Journal: Molecular Oncology

Article Title: FGFR1 clustering with engineered tetravalent antibody improves the efficiency and modifies the mechanism of receptor internalization

doi: 10.1002/1878-0261.12740

Figure Lengend Snippet: Characterization of anti‐FGFR1 engineered antibodies of different valency. (A) The schematic representation of structures of tetravalent (T‐Fc) and bivalent (B‐Fc) anti‐FGFR1 engineered antibodies. Fc region of IgG (CH2 and CH3 domains) is labeled in gray, and scFv proteins (VH and VL fusions) are marked in blue. Antibody regions recognizing epitopes within FGFR1 are marked in orange. (B) Expression and purification of T‐Fc and B‐Fc. Levels and purity of T‐Fc at different stages of protein expression and purification process were monitored with SDS/PAGE and western blotting with antibodies recognizing Fc fragment. (C) B‐Fc and T‐Fc are specific toward FGFR1. The extracellular regions of FGFR1, VEGFR2, and PDGFR were immobilized on SPR sensors and tested for the interaction with B‐Fc, T‐Fc, and commercial anti‐VEGFR2 and anti‐PDGFR antibodies with SPR. (D) Selectivity tests of B‐Fc and T‐Fc against FGFRs. The extracellular regions of FGFR2, FGFR3, and FGFR4 were immobilized on SPR sensors and tested for the interaction with B‐Fc, T‐Fc, and FGF1 as a control using SPR. (E) Engineered antibodies bind the D1 domain of the receptor. The full‐length extracellular domain of FGFR1 (D1‐D2‐D3), FGFR1 variant lacking the D1 domain (FGFR1 D2‐D3), and recombinant D1 domain (FGFR1 GST‐D1) were immobilized on SPR sensors and tested for interaction with B‐Fc and T‐Fc using SPR.

Article Snippet: Human osteosarcoma cell line (U2OS) was obtained from American Type Culture Collection (ATCC), and U2OS cells stably expressing FGFR1 (U2OS‐R1) were a kind gift from Dr. E.M. Haugsten from the Norwegian Radium Hospital.

Techniques: Labeling, Expressing, Purification, SDS Page, Western Blot, Control, Variant Assay, Recombinant

Journal: Molecular Oncology

Article Title: FGFR1 clustering with engineered tetravalent antibody improves the efficiency and modifies the mechanism of receptor internalization

doi: 10.1002/1878-0261.12740

Figure Lengend Snippet: B‐Fc and T‐Fc bind FGFR1 with high affinity. (A, B) SPR‐determined kinetic parameters of the interaction between B‐Fc and T‐Fc, and FGFR1, respectively. The extracellular region of FGFR1 was immobilized on SPR sensors and incubated with various concentrations of B‐Fc and T‐Fc. K D , k on , and k off values are presented. (C) BLI comparison of B‐Fc and T‐Fc interaction with FGFR1. The extracellular region of FGFR1 was immobilized on BLI sensors and incubated either with B‐Fc or T‐Fc. The association and dissociation profiles were measured. (D) Upper panel, B‐Fc and T‐Fc interaction with FGFR1 on model cells. U2OS‐R1 cells stably producing FGFR1 were incubated with B‐Fc or T‐Fc on ice to prevent internalization of receptor–antibody complexes, or briefly at room temperature. Nuclei were labeled with NucBlue Live; cells were washed, and fixed; and bound antibodies were visualized with Zenon AF‐488 using fluorescence microscopy. Scale bars represent 20 µm. Lower panel, quantification of T‐Fc and B‐Fc cell binding at room temperature performed using zen 2.3 software based on three independent experiments. The signal of B‐Fc was set to 100%, and average intensity of T‐Fc in relation to B‐Fc ±SD was shown. (E) BN‐PAGE analysis of FGFR1 complexes with engineered antibodies. FGFR1‐Fc (0.1 μ m ) was incubated with B‐Fc (0.2 μ m , 1 μ m ) and T‐Fc (0.2 μ m , 1 μ m ), and proteins were separated on 4–10% BN‐PAGE gels and detected by western blotting.

