Journal: FEBS letters

Article Title: Differential effects of the N-terminal helix of FGF8b on the activity of a small-molecule FGFR inhibitor in cell culture and for the extracellular domain of FGFR3c in solution.

doi: 10.1002/1873-3468.14976

Figure Lengend Snippet: Fig. 1. Activity of SSR against FGF2 and FGF8b. (A–D) Fluorescence intensity analysis of U-2 OS cells immunolabeled for pMAPK (pY202/ 204) (A, B) or pPLCγ1 (pY783) (C, D) after stimulation with FGF2 (A, C) and FGF8b (B, D) and in the presence and absence of 25 μM SSR. The statistical significance was evaluated using the Mann–Whitney U test (*P < 0.05; **P < 0.01; ***P < 0.001; n.s., non-significant). Number of biologically independent replicates is n = 4 for A, B, and D; and 3 for panel C. (E) Representative microscopic images of proliferating bEnd.3 cells (EdU+, green; upper panel) and total bEnd.3 cells (nuclear marker DAPI, gray; bottom panel). Length of the scale bar equals 100 μM. (F) Percentage of proliferating human bEnd.3 cells upon stimulation by FGF2 and FGF8b in the presence and absence of either 50 nM or 100 μM SSR. BSA was used as a negative control. Error bars represent the SEM. Number of biologically independent replicates is n = 3.

Article Snippet: The human osteosarcoma cell line U-2 OS (CLS Cell Lines Service GmbH, Eppelheim, Germany; cat. no. 300364) was grown in growth medium consisting of high glucose DMEM/F-12 (Gibco, Grand Island, NY, USA; cat. no. 21041025) and supplemented with 1% GlutaMAX (Gibco; cat. no. 35050061), 1 unit mL 1 penicillin, 1 μg mL 1 streptomycin (Gibco; cat. no. 10378016), and 10% FBS (Satorius, Goettingen, Germany) at 37 °C and 5% CO2.

Techniques: Activity Assay, Fluorescence, Immunolabeling, MANN-WHITNEY, Marker, Negative Control

Journal: Molecular and Cellular Biology

Article Title: LDB1 Enforces Stability on Direct and Indirect Oncoprotein Partners in Leukemia

doi: 10.1128/MCB.00652-19

Figure Lengend Snippet: Halo-tagged proteins expressed from recombinant lentiviruses have physiological abundance and localization and can be precisely quantified in lysates or live cells. (A) Cell lysates were prepared from untransduced Jurkat cells; Jurkat cells transduced with Halo-LMO2; untransduced K562 cells; K562 cells transduced with Halo-LMO2; and KOPT-K1, LOUCY, and U937 cells. Lysates were subjected to 4% to 15% SDS-PAGE, transferred, and blotted with anti-LDB1, anti-LMO2 monoclonal antibody, anti-Halo, anti-TAL1, and anti-VCP (gel loading control). Molecular weight standards were run on the same gel and are shown at the right. (B) The top panel shows an immunoblot of lysates prepared from U2OS with a Halo knock-in at CTCF (lane 1) or lysates from Jurkat cells transduced with the constructs shown in the grid above (lanes 2 to 14). Empty vector control is shown as EBFPII-Hygro (lane 2). Lanes 2 to 9 show lysates from Jurkat cells lentivirally expressing Halo-CTCF at increasing MOIs. Lanes 10 to 14 show lysates from Jurkat cells lentivirally expressing Halo-SOX2 at increasing MOIs. The top panel shows gradient SDS-PAGE, transfer, and blotting with anti-CTCF antibody. The middle panel shows direct in-gel Halo fluorescence. Live cells prepared as described for panel A were labeled with cell-permeative fluorescent ligand R110, lysed, and subjected to gradient SDS-PAGE. The gel was visualized for green fluorescence as described in Materials and Methods. (C) Live Jurkat cells expressing Halo-tagged proteins were labeled with R110 Halo ligand, washed, and subjected to flow cytometry. Histograms show FITC fluorescence of the various cells in comparison to the labeled U2OS cells, which have a Halo tag knocked into the CTCF gene. (D) Table showing calculated copy numbers of Halo-tagged proteins based on the absolute values derived from the Halo knock-in cell line. Lentiviral Halo CTCF and Halo SOX2 were quantified from cell populations infected at intermediate and low MOIs, respectively. (E) Confocal microscopy of Jurkat cells lentivirally expressing Halo-LMO2 and labeled with Halo ligand, R110, and nuclear stain (Syto 17 Red). The right panel shows a merged image. Voxel quantification showed that 95% of Halo-LMO2 was nuclear.

