Journal: Nucleic Acids Research

Article Title: Cancer-causing BRCA2 missense mutations disrupt an intracellular protein assembly mechanism to disable genome maintenance

doi: 10.1093/nar/gkab308

Figure Lengend Snippet: Clustering of pathogenic BRCA2 missense mutations within the DBD. ( A ) Clinical significance of BRCA2 mutations by mutation type. The graph shows 9085 variants classified by molecular consequence (mutation type) and clinical significance, out of a total of 12 684 BRCA2 variants listed in the ClinVar database (version of 2020.07.27). ( B ) Clinical significance of 143 BRCA2 missense mutations reviewed by the expert panel are shown. Pathogenic mutations clustered in the DBD are marked with a blue circle. The schematic shows human BRCA2 protein structure. Amino acid residue numbers are marked. HD, helical domain: OB, oligonucleotide/oligosaccharide binding; TD, tower domain; NLS, nuclear localisation signal. ( C ) Location of each pathogenic mutation within the BRCA2 DBD.

Article Snippet: Primary antibodies used were α-BRCA2 (Ab-1, clone 2B, Merck Millipore OP95, 1:500; clone 5.23, Merck Millipore, 05-666, 1:2000), α-RAD51 (clone 14B4, GeneTex, GTX70230, 1:1000), α-GFP (clone JL-8, Clontech, 632381,1:1000), α-mCherry (α-dsRed, Clontech, 632496, 1:1000), α-Myc (clone 9E10, Santa Cruz, sc-40, 1:500), and α-MEK2 (BD Transduction, 610235, 1:1000).

Techniques: Mutagenesis, Residue, Binding Assay

Journal: Nucleic Acids Research

Article Title: Cancer-causing BRCA2 missense mutations disrupt an intracellular protein assembly mechanism to disable genome maintenance

doi: 10.1093/nar/gkab308

Figure Lengend Snippet: Pathogenic BRCA2 mutations in the DBD affect DSS1 binding and nuclear localization. ( A ) List of pathogenic (red) or benign (blue) mutations in the DBD analysed in this study with a schematic of the expression construct, GFP-BRCA2 CT . ( B and C ) Interaction between the DBD mutants and DSS1. Wild-type or mutant forms of GFP-BRCA2 CT were transiently expressed with Myc-DSS1 in 293T cells. Interaction was detected by anti GFP-IP and western blotting with anti-Myc antibody. The graph ( C ) shows DSS1-binding activity of the mutants normalised to the WT value. Binding activity was assessed by quantifying the intensity of the Myc-DSS1 bands pulled-down by IP. Data are presented as mean ± s.e. from three repeats. Statistical significance was assessed by 1-way ANOVA test, followed by Dunnett's multiple comparison test (compared to WT). (D and E) Nucleocytoplasmic distribution of the DBD mutants. HeLa cells transiently expressing GFP-BRCA2 CT fragments were fixed with PFA before imaging. The graph ( E ) shows the GFP intensity ratio (nuclear/cytoplasmic, mean ± s.e.). More than 100 cells per sample were analysed. Statistical significance was assessed by the Kruskal-Wallis test, followed by Dunnett's multiple comparison test (compared to WT). A representative result from two independent experiments is shown. ( D ) shows representative microscopic images. Scale bar, 10μm.

Article Snippet: Primary antibodies used were α-BRCA2 (Ab-1, clone 2B, Merck Millipore OP95, 1:500; clone 5.23, Merck Millipore, 05-666, 1:2000), α-RAD51 (clone 14B4, GeneTex, GTX70230, 1:1000), α-GFP (clone JL-8, Clontech, 632381,1:1000), α-mCherry (α-dsRed, Clontech, 632496, 1:1000), α-Myc (clone 9E10, Santa Cruz, sc-40, 1:500), and α-MEK2 (BD Transduction, 610235, 1:1000).

Techniques: Binding Assay, Expressing, Construct, Mutagenesis, Western Blot, Activity Assay, Comparison, Imaging

Journal: Nucleic Acids Research

Article Title: Cancer-causing BRCA2 missense mutations disrupt an intracellular protein assembly mechanism to disable genome maintenance

doi: 10.1093/nar/gkab308

Figure Lengend Snippet: The BRCA2-DSS1 interaction antagonizes intracellular BRCA2 oligomerization. ( A ) Self-oligomerization of BRCA2 CT is suppressed by Myc-DSS1. Wildtype or D2723H mutant forms of BRCA2 CT , tagged with either GFP or mCherry, were transiently expressed in 293T cells with or without Myc-DSS1. Interaction was detected by anti GFP-IP and western blotting for mCherry-BRCA2 CT and endogenous BRCA2. ( B ) Self-oligomerization of BRCA2 CT fragments in the BRCA2 Knock-out (KO) HeLa cells. BRCA2 CT fragments tagged with either GFP or mCherry were co-expressed in BRCA2 KO HeLa cells. Their interaction was detected by anti GFP-IP and western blotting for mCherry-BRCA2 CT . GFP vector alone serves as a negative control. ( C ) GST-pull down assay. Purified GST-BRCA2 CT fragments bound to the Glutathione Sepharose 4B beads were incubated with purified GFP-BRCA2 CT fragments. The beads were washed, and bound proteins were eluted, before separation by SDS-PAGE. Interactions were detected by western blotting for GFP-BRCA2 CT . GST alone serves as a negative control. ( D ) Native gel electrophoresis of GFP-BRCA2 CT . Extracts from HeLa cells transiently expressing GFP-BRCA2 CT forms with or without Myc-DSS1 were analysed by native gel electrophoresis. The bands corresponding to GFP-BRCA2 CT oligomers, GFP-BRCA2 CT /Myc-DSS1 complex and GFP-BRCA2 CT are marked with arrows.

Article Snippet: Primary antibodies used were α-BRCA2 (Ab-1, clone 2B, Merck Millipore OP95, 1:500; clone 5.23, Merck Millipore, 05-666, 1:2000), α-RAD51 (clone 14B4, GeneTex, GTX70230, 1:1000), α-GFP (clone JL-8, Clontech, 632381,1:1000), α-mCherry (α-dsRed, Clontech, 632496, 1:1000), α-Myc (clone 9E10, Santa Cruz, sc-40, 1:500), and α-MEK2 (BD Transduction, 610235, 1:1000).

Techniques: Mutagenesis, Western Blot, Knock-Out, Plasmid Preparation, Negative Control, Pull Down Assay, Purification, Incubation, SDS Page, Nucleic Acid Electrophoresis, Expressing

Journal: Nucleic Acids Research

Article Title: Cancer-causing BRCA2 missense mutations disrupt an intracellular protein assembly mechanism to disable genome maintenance

doi: 10.1093/nar/gkab308

Figure Lengend Snippet: Pathogenic