Article Snippet: Human osteosarcoma cell line (U2OS) was obtained from American Type Culture Collection (ATCC), and U2OS cells stably expressing FGFR1 (U2OS‐R1) were a kind gift from Dr. E.M. Haugsten from the Norwegian Radium Hospital.

Techniques: Incubation, Comparison, Stable Transfection, Labeling, Fluorescence, Microscopy, Binding Assay, Software, Western Blot

Journal: Molecular Oncology

Article Title: FGFR1 clustering with engineered tetravalent antibody improves the efficiency and modifies the mechanism of receptor internalization

doi: 10.1002/1878-0261.12740

Figure Lengend Snippet: The impact of engineered antibodies on the interaction of FGFR1 with partner proteins. (A) B‐Fc and T‐Fc are unable to activate FGFR1. Serum‐starved NIH3T3 cells were incubated with FGF1 (positive control) or with different concentrations of B‐Fc and T‐Fc. Cells were lysed and activation of FGFR1, and receptor‐downstream signaling was assessed with western blotting (WB). The level of tubulin served as a loading control. Bottom panels: quantification of signaling experiments performed with image lab software . Average values ±SD from at least three independent experiments are shown. The statistical significance was calculated using the t ‐test; * P < 0.05, ** P < 0.005, n.s.—not significant. (B) B‐Fc and T‐Fc have no impact on FGFR1 activation by FGF1. Serum‐starved NIH3T3 cells were incubated with FGF1 alone or in combination with B‐Fc and T‐Fc. Cells were lysed and activation of FGFR1, and receptor‐downstream signaling was assessed with western blotting. The level of tubulin served as a loading control. Bottom panels: quantification of signaling experiments performed with image lab software . Average values ±SD from at least three independent experiments are shown. The statistical significance was calculated using the t ‐test; * P < 0.05, ** P < 0.005, n.s.—not significant. (C) The effect of engineered antibodies on FGF1/FGFR1 interaction. The extracellular domain of FGFR1 was immobilized on BLI sensors and either left untreated or incubated with the saturating concentrations of B‐Fc (left graph) or T‐Fc (right graph). Subsequently, sensors were incubated with FGF1 to assess the impact of antibodies on FGF1/FGFR1 interaction. (D) B‐Fc and T‐Fc have no impact on galectin‐1/FGFR1 interaction. The extracellular domain of FGFR1 was immobilized on BLI sensors and either left untreated or incubated with the saturating concentrations of B‐Fc (left graph) or T‐Fc (right graph). Subsequently, sensors were incubated with galectin‐1 to assess the impact of antibodies on galectin‐1/FGFR1 interaction. (E) T‐Fc partially inhibits binding of galectin‐3 to FGFR1. The extracellular domain of FGFR1 was immobilized on BLI sensors and either left untreated or incubated with the saturating concentrations of B‐Fc (left graph) or T‐Fc (right graph). Subsequently, sensors were incubated with galectin‐3 to assess the impact of antibodies on galectin‐3/FGFR1 interaction.

Article Snippet: Human osteosarcoma cell line (U2OS) was obtained from American Type Culture Collection (ATCC), and U2OS cells stably expressing FGFR1 (U2OS‐R1) were a kind gift from Dr. E.M. Haugsten from the Norwegian Radium Hospital.

Techniques: Incubation, Positive Control, Activation Assay, Western Blot, Control, Software, Binding Assay

Journal: Molecular Oncology

Article Title: FGFR1 clustering with engineered tetravalent antibody improves the efficiency and modifies the mechanism of receptor internalization