Article Snippet: Catalog no. S7579 pBluescript SK Stratagene Iscove's modified Dulbecco's medium (IMDM) Gibco Catalog no. 12200-036 RPMI 1640 Gibco Catalog no. 31800-022 Penicillin-streptomycin solution 10× Corning Catalog no. 30-022-CI Geneticin Gibco Catalog no. 10131-027 0.05% trypsin, 0.53 mM EDTA 1× [−]sodium bicarbonate Corning Catalog no. 20116004 Puromycin dihydrochloride Fisher Bioreagents Catalog no. BP2956-100 Pierce protease inhibitor tablets Thermo Scientific Catalog no. A32965 Hygromycin B-PBS (50 mg/ml) Invitrogen Catalog no. 10687010 Anti-FLAG M2 resin Sigma Catalog no. A2220 Protein A/G resin Santa Cruz Polyvinylidene difluoride (PVDF) membrane GE Catalog no. 10600022 SuperSignal PicoWest Plus Thermo/Pierce Catalog no. 1863099 Experimental models: cell lines Human: HEK 293 ATCC Human: Jurkat ATCC Human: K562 ATCC Human: KOPTK1 ATCC Human: LOUCY ATCC Human: U937 ATCC Human: U2OS C32 Halo-CTCF Hansen et al. ( 27 ) U2OS endogenous knock-in cell line where all endogenous copies of CTCF have been N-terminally tagged with FLAG-HaloTag; clone 32 Software and algorithms Flowjo 10.3 analysis software FlowJo, LLC Contact corresponding author for URL Ideas software Amnis Corporation Contact corresponding author for URL ImageLab 5.2.1 BioRad Contact corresponding author for URL Imaris Bitplane Inc. Other CytoFLEX benchtop cytometer Beckman Contact corresponding author for URL Leica TCS SP8 confocal imaging system Leica Contact corresponding author for URL ImageStream Mk II Amnis Contact corresponding author for URL Open in a separate window Reagents and resources Experimental model and subject details. (i) Cell lines.

Techniques: Recombinant, Transduction, SDS Page, Control, Molecular Weight, Western Blot, Knock-In, Construct, Plasmid Preparation, Expressing, Fluorescence, Labeling, Flow Cytometry, Comparison, Derivative Assay, Infection, Confocal Microscopy, Staining

Journal: Molecular and Cellular Biology

Article Title: LDB1 Enforces Stability on Direct and Indirect Oncoprotein Partners in Leukemia

doi: 10.1128/MCB.00652-19

Figure Lengend Snippet: Reagents and resources

Article Snippet: Catalog no. S7579 pBluescript SK Stratagene Iscove's modified Dulbecco's medium (IMDM) Gibco Catalog no. 12200-036 RPMI 1640 Gibco Catalog no. 31800-022 Penicillin-streptomycin solution 10× Corning Catalog no. 30-022-CI Geneticin Gibco Catalog no. 10131-027 0.05% trypsin, 0.53 mM EDTA 1× [−]sodium bicarbonate Corning Catalog no. 20116004 Puromycin dihydrochloride Fisher Bioreagents Catalog no. BP2956-100 Pierce protease inhibitor tablets Thermo Scientific Catalog no. A32965 Hygromycin B-PBS (50 mg/ml) Invitrogen Catalog no. 10687010 Anti-FLAG M2 resin Sigma Catalog no. A2220 Protein A/G resin Santa Cruz Polyvinylidene difluoride (PVDF) membrane GE Catalog no. 10600022 SuperSignal PicoWest Plus Thermo/Pierce Catalog no. 1863099 Experimental models: cell lines Human: HEK 293 ATCC Human: Jurkat ATCC Human: K562 ATCC Human: KOPTK1 ATCC Human: LOUCY ATCC Human: U937 ATCC Human: U2OS C32 Halo-CTCF Hansen et al. ( 27 ) U2OS endogenous knock-in cell line where all endogenous copies of CTCF have been N-terminally tagged with FLAG-HaloTag; clone 32 Software and algorithms Flowjo 10.3 analysis software FlowJo, LLC Contact corresponding author for URL Ideas software Amnis Corporation Contact corresponding author for URL ImageLab 5.2.1 BioRad Contact corresponding author for URL Imaris Bitplane Inc. Other CytoFLEX benchtop cytometer Beckman Contact corresponding author for URL Leica TCS SP8 confocal imaging system Leica Contact corresponding author for URL ImageStream Mk II Amnis Contact corresponding author for URL Open in a separate window Reagents and resources Experimental model and subject details. (i) Cell lines.

Techniques: Recombinant, Staining, Modification, Protease Inhibitor, Membrane, Knock-In, Software, Cytometry, Imaging

Journal: bioRxiv

Article Title: High-throughput mapping of 6,888 RAD51D variants identifies distinct biochemical functions needed for homologous recombination and olaparib response

doi: 10.64898/2026.01.11.698865

Figure Lengend Snippet: (a) ClinVar classifications of RAD51D missense variants each year. P/LP = Pathogenic/Likely Pathogenic, B/LB = Benign/Likely Benign, VUS/CON = unknown significance/conflicting reports. ( b-c ) RAD51D forms an obligate heterodimer with XRCC2 to form the ( b ) BCDX2 (PDB: 8GBJ) or ( c ) XRCC3 (X3CDX2) complexes (PDB: 9SVX) through its interaction with RAD51C. ( d ) Schematic of pooled RAD51D variant function assay. RAD51D mutant library cell population is treated with 250 nM olaparib over several passages, selecting for cells with normal RAD51D function. RAD51D variants are quantified within the starting (P0) and final (P2) cell populations by sequencing and scored by their relative enrichment in P2 versus P0.

