and without (green) doxycycline-induced ATRX expression, were transfected with the indicated siRNAs. They were irradiated with 2 Gy, collected after 16h, and processed to obtain chromosome spreads. SCEs per spread were quantified and normalized to 70 chromosomes. Results from individual experiments, each derived from 40 cells, are indicated (top), and red horizontal lines indicate the mean. The bar graph shows the IR-induced SCE numbers (total SCEs after background subtraction) (bottom). IR-induced SCE data show mean ± SEM, and results from individual experiments are indicated. 80-120 spreads and >7,000 chromosomes per condition were analysed from 2-3 independent experiments. (D) Model illustrating the interplay between RAD51AP1 and RAD54B, which promote SDSA, and RAD54L and ATRX, which facilitate dHJ formation.

Article Snippet: U2OS (ATCC), U2OS DR-GFP, HeLa-S3 cells (ATCC), HeLa EV, HeLa RAD54L, HeLa RAD54B KO, HeLa RAD51AP1 KO, HeLa ATRX KO and HeLa pGC cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma), supplemented with 10% fetal bovine serum (FBS; Bio&SELL), 1% non-essential amino acid (NEAA; Sigma-Aldrich) and 1% penicillin/streptomycin (Sigma-Aldrich).

Techniques: Expressing, Transfection, Irradiation, Derivative Assay, Knockdown, Western Blot

Journal: bioRxiv

Article Title: PARP inhibitor synthetic lethality reveals homologous recombination sub-pathway architecture

doi: 10.64898/2026.03.06.709881

Figure Lengend Snippet: (A) (Top) U2OS cells were transfected with the indicated siRNAs, and γH2AX foci were enumerated 10 h following 2 Gy IR in EdU-negative G2 cells. Spontaneous foci were subtracted. All data show mean ± SEM (n = 3-9). Results from individual experiments, each derived from 40 cells, are indicated. (Bottom) Knockdown of TOP3A, RECQ5, RAD51AP1 and RAD54L was confirmed by western blotting. (B) U2OS cells were transfected with the indicated siRNAs, and γH2AX foci were enumerated after treatment with 1 µM olaparib for 24 h in EdU-negative G2 cells. Spontaneous foci were subtracted. All data show mean ± SEM (n = 3). Results from individual experiments, each derived from 40 cells, are indicated. (C) U2OS DR-GFP cells were transfected with the indicated siRNAs, followed by transfection with the I-SceI expression plasmid to induce site-specific DSBs. The percentage of GFP-positive cells, representing HR repair events, was measured by flow cytometry. All data show mean ± SEM (n = 3-7). Results from individual experiments are indicated. (D) U2OS cells were transfected with siCTRL, siBRCA2 and siTOP3A, irradiated, and RAD51 foci were enumerated 2 h following 2 Gy IR in EdU-negative G2 cells (left). Spontaneous foci were subtracted. All data show mean ± SEM (n = 3). Results from individual experiments, each derived from 40 cells, are indicated. Knockdown of BRCA2 and TOP3A was confirmed by western blotting (right). (E) U2OS cells were transfected with the indicated siRNAs, and γH2AX foci were enumerated 10 h following 2 Gy IR in EdU-negative G2 cells (left). Spontaneous foci were subtracted. All data show mean ± SEM (n = 2-4). Results from individual experiments, each derived from 40 cells, are indicated. Knockdown of BLM was confirmed by western blotting (right). *P < 0.05; **P < 0.01; ***P < 0.001; ns: not significant (two-tailed t test).

Article Snippet: U2OS (ATCC), U2OS DR-GFP, HeLa-S3 cells (ATCC), HeLa EV, HeLa RAD54L, HeLa RAD54B KO, HeLa RAD51AP1 KO, HeLa ATRX KO and HeLa pGC cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma), supplemented with 10% fetal bovine serum (FBS; Bio&SELL), 1% non-essential amino acid (NEAA; Sigma-Aldrich) and 1% penicillin/streptomycin (Sigma-Aldrich).

Techniques: Transfection, Derivative Assay, Knockdown, Western Blot, Expressing, Plasmid Preparation, Flow Cytometry, Irradiation, Two Tailed Test

Journal: bioRxiv

Article Title: PARP inhibitor synthetic lethality reveals homologous recombination sub-pathway architecture

doi: 10.64898/2026.03.06.709881

Figure Lengend Snippet: (A) Representative images of γH2AX foci in U2OS ATRX G2 cells following transfection with the indicated siRNAs and subsequent irradiation are shown. (B) U2OS ATRX cells, with (blue) and without (green) doxycycline-induced ATRX expression, were transfected with the indicated siRNAs, and γH2AX foci were enumerated at 12 h following 2 Gy IR in EdU-negative G2 cells. Spontaneous foci were subtracted. Striped bars indicate TOP3A depletion. All data show mean ± SEM (n = 3). Results from individual experiments, each derived from 40 cells, are indicated. Knockdown was confirmed by western blotting. (C) U2OS ATRX cells, with (blue) and without (green) doxycycline-induced ATRX expression, were transfected with the indicated siRNAs. They were irradiated with 2 Gy, collected after 16h, and processed to obtain chromosome spreads. SCEs per spread were quantified and normalized to 70 chromosomes. Results from individual experiments, each derived from 40 cells, are indicated (left), and red horizontal lines indicate the mean. The bar graph shows the IR-induced SCE numbers (total SCEs after background subtraction) (right). IR-induced SCE data show mean ± SEM, and results from individual experiments are indicated. 120 spreads and >7,000 chromosomes per condition were analysed from three independent experiments. (D) U2OS cells were transfected with siCTRL, siRAD54L and/or siTOP3A, labeled with EdU, irradiated with 4 Gy, and then incubated with BrdU. Representative images of BrdU foci reflecting DNA repair synthesis in G2 cells are shown. (E) BrdU foci were enumerated in EdU-negative G2 cells. Spontaneous foci were subtracted. All data show mean ± SEM (n = 3). Results from individual experiments, each derived from 40 cells, are indicated. **P < 0.01 (two-tailed t test). (F) Model illustrating the regulation of HR sub-pathway usage by TOP3A in intrinsically ATRX-deficient U2OS cells. TOP3A acts as a positive SDSA-promoting HR factor together with RAD51AP1 and RAD54B to facilitate SDSA usage. Additionally, TOP3A indirectly inhibits the usage of the dHJ sub-pathway in ATRX-deficient cells. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns: not significant (two-tailed t test).