doi: 10.1002/1878-0261.12740

Figure Lengend Snippet: The differential influence of T‐Fc and B‐Fc on FGFR1 internalization. (A) Engineered antibodies are internalized via FGFR1‐dependent endocytosis. U2OS‐R1 cells stably expressing FGFR1 and U2OS cells (control cell line with negligible level of FGFR1) were incubated with 100 n m of B‐Fc and T‐Fc for 30 min. Nuclei were stained with NucBlue Live, cells were fixed, and internalized antibodies were visualized with Zenon AF‐488 using wide‐field fluorescence microscope. Scale bar represents 20 µm. (B) Internalized T‐Fc and B‐Fc are present in endosomes. U2OSR1 cells were incubated with 100 n m B‐Fc and T‐Fc for 15 min, cells were fixed, internalized antibodies were labeled with Zenon AF‐488, and early endosome marker protein EEA1 was detected with immunolabeling. Cells were analyzed with confocal microscopy. Scale bar represents 50 µm. (C) Confocal microscopy analysis of the kinetics of B‐Fc and T‐Fc internalization. U2OSR1 cells were incubated with 100 n m B‐Fc and T‐Fc for different time periods, and internalized antibodies were labeled with Zenon AF‐488 and analyzed with confocal microscopy. Scale bar represents 50 µm. (D) Quantification of B‐Fc and T‐Fc internalization (expressed as integral fluorescence intensity in arbitrary units, AU) using the harmony software. Mean values of three independent experiments of integral intensity of Zenon AF‐488 vesicles ±SEM are shown. T ‐test was used to assess the statistical significance of measured differences in internalization; * P < 0.05, ** P < 0.005. (E) Engineered antibodies induce FGFR1 degradation. U2OS‐R1 cells were serum starved, treated with cycloheximide to inhibit synthesis of new FGFR1 pool, and incubated with equimolar concentrations of B‐Fc and T‐Fc for various time points, or left untreated (control). Cells were lysed, and the level of FGFR1 was determined with western blotting (WB). Tubulin detection was used as an indication of equal loading. Representative results from four independent experiments are shown. (F) Quantitative analyses of FGFR1 degradation (Fig. ) upon stimulation with engineered antibodies. FGFR1 band intensities were quantified and corrected for loading differences (intensity of tubulin bands). Average values from four independent experiments ±SD are shown. T ‐test was used to assess the statistical significance of measured differences in FGFR1 levels,* P < 0.05, ** P < 0.005, n.s.—not significant.

Article Snippet: Human osteosarcoma cell line (U2OS) was obtained from American Type Culture Collection (ATCC), and U2OS cells stably expressing FGFR1 (U2OS‐R1) were a kind gift from Dr. E.M. Haugsten from the Norwegian Radium Hospital.

Techniques: Stable Transfection, Expressing, Control, Incubation, Staining, Fluorescence, Microscopy, Labeling, Marker, Immunolabeling, Confocal Microscopy, Software, Western Blot

Journal: Molecular Oncology

Article Title: FGFR1 clustering with engineered tetravalent antibody improves the efficiency and modifies the mechanism of receptor internalization

doi: 10.1002/1878-0261.12740

Figure Lengend Snippet: Hypothetical model of the effect of differential FGFR1 clustering on the receptor endocytosis. FGFR1 dimerization, either with FGF1 or bivalent antibodies, like B‐Fc, induces CME of the receptor. CME initiation is independent of FGFR1 activation. Clustering of FGFR1 into large structures on the plasma membrane with tetravalent T‐Fc largely improves the cellular uptake of FGFR1–antibody complexes. Furthermore, FGFR1 clustering changes the mechanism of the receptor endocytosis by engaging dynamin‐dependent CIE pathways. Similarly to CME, CIE of FGFR1 does not require receptor activation.

Article Snippet: Human osteosarcoma cell line (U2OS) was obtained from American Type Culture Collection (ATCC), and U2OS cells stably expressing FGFR1 (U2OS‐R1) were a kind gift from Dr. E.M. Haugsten from the Norwegian Radium Hospital.

Techniques: Activation Assay, Clinical Proteomics, Membrane

Journal: Science Advances

Article Title: Arginine methylation expands the regulatory mechanisms and extends the genomic landscape under E2F control

doi: 10.1126/sciadv.aaw4640

Figure Lengend Snippet: ( A ) Schematic representation of E2F1, highlighting the region of the protein targeted by PRMT1 and PRMT5. The arginine methylation-defective E2F1 derivatives [R109K and R111/113 K (KK)] used to generate U2OS stable cell lines for RNA-seq analysis are also indicated (i). An immunoblot displaying E2F1 protein expression in U2OS stable cells after 24 hours of doxycycline (1 μg/ml) treatment is also included (ii). See also fig. S1 (A to D). ( B ) Venn diagrams showing the crossover of genes up- or down-regulated over twofold (adjusted P value threshold < 0.01) in each cell line condition with respect to the pTRE empty vector cell line, filtered for genes containing an E2F1 motif in their proximal promoter region (−900 to +100). These data were generated from three independent biological samples.