Article Snippet: Human osteosarcoma U2OS SCR (sister chromatid recombination) #18 wild-type (gifted from Mauro Modesti; ) and RAD51D CRISPR knockout (KO; clone #4, purchased from DSMZ, no. ACC835) were cultured in Corning DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin antibiotic (50 U/mL).

Techniques: Variant Assay, Functional Assay, Mutagenesis, Sequencing

Journal: bioRxiv

Article Title: High-throughput mapping of 6,888 RAD51D variants identifies distinct biochemical functions needed for homologous recombination and olaparib response

doi: 10.64898/2026.01.11.698865

Figure Lengend Snippet: Histograms of RAD51D variant abundance, log10(counts/million counts), within pre-selection libraries for each mutagenesis tile. Cutoff line is at 100 cpm (1/10,000).

Article Snippet: Human osteosarcoma U2OS SCR (sister chromatid recombination) #18 wild-type (gifted from Mauro Modesti; ) and RAD51D CRISPR knockout (KO; clone #4, purchased from DSMZ, no. ACC835) were cultured in Corning DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin antibiotic (50 U/mL).

Techniques: Variant Assay, Selection, Mutagenesis

Journal: bioRxiv

Article Title: High-throughput mapping of 6,888 RAD51D variants identifies distinct biochemical functions needed for homologous recombination and olaparib response

doi: 10.64898/2026.01.11.698865

Figure Lengend Snippet: ( a ) Distributions of function scores by variant class; nonsense variants in codons 1–304 are perfectly separated from synonymous variants. Vertical lines denote cutoffs for function classifications (LOF, loss-of-function; INT, intermediate; NEU, neutral). ( b ) Per-residue mean missense function scores, grouped by residue surface area exposure status and secondary structure. Unique LOF exposed residues are labeled. ( c ) Variant-to-function heatmap across RAD51D, shaded by function score (white, WT-like; red, null-like) for each mutant amino acid (rows) at each codon position (columns). Variants that do not pass multiple testing correction (lfsr>0.01) are shaded from white to gray, WT residues are boxed, and dark gray denotes missing data. Tracks above heatmaps, from top to bottom: conservation score, protein secondary structure, key domains. Asp (D) or Glu (E) are shown in boldface to highlight stronger effects of these substitutions at some sites. ( d ) Function scores separate pathogenic from benign variants among single-residue variants reported in ClinVar, and provide evidence for missense VUS and those with conflicting reports (all variants plotted SpliceAI score <0.2). Point color denotes variant type, and filled/open points denote statistical significance (i.e. filled: lfsr ≤0.01). ( e ) Precision-recall curve showing classification performance between a set of 211 SNVs with confident P/LP and B/LB variant classification, included in ( a ).

Article Snippet: Human osteosarcoma U2OS SCR (sister chromatid recombination) #18 wild-type (gifted from Mauro Modesti; ) and RAD51D CRISPR knockout (KO; clone #4, purchased from DSMZ, no. ACC835) were cultured in Corning DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin antibiotic (50 U/mL).

Techniques: Variant Assay, Residue, Labeling, Mutagenesis

Journal: bioRxiv

Article Title: High-throughput mapping of 6,888 RAD51D variants identifies distinct biochemical functions needed for homologous recombination and olaparib response

doi: 10.64898/2026.01.11.698865

Figure Lengend Snippet: ( a ) Surface representation of RAD51D within the BCDX2 complex (PDB: 8GBJ). Surface residues are colored according to either red (50% pathogenicity) or yellow (20% pathogenicity). Cartoon representations of XRCC2 (purple), RAD51D (light blue), RAD51C (green), and RAD51B (red) are shown consistently throughout. Bound ATP molecules are represented as yellow-orange sticks. ( b ) Specific loss-of-function variants (red, 50% pathogenicity) listed above or (yellow, 20% pathogenicity) listed below the RAD51D schematic are shown with the indicated interaction interfaces indicated. ( c-g ) Binding sites for RAD51C ( c ), ATP site 1 ( d ), XRCC2 ( e ), ssDNA ( f ), and ATP site 2 ( g ).

Article Snippet: Human osteosarcoma U2OS SCR (sister chromatid recombination) #18 wild-type (gifted from Mauro Modesti; ) and RAD51D CRISPR knockout (KO; clone #4, purchased from DSMZ, no. ACC835) were cultured in Corning DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin antibiotic (50 U/mL).