Article Snippet: U2OS (ATCC), U2OS DR-GFP, HeLa-S3 cells (ATCC), HeLa EV, HeLa RAD54L, HeLa RAD54B KO, HeLa RAD51AP1 KO, HeLa ATRX KO and HeLa pGC cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma), supplemented with 10% fetal bovine serum (FBS; Bio&SELL), 1% non-essential amino acid (NEAA; Sigma-Aldrich) and 1% penicillin/streptomycin (Sigma-Aldrich).

Techniques: Transfection, Irradiation, Expressing, Derivative Assay, Knockdown, Western Blot, Labeling, Incubation, Two Tailed Test

Journal: bioRxiv

Article Title: PARP inhibitor synthetic lethality reveals homologous recombination sub-pathway architecture

doi: 10.64898/2026.03.06.709881

Figure Lengend Snippet: (A) HeLa and U2OS cells were transfected with the indicated siRNAs, irradiated with 2 Gy, and γH2AX foci were enumerated at 8 h (HeLa) or 10 h (U2OS) post-irradiation in EdU-negative G2 cells. Spontaneous foci were subtracted. All data show mean ± SEM (n = 2-3). Results from individual experiments, each derived from 40 cells, are indicated. Knockdown of HIRA was confirmed by western blotting (right). (B) HeLa pGC cells were transfected with the indicated siRNAs, followed by transfection with the I-SceI expression plasmid to induce site-specific DSBs. The percentage of GFP-positive cells, representing HR repair events, was measured by flow cytometry. All data show mean ± SEM (n = 3). Results from individual experiments are indicated. (C) U2OS cells were transfected with the siHIRA and RAD51 foci were enumerated 2 h following 2 Gy IR in EdU-negative G2 cells. Spontaneous foci were subtracted. All data show mean ± SEM (n = 2). Results from individual experiments, each derived from 40 cells, are indicated. *P < 0.05; **P < 0.01; ***P < 0.001; ns: not significant (two-tailed t test).

Article Snippet: U2OS (ATCC), U2OS DR-GFP, HeLa-S3 cells (ATCC), HeLa EV, HeLa RAD54L, HeLa RAD54B KO, HeLa RAD51AP1 KO, HeLa ATRX KO and HeLa pGC cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma), supplemented with 10% fetal bovine serum (FBS; Bio&SELL), 1% non-essential amino acid (NEAA; Sigma-Aldrich) and 1% penicillin/streptomycin (Sigma-Aldrich).

Techniques: Transfection, Irradiation, Derivative Assay, Knockdown, Western Blot, Expressing, Plasmid Preparation, Flow Cytometry, Two Tailed Test

Journal: bioRxiv

Article Title: PARP inhibitor synthetic lethality reveals homologous recombination sub-pathway architecture

doi: 10.64898/2026.03.06.709881

Figure Lengend Snippet: (A) U2OS ATRX cells, with (blue) and without (green) doxycycline-induced ATRX expression, were transfected with the indicated siRNAs, and γH2AX foci were enumerated at 12 h following 2 Gy IR in EdU-negative G2 cells. Spontaneous foci were subtracted. All data show mean ± SEM (n = 3). Results from individual experiments, each derived from 40 cells, are indicated. Knockdown was confirmed by western blotting. (B) U2OS DR-GFP reporter cells carry a GFP cassette that is disrupted by the I-SceI sequence and a second truncated GFP cassette. I-SceI-induced HR repair events result in the expression of functional GFP. Cells were transfected with the indicated siRNAs followed by the I-SceI plasmid transfection, and GFP-positive cells were measured by flow cytometry. All data show mean ± SEM (n = 3). Results from individual experiments are indicated. (C) Model illustrating the regulation of HR sub-pathway usage in intrinsically ATRX-deficient U2OS cells. In addition to RAD51AP1 and RAD54B, HIRA contributes to SDSA, but not to the dHJ sub-pathway, through its H3.3 deposition function. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns: not significant (two-tailed t test).

Article Snippet: U2OS (ATCC), U2OS DR-GFP, HeLa-S3 cells (ATCC), HeLa EV, HeLa RAD54L, HeLa RAD54B KO, HeLa RAD51AP1 KO, HeLa ATRX KO and HeLa pGC cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma), supplemented with 10% fetal bovine serum (FBS; Bio&SELL), 1% non-essential amino acid (NEAA; Sigma-Aldrich) and 1% penicillin/streptomycin (Sigma-Aldrich).

Techniques: Expressing, Transfection, Derivative Assay, Knockdown, Western Blot, Sequencing, Functional Assay, Plasmid Preparation, Flow Cytometry, Two Tailed Test

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