Article Snippet: U2OS cells (HTB-96, American Type Culture Collection; RRID: CVCL_0042) were plated on coverslips and transfected for 48 hours with the indicated plasmids, or U2OS-Tet-ON cells were induced to express WT E2F1, E2F1-KK, or E2F1-R109K for 24 hours as appropriate.

Techniques: Methylation, Stable Transfection, RNA Sequencing, Western Blot, Expressing, Plasmid Preparation, Generated

Journal: Science Advances

Article Title: Arginine methylation expands the regulatory mechanisms and extends the genomic landscape under E2F control

doi: 10.1126/sciadv.aaw4640

Figure Lengend Snippet: ( A ) U2OS cells were lysed in RIP lysis buffer, containing ribonuclease A (RNase A; 20 μg/ml) where indicated. Cell extracts were immunoprecipitated with E2F1 antibody, and coimmunoprecipitated RNA was reverse-transcribed before quantitative polymerase chain reaction (qPCR) analysis with primers against U6 (i) and U4 (ii) snRNAs as indicated. Input protein levels were determined by immunoblot (iii). n = 2. ( B ) U2OS cells were treated with 5 μM PRMT5 inhibitor (P5 inh), as indicated, before performing an anti-E2F1 RIP. Coimmunoprecipitated U6 (i) and U4 (ii) snRNAs were identified with specific primers by quantitative reverse transcription PCR (qRT-PCR). Input protein levels were determined by immunoblot (iii). n = 3. ( C ) An anti-E2F1 RIP was performed on U2OS cells, and coimmunoprecipitated U1 snRNA was detected by qRT-PCR. n = 2. ( D ) An anti-E2F1 RIP was performed on extracts prepared from U2OS or U2OS E2F1 CRISPR cell lines as indicated. Immunoprecipitated RNA was analyzed by qRT-PCR using primers specific to U1 (i), U6 (ii), or U5 (iii) snRNAs. Input protein levels are also displayed (iv). n = 2. ( E ) HCT116 cells were treated with 5 μM PRMT5 inhibitor, where indicated, before performing an anti-E2F1 RIP. Coimmunoprecipitated U1 (i) and U6 (ii) snRNA were detected by qRT-PCR. Input protein levels are also displayed (iii). n = 2. ( F ) As described above, although the experiment was performed in MCF7 cells. ( G ) U2OS cells were transfected with 1 μg of plasmid encoding WT E2F1, DNA binding domain mutant constructs (L132E and R166H) or empty vector (−) as indicated. Forty-eight hours later, cell extracts were used for ChIP analysis with the anti–hemagglutinin (HA) antibody. Immunoprecipitated chromatin was analyzed by qPCR using primers targeting the indicated promoters, where albumin served as the non-E2F target gene control (i to iii). Input protein levels are shown in (H). n = 2. See also fig. S4B. ( H ) U2OS cells were transfected as above. Forty-eight hours later, cell extracts were used for RIP analysis with anti-HA antibody. Immunoprecipitated RNA was analyzed by qRT-PCR using primers specific to U6 snRNA (i) or actin RNA (ii). Input protein levels were determined by immunoblot (iii). n = 3.

Article Snippet: U2OS cells (HTB-96, American Type Culture Collection; RRID: CVCL_0042) were plated on coverslips and transfected for 48 hours with the indicated plasmids, or U2OS-Tet-ON cells were induced to express WT E2F1, E2F1-KK, or E2F1-R109K for 24 hours as appropriate.

Techniques: Lysis, Immunoprecipitation, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Quantitative RT-PCR, CRISPR, Transfection, Plasmid Preparation, Binding Assay, Mutagenesis, Construct, Control