Techniques: Binding Assay

Journal: bioRxiv

Article Title: High-throughput mapping of 6,888 RAD51D variants identifies distinct biochemical functions needed for homologous recombination and olaparib response

doi: 10.64898/2026.01.11.698865

Figure Lengend Snippet: ( a ) Intolerance of negatively charged (i.e. Asp (D) and Glu (E)) residues throughout RAD51D, as measured by the difference between the mean function scores of D/E and non-D/E mutations. Residues with a difference ±0.5 are labeled. ( b ) Mean function scores of residues making contact with other proteins, ATP, or DNA via H-bonds or salt bridges (noted with circles or starbursts, respectively) in the BCDX2 complex (as predicted in PDB: 8GBJ). ( c ) AlphaMissense pathogenicity scores compared to MAVE function scores. AlphaMissense scores were binned as benign, pathogenic, and ambiguous using the published cutoff values. Counts of each plotted category are reported in the table.

Article Snippet: Human osteosarcoma U2OS SCR (sister chromatid recombination) #18 wild-type (gifted from Mauro Modesti; ) and RAD51D CRISPR knockout (KO; clone #4, purchased from DSMZ, no. ACC835) were cultured in Corning DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin antibiotic (50 U/mL).

Techniques: Labeling

Journal: bioRxiv

Article Title: High-throughput mapping of 6,888 RAD51D variants identifies distinct biochemical functions needed for homologous recombination and olaparib response

doi: 10.64898/2026.01.11.698865

Figure Lengend Snippet: ( a ) Schematic of sister chromatid exchange assay to measure homologous recombination. In this assay, a cassette with a non-functional copy of GFP is integrated into RAD51D CRISPR/Cas9 U2OS cells. This GFP has a unique I-SceI restriction cut site. A DSB can be induced by expression of a plasmid expressing the I-SceI restriction enzyme. GFP expression is restored by use of a homologous template provided on the cassette following homologous recombination. ( b-c ) A plasmid with indicated synonymous or truncation variant was transiently transfected RAD51D CRISPR/Cas9 U2OS cells with a plasmid coding for the I-SceI restriction enzyme. The percentage of GFP+ cells was measured after three days, indicating a recombination event using a GFP fragment on the cassette. The HR proficiency threshold was determined based on comparison with the range of synonymous variants (green bars) to a wild-type RAD51D expressing plasmid (HR >0.75). The threshold for loss of HR function was calculated using the range of truncation variants compared to a wild-type RAD51D expressing plasmid, as <0.6 (indicated in red for the variants). Note that a subset of those variants analyzed here are replotted in as representative variants. The experiment was performed three to seven times with standard deviations plotted. An empty vector was used as a negative control.

Article Snippet: Human osteosarcoma U2OS SCR (sister chromatid recombination) #18 wild-type (gifted from Mauro Modesti; ) and RAD51D CRISPR knockout (KO; clone #4, purchased from DSMZ, no. ACC835) were cultured in Corning DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin antibiotic (50 U/mL).

Techniques: Homologous Recombination, Functional Assay, CRISPR, Expressing, Plasmid Preparation, Variant Assay, Transfection, Comparison, Negative Control

Journal: bioRxiv

Article Title: High-throughput mapping of 6,888 RAD51D variants identifies distinct biochemical functions needed for homologous recombination and olaparib response

doi: 10.64898/2026.01.11.698865

Figure Lengend Snippet: (a) The HR proficiency of 70 RAD51D variants was tested using the sister chromatid recombination assay calibrated against synonymous and truncation variants. Loss of HR function was calculated based on the range of truncation variants (indicated in red) as <0.6 (missense LOF in light). HR proficient variants (gray), were determined based on comparison with the range of synonymous variants (green) if they exhibited HR >0.75. Variants with intermediate HR proficiency, in the range of 0.6–0.75, were color-coded in yellow. See for all synonymous and truncation variants tested, and note that a subset of those variants analyzed are replotted here as representative variants. The experiment was performed 4–9 times and plotted as mean values ± s.d. ( b-e ) Olaparib and cisplatin sensitivity of breast/ovarian cancer identified RAD51D variants with reduced HR. Representative images of U2OS cell lines stably expressing WT or the indicated RAD51D variant that were treated with increasing concentrations of Olaparib ( b ) or cisplatin ( d ). ( c & e ) Clonogenic survival assays were quantified by percent colony area and normalized to the area of untreated or vehicle control. Means of 4–12 trials are plotted ± s.d., and drug concentrations with colony area <0.001 are omitted. ( f ) RAD51D variants with reduced HR are expressed. Western blot analysis of U2OS cell lines stably expressing WT or the indicated RAD51D variants. RAD51D protein expression was assessed using an anti-RAD51D antibody, and equal protein loading was assessed using an anti-Tubulin antibody. Note that L4H exhibits reduced protein expression. Experiment performed in triplicate. ( g ) Structures of the RAD51 paralog pentamer, XRCC3 complex, (PDB: 9SVX) and the BCDX2 complex (PDB: 8GBJ). Cartoon representations of XRCC2 (purple), RAD51D (light blue), RAD51C (green), XRCC3 (yellow), RAD51 (orange), and RAD51B (red) are shown consistently throughout. Bound ATP molecules are represented as yellow-orange sticks. ssDNA is shown in orange. ( h-i ) Variants causing complete (red, h ) or intermediate (yellow, i ) deficiency in homologous recombination are highlighted on RAD51D. The interacting surfaces of RAD51C and XRCC2 are outlined with black dashed lines. The contact interfaces are identical between the XRCC3 and BCDX2 complex. ( j-k ) Variants that disrupt RAD51C-RAD51D interactions are shown as red sticks, mapped onto the BCDX2 complex structure.

Article Snippet: Human osteosarcoma U2OS SCR (sister chromatid recombination) #18 wild-type (gifted from Mauro Modesti; ) and RAD51D CRISPR knockout (KO; clone #4, purchased from DSMZ, no. ACC835) were cultured in Corning DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin antibiotic (50 U/mL).

Techniques: Recombination Assay, Comparison, Stable Transfection, Expressing, Variant Assay, Control, Western Blot, Homologous Recombination

Journal: bioRxiv

Article Title: High-throughput mapping of 6,888 RAD51D variants identifies distinct biochemical functions needed for homologous recombination and olaparib response

doi: 10.64898/2026.01.11.698865

Figure Lengend Snippet: ( a-b ) Interaction proficiency of RAD51D VUS was measured via ( a ) yeast 2-hybrid (Y2H) of pGAD-RAD51D or its variant, expressed in a GAL4 DNA activating domain expressing plasmid with pGBD-XRCC2 expressed in the GAL4 DNA binding domain expressing plasmid and ( b ) pGAD-RAD51D or its variant, expressed in the pGAD (GAL4 DNA activating domain) plasmid with pGBD-RAD51C (GAL4 DNA binding domain) plasmid with pADH1-RAD51B via yeast 3-hybrid (Y3H). RAD51B serves to stabilize RAD51C protein levels. Empty vectors are used as a negative control. Quantification of yeast growth from three experiments is plotted as mean ± s.d relative to the WT control. Representative images for variants and controls are shown. Variants with <50% interaction are classified as deficient and plotted in red. ( c ) RAD51D variants with reduced interaction with XRCC2 or RAD51C are expressed. Western blot analysis of protein extract from yeast cells in (a) expressing WT RAD51D or the indicated RAD51D variants was assessed using an anti-RAD51D antibody, and equal protein loading was assessed by measuring the amount of Kar2 using an anti-Kar2 antibody. Note that a subset of variants has reduced protein expression relative to WT RAD51D. Experiment performed in triplicate. See for Western blots of all variants. ( d-f ) MAVE function scores vs individual variant assay results of (d) HR activity, (e) XRCC2 binding, and (f) RAD51C binding. Missense variant point shape denotes functional status (LOF/INT/NEU) based upon MAVE result.

Article Snippet: Human osteosarcoma U2OS SCR (sister chromatid recombination) #18 wild-type (gifted from Mauro Modesti; ) and RAD51D CRISPR knockout (KO; clone #4, purchased from DSMZ, no. ACC835) were cultured in Corning DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin antibiotic (50 U/mL).

Techniques: Variant Assay, Expressing, Plasmid Preparation, Binding Assay, Negative Control, Control, Western Blot, Activity Assay, Functional Assay

Journal: bioRxiv

Article Title: High-throughput mapping of 6,888 RAD51D variants identifies distinct biochemical functions needed for homologous recombination and olaparib response

doi: 10.64898/2026.01.11.698865

Figure Lengend Snippet: ( a ) Western blot analysis of protein extract from U2OS cells expressing wild-type RAD51D or the indicated RAD51D variants was assessed using an anti-RAD51D antibody, and equal protein loading was assessed using an anti-tubilin antibody. Note that a subset of variants has reduced protein expression relative to WT RAD51D. Experiment performed in triplicate. ( b ) HR analysis of RAD51D variants that are stably expressed in the RAD51D KO cell line used for the clonogenic survival assays shown in .

Article Snippet: Human osteosarcoma U2OS SCR (sister chromatid recombination) #18 wild-type (gifted from Mauro Modesti; ) and RAD51D CRISPR knockout (KO; clone #4, purchased from DSMZ, no. ACC835) were cultured in Corning DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin antibiotic (50 U/mL).

Techniques: Western Blot, Expressing, Stable Transfection

Journal: bioRxiv

Article Title: High-throughput mapping of 6,888 RAD51D variants identifies distinct biochemical functions needed for homologous recombination and olaparib response

doi: 10.64898/2026.01.11.698865

Figure Lengend Snippet: Western blot analysis of protein extract from yeast cells expressing wild-type RAD51D or the indicated RAD51D variants was assessed using an anti-RAD51D antibody, and equal protein loading was assessed using an anti-KAR2 antibody. Note that a subset of variants has reduced protein expression relative to WT RAD51D. Experiment performed in triplicate.