Journal: Science Advances

Article Title: Arginine methylation expands the regulatory mechanisms and extends the genomic landscape under E2F control

doi: 10.1126/sciadv.aaw4640

Figure Lengend Snippet: ( A ) Schematic representation of exon structure for the SENP7 gene. Each alternatively spliced transcript expressed from this gene is displayed, with primer binding sites used to detect specific transcript variants in subsequent experiments indicated with black arrows. Note that forward primers were designed to span exon junctions. Mining of the RIP-seq dataset for exon spanning peaks identified reads around exons 4 and 7 (indicated by the red numbering), which occurs in SENP7 transcript V5 (highlighted in red text). ( B ) Anti-E2F1 RIP with U2OS cells treated with siRNA against E2F1, TSN, or nontargeting (NT) control, as indicated, for 72 hours. Cells were then immunoprecipitated with E2F1 antibody, and coimmunoprecipitated RNA was reverse-transcribed before qPCR analysis with primers against specific SENP7 transcript variants as indicated. n = 3. ( C ) HCT116 cells were treated with 5 μM PRMT5 inhibitor, where indicated, before performing an anti-E2F1 RIP. Coimmunoprecipitated SENP7 V5 transcripts were analyzed by qRT-PCR. Input protein levels are the same as those displayed in . n = 2. DMSO, dimethyl sulfoxide. ( D ) U2OS cells were treated for 72 hours with 5 μM PRMT5 inhibitor. RNA was then isolated from cells and analyzed by qRT-PCR using primers targeting specific SENP7 transcript variants or total SENP7 RNA. Average (mean) fold change of each RNA species as compared to untreated U2OS cells was calculated and displayed with SE. Statistical analysis for each condition compared to untreated U2OS cells is also displayed over each bar (i). An immunoblot to demonstrate input protein levels is also included (ii). n = 3. ( E ) As described above, although the experiment was performed in HCT116 cells. n = 4. ns, not significant. ( F ) Examination of the promoter region of the SENP7 gene (–2 to +1 kb) identified an E2F1 DNA binding motif within +450 bp of the transcription start site, lying within the first intron (E2F1 motif marked in red) (i). An E2F1 ChIP was performed in the HCT116 E2F1 CRISPR and MCF7 TSN CRISPR cell lines. Immunoprecipitated chromatin was analyzed using primers spanning the identified E2F DNA binding motif in SENP7 or against the known E2F motif in the promoter sequence of CDC6 (ii). An immunoblot is included to demonstrate input protein levels (iii). n = 3

Article Snippet: U2OS cells (HTB-96, American Type Culture Collection; RRID: CVCL_0042) were plated on coverslips and transfected for 48 hours with the indicated plasmids, or U2OS-Tet-ON cells were induced to express WT E2F1, E2F1-KK, or E2F1-R109K for 24 hours as appropriate.

Techniques: Binding Assay, Control, Immunoprecipitation, Reverse Transcription, Quantitative RT-PCR, Isolation, Western Blot, CRISPR, Sequencing

Journal: Science Advances

Article Title: Arginine methylation expands the regulatory mechanisms and extends the genomic landscape under E2F control

doi: 10.1126/sciadv.aaw4640

Figure Lengend Snippet: ( A ) Schematic representation of exon structure for the MECOM gene. Each alternatively spliced transcript expressed from this gene is displayed, with primer binding sites used to detect specific transcript variants in subsequent experiments indicated with black arrows. Note that forward primers were designed to span exon junctions. Mining of the RIP-seq dataset for exon spanning peaks identified reads spanning exons 1 and 3 (indicated by the red numbering), which occurs in MECOM transcript V7 (highlighted in red text). ( B ) U2OS (i), MCF7 (ii), or HCT116 cells (iii) were treated with 5 μM PRMT5 inhibitor as indicated. An anti-E2F1 RIP was then performed, and coimmunoprecipitated MECOM transcript variant V7 was analyzed by qRT-PCR using specific primers. Input protein levels for the U2OS experiment are also included (iv), while the input protein levels for HCT116 and MCF7 cells are the same as those displayed in . n = 2. ( C ) Examination of the promoter region of the MECOM gene identified an E2F1 DNA binding motif lying within the first intron of V7 or the second intron of V4 (E2F1 motif marked in red) (i). An E2F1 ChIP was performed in HCT116 or HCT116 E2F1 CRISPR cell lines. Immunoprecipitated chromatin was analyzed using primers spanning the identified E2F DNA binding motif in MECOM or against the known E2F motif in the promoter sequence of CDC6 (ii). Input protein levels are the same as those displayed in . n = 3. ( D ) U2OS cells (i) or HCT116 cells (iii) were treated with 5 μM PRMT5 inhibitor, where indicated. RNA was then isolated from cells and analyzed by qRT-PCR using primers targeting specific MECOM transcript variants or total MECOM RNA. Average (mean) fold change of each RNA species as compared to untreated U2OS/HCT116 cells was calculated and displayed with SE. Statistical analysis for each condition compared to untreated cells is also displayed over each bar. Input protein levels for U2OS cells are also displayed (ii), while the input protein levels for HCT116 cells are the same as those displayed in . n = 4.