Article Snippet: Human osteosarcoma U2OS SCR (sister chromatid recombination) #18 wild-type (gifted from Mauro Modesti; ) and RAD51D CRISPR knockout (KO; clone #4, purchased from DSMZ, no. ACC835) were cultured in Corning DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin antibiotic (50 U/mL).

Techniques: Western Blot, Expressing

Journal: bioRxiv

Article Title: High-throughput mapping of 6,888 RAD51D variants identifies distinct biochemical functions needed for homologous recombination and olaparib response

doi: 10.64898/2026.01.11.698865

Figure Lengend Snippet: ( a ) Pull-down analysis of WT BC (RAD51B-His/RAD51C) and DX2 (RAD51D/XRCC2-FLAG) sub-complexes with indicated RAD51D variants using anti-FLAG resin. ( b ) ssDNA binding of BCDX2 paralog complexes reconstituted by mixing WT BC and DX2 sub-complexes with indicated RAD51D variants. ( c ) Pull-down analysis of WT CX3 (RAD51C-His/XRCC3-STREP) and DX2 (RAD51D/XRCC2-FLAG) sub-complexes with indicated RAD51D variants using anti-FLAG resin. ( d ) ATPase analysis of 0.5 μM WT BC and DX2 sub-complexes with indicated RAD51D variants in the presence of ssDNA after 60 min incubation. Pi indicates released inorganic phosphate after hydrolysis. ( e ) ssDNA binding of X3CDX2 paralog complexes reconstituted by mixing WT CX3 and DX2 sub-complexes with indicated RAD51D variants. For ( b, d-e ), results from three independent experiments were plotted as mean values ± s.d.

Article Snippet: Human osteosarcoma U2OS SCR (sister chromatid recombination) #18 wild-type (gifted from Mauro Modesti; ) and RAD51D CRISPR knockout (KO; clone #4, purchased from DSMZ, no. ACC835) were cultured in Corning DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin antibiotic (50 U/mL).

Techniques: Binding Assay, Incubation

Journal: bioRxiv

Article Title: High-throughput mapping of 6,888 RAD51D variants identifies distinct biochemical functions needed for homologous recombination and olaparib response

doi: 10.64898/2026.01.11.698865

Figure Lengend Snippet: ( a ) Summary of cellular findings. Schematic of RAD51D (1-328 aa) is shown with the Walker A and B motifs (green), and DNA binding loops (blue) indicated. The variants analyzed are shown above with the results from the cellular studies (MAVE variant-function map; ), RAD51D Y2H interaction with XRCC2 and its Y3H interaction with RAD51C , SCR recombination (HR), as well as olaparib and cisplatin clonogenic survival assays are summarized based on functional score. Loss-of-function is shown in red, an intermediate function is shown in yellow, and wild-type function is indicated in green. ( b ) Summary of biochemical findings. Variants included in biochemical analysis for BCDX2 or XRCC3 (X3CDX2) complex formation, DNA binding, and ATPase restrain from are shown. ( c ) Model for BCDX2 and X3CDX2 function in DSB repair. Upon DSB formation, the 3’ ssDNA end is resected and coated by RPA. The BCDX2 and X3CDX2 complexes facilitate the displacement of RPA and the loading of RAD51 onto ssDNA. This is achieved by the regulation of BC ATPase activity by DX2, which facilitates its binding to ssDNA. CX3 in complex with DX2 then targets RAD51 to ssDNA. The combined functions of the BCDX2 and X3CDX2 complexes are necessary to facilitate RAD51 filament assembly and subsequent RAD51-mediated homology search and strand exchange activities.

Article Snippet: Human osteosarcoma U2OS SCR (sister chromatid recombination) #18 wild-type (gifted from Mauro Modesti; ) and RAD51D CRISPR knockout (KO; clone #4, purchased from DSMZ, no. ACC835) were cultured in Corning DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin antibiotic (50 U/mL).

Techniques: Binding Assay, Variant Assay, Functional Assay, Activity Assay

Journal: Nature methods

Article Title: Deep learning enables fast and dense single-molecule localization with high accuracy

doi: 10.1038/s41592-021-01236-x

Figure Lengend Snippet: a) DECODE can reduce acquisition times by one order of magnitude. The same sample of microtubules, labeled with anti-α-tubulin primary and AF647 secondary antibodies, imaged with different UV activation intensities to result in different emitter densities per frame, between 0.08 and 0.86μm −2 and acquisition times between 93 and 1120 s, while keeping the total number of localizations the same. For high-density activation, we show a comparison with CSpline. b) Fourier Ring Correlation curves for DECODE and CSpline for different emitter densities. c) Resolution estimates obtained using the Fourier Ring Correlation and 0.143 criterion across densities for both methods. d) Fast live-cell SMLM on the Golgi apparatus labeled with a -mannosidase II-mEos3.2. See Supplementary Movie 1. e) Fast live-cell SMLM on the endoplasmic reticulum labeled with calnexin-mEos3.2. See Supplementary Movie 2 and . f) Fast live-cell SMLM on the nuclear pore complex protein Nup96-mMaple acquired in 3 seconds. g) DECODE enables ultra-high labeling densities. Microtubules labeled with a high concentration of anti-α and anti-β-tubulin primary and Alexa Fluor 647 secondary antibodies. g1, g2) Magnified regions as indicated in g. Data acquired with high-density labeling shows continuous structures. As a comparison, the same sample was acquired after pre-bleaching of the fluorophores to reach the single-molecule blinking regime. Here, single labels are resolved in the superresolution reconstruction and lead to a sparse decoration of the microtubules. g3, g4) Side view reconstructions of regions as indicated in g1, g2 resolving the hollow, cylinder-like structure of immunolabeled microtubules. h) Representative raw camera frames for the high-density and single-emitter acquisitions, respectively. Scale bars: 10μm (f inset, h), 1 μm (a, d, e, f, g, g1, g2), 100nm (g3, g4).

Article Snippet: The pulse length of the 405nm laser was adjusted manually to maintain a high emitter density and to allow imaging of all fluorophores in the field of view in about 1 min. For the acquisition of live-cell data of Nup96-mMaple , coverslips containing Nup96-mMaple cells (catalog no. 300461; CLS Cell Line Service, Eppelheim, Germany) were rinsed twice with warm PBS before they were mounted in 1mL growth medium containing 20 mM HEPES buffer and imaged directly.

Techniques: Labeling, Activation Assay, Comparison, Concentration Assay, Immunolabeling

Journal: Cancer Research

Article Title: Functional and Clinical Characterization of Variants of Uncertain Significance Identifies a Hotspot for Inactivating Missense Variants in RAD51C

doi: 10.1158/0008-5472.CAN-22-2319

Figure Lengend Snippet: Evaluation of 173 RAD51C missense variants by HDR assay. A, DR-GFP reporter assay showing the range of HDR activity for 173 missense variants in CL-V4B cells, measured as fold change in GFP-positive cells (normalized to 1–5 scale, WT = 5 and p.Leu138Phe = 1). Neutral (>2.5 scale, gray bars), deleterious (<1.25 scale, red bars), and intermediate effects (>1.25, <2.5 scale, light gray bars). Purple, L138F and C135Y deleterious controls. Amino acid changes in one letter code are labeled on the x -axis in clusters (black, blue, and orange) in order of presentation on the bar chart. Error bars, SEM of three independent experiments. B, Illustration of the location of missense variants within the RAD51C linear sequence identifying a deleterious variant hotspot. Key functional domains of RAD51C are indicated and neutral (blue), deleterious (orange), and intermediate (green) variants are shown at top. C, Circos plot of the RAD51C variants and functional assay (HDR, cisplatin and olaparib sensitivity, binding to XRCC3, RAD51D, and XRCC2) results. RAD51C variants are indicated by residue position in the outer ring. Track 1 shows the final score based on all functional assays. For HDR, variants were classified as neutral (light blue; ≥51.1% relative to WT), intermediate (green; 48.7%–26.8%), or deleterious (orange; ≤22.7%). Cisplatin sensitivity was classified as neutral (≥83.5% relative to WT), intermediate (43%–82%), or deleterious (≤13.1%). Olaparib sensitivity was classified as neutral (≥ 80.4% relative to WT), intermediate (78.7%–51.7%), or deleterious (≤42%). Dark blue, interactions with XRCC3, RAD51D, and XRCC2; yellow, partial interaction; red, no interaction.

Article Snippet: The landing pad genomic locus was integrated into U2OS RAD51C −/− cells [Leibniz Institute (U2OS#18-RAD51C-15; DSMZ ACC 834)] as previously described ( ).

Techniques: Reporter Assay, Activity Assay, Labeling, Sequencing, Variant Assay, Functional Assay, Binding Assay, Residue

Journal: Cancer Research

Article Title: Functional and Clinical Characterization of Variants of Uncertain Significance Identifies a Hotspot for Inactivating Missense Variants in RAD51C

doi: 10.1158/0008-5472.CAN-22-2319

Figure Lengend Snippet: Influence of RAD51C missense variants on response to cisplatin and olaparib treatment. Relative IC 50 values (normalized to 1–15 scale, WT = 15 and p.Cys135Tyr = 1) from an MTS assay of CL-V4B cells, transduced with selected RAD51C variant lentivirus, 5 days after treatment with varying doses of cisplatin ( A ) and olaparib ( B ). Variants defined as neutral (gray), intermediate (light gray), and deleterious (red) by the HDR assay are shown. Purple, L138F and C135Y deleterious controls. Error bars, SEM of three independent experiments.