Article Snippet: U2OS cells (HTB-96, American Type Culture Collection; RRID: CVCL_0042) were plated on coverslips and transfected for 48 hours with the indicated plasmids, or U2OS-Tet-ON cells were induced to express WT E2F1, E2F1-KK, or E2F1-R109K for 24 hours as appropriate.

Techniques: Binding Assay, Variant Assay, Quantitative RT-PCR, CRISPR, Immunoprecipitation, Sequencing, Isolation

Journal: Science Advances

Article Title: Arginine methylation expands the regulatory mechanisms and extends the genomic landscape under E2F control

doi: 10.1126/sciadv.aaw4640

Figure Lengend Snippet: ( A ) U2OS cells were treated with 5 μM PRMT5 inhibitor for 72 hours, where indicated, before ChIP analysis with anti-SUMO2/3–specific or control antibodies. Immunoprecipitated chromatin was analyzed using primers specific for the E2F site in the p73 promoter (i). An RT-PCR was also performed to monitor the levels of p73 transcripts in the cell (ii). An immunoblot for H4R3me2s is included to demonstrate the activity of the PRMT5 inhibitor (iii). n = 3. See also fig. S4 (F and G). ( B ) As described above, although cells were treated with the PRMT5 inhibitor for 24 or 48 hours as indicated. ChIP analysis was performed with anti-HP1α–specific or control antibodies (i). An immunoblot for H4R3me2s is included to demonstrate the activity of the PRMT5 inhibitor (ii). n = 2. ( C ) U2OS cells were transfected with SENP7 siRNA or nontargeting siRNA (siNT) for 96 hours as indicated. Cells were then prepared for ChIP analysis as described above (i). An immunoblot is included to demonstrate input protein levels (ii). n = 4. ( D ) ChIP analysis as described above, although U2OS cells were transfected with siRNA targeting E2F1, SENP7, or a combination of the two (siE2F1 + siSENP7). n = 3. ( E ) U2OS cells were transfected with siRNA targeting SENP7 or nontargeting siRNA for 96 hours, as indicated. Cells were subsequently transfected for 48 hours with an empty vector or a plasmid expressing Flag-tagged SENP7 V5. Cells were then prepared for ChIP analysis as described above (i). An immunoblot is included to demonstrate input protein levels (ii). n = 3. ( F ) U2OS cells were transfected with p73–luciferase (luc) or CDC6-luciferase reporter plasmids for 48 hours, along with empty vector (vec) or Flag-tagged SENP7 V5. Reporter activity was measured, and immunoblots were performed to monitor input protein levels. n = 2. ( G ) Model diagram where PRMT5-mediated methylation of chromatin-associated E2F1 mediates its interaction with p100/TSN, which permits the E2F1 complex to associate with a subset of RNAs, some being derived from E2F-target genes. By regulating the activity of the splicing machinery, it is proposed that the E2F1-p100/TSN complex can influence the alternative splicing of these RNAs. In the absence of E2F1 methylation (either under conditions of PRMT5 inhibitor treatment or in cells expressing E2F1-meR point mutants), a p100/TSN-dependent interaction with the splicing machinery is lost, and changes to alternative splicing of a subset of RNAs result.

Article Snippet: U2OS cells (HTB-96, American Type Culture Collection; RRID: CVCL_0042) were plated on coverslips and transfected for 48 hours with the indicated plasmids, or U2OS-Tet-ON cells were induced to express WT E2F1, E2F1-KK, or E2F1-R109K for 24 hours as appropriate.

Techniques: Control, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Activity Assay, Transfection, Plasmid Preparation, Expressing, Luciferase, Methylation, Derivative Assay, Alternative Splicing

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 ) 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