Article Snippet: The landing pad genomic locus was integrated into U2OS RAD51C −/− cells [Leibniz Institute (U2OS#18-RAD51C-15; DSMZ ACC 834)] as previously described ( ).

Techniques: MTS Assay, Transduction, Variant Assay

Journal: Cancer Research

Article Title: Functional and Clinical Characterization of Variants of Uncertain Significance Identifies a Hotspot for Inactivating Missense Variants in RAD51C

doi: 10.1158/0008-5472.CAN-22-2319

Figure Lengend Snippet: Drug sensitivity of human U2OS landing pad cells. A, Olaparib sensitivity associated with RAD51C variants. Cell survival of U2OS cells expressing RAD51C variants was quantified after 4 days of treatment and calculated relative to mock-treated cells. Mean ± SEM was calculated from three independent experiments, each performed in triplicate. B, RAD51C protein levels in stable U2OS landing pad cell lines. Expression of RAD51C WT and variant proteins in U2OS cells was determined by Western blot analysis after 24 hours of doxycycline induction.

Article Snippet: The landing pad genomic locus was integrated into U2OS RAD51C −/− cells [Leibniz Institute (U2OS#18-RAD51C-15; DSMZ ACC 834)] as previously described ( ).

Techniques: Expressing, Variant Assay, Western Blot

Journal: Cancer Research

Article Title: Functional and Clinical Characterization of Variants of Uncertain Significance Identifies a Hotspot for Inactivating Missense Variants in RAD51C

doi: 10.1158/0008-5472.CAN-22-2319

Figure Lengend Snippet: Characterization of U20S landing pad cells expressing RAD51C variants. A, Colony formation assays of RAD51C variants. B, RAD51 foci quantification and representative microscopy images for each variant. RAD51 foci formation was quantified after exposure of landing pad cells to 5 Gy of γ-irradiation. Each dot represents a geminin (S/G 2 phase)-positive cell and the bars designate the mean number of foci in at least 1,000 cells obtained in at least three independent experiments. The mean change percentage was calculated for each variant relative to the WT.

Article Snippet: The landing pad genomic locus was integrated into U2OS RAD51C −/− cells [Leibniz Institute (U2OS#18-RAD51C-15; DSMZ ACC 834)] as previously described ( ).

Techniques: Expressing, Microscopy, Variant Assay, Irradiation

Journal: Cancer Research

Article Title: Functional and Clinical Characterization of Variants of Uncertain Significance Identifies a Hotspot for Inactivating Missense Variants in RAD51C

doi: 10.1158/0008-5472.CAN-22-2319

Figure Lengend Snippet: Coimmunoprecipitation analysis of RAD51C CX3 and BCDX2 complexes. Western blotting of coimmunoprecipitated Flag-tagged RAD51C variant proteins with XRCC3 and RAD51D, XRCC2 paralogs 48 hours after transfection of HEK293T cells with FLAG-tagged RAD51C variant expression plasmids. IP, immunoprecipitation; TCL, total cell lysate.

Article Snippet: The landing pad genomic locus was integrated into U2OS RAD51C −/− cells [Leibniz Institute (U2OS#18-RAD51C-15; DSMZ ACC 834)] as previously described ( ).

Techniques: Western Blot, Variant Assay, Transfection, Expressing, Immunoprecipitation

Journal: Cancer Research

Article Title: Functional and Clinical Characterization of Variants of Uncertain Significance Identifies a Hotspot for Inactivating Missense Variants in RAD51C

doi: 10.1158/0008-5472.CAN-22-2319

Figure Lengend Snippet: Evaluation of RAD51C deleterious variants in RAD51C 3D structure prediction model. A, Locations of 30 neutral and 30 deleterious variants in the 3D model of two ATP-bound RAD51C monomers (yellow and green). The locations of neutral and deleterious variants are shown with green and red dots, respectively. B, Magnified view of the 11 deleterious variants predicted to disrupt the binding of ATP.

Article Snippet: The landing pad genomic locus was integrated into U2OS RAD51C −/− cells [Leibniz Institute (U2OS#18-RAD51C-15; DSMZ ACC 834)] as previously described ( ).

Techniques: Binding Assay

Journal: Cancer Research

Article Title: Functional and Clinical Characterization of Variants of Uncertain Significance Identifies a Hotspot for Inactivating Missense Variants in RAD51C

doi: 10.1158/0008-5472.CAN-22-2319

Figure Lengend Snippet: Association of functionally characterized RAD51C variants with breast and ovarian cancer risk.

Article Snippet: The landing pad genomic locus was integrated into U2OS RAD51C −/− cells [Leibniz Institute (U2OS#18-RAD51C-15; DSMZ ACC 834)] as previously described ( ).

Techniques: Variant Assay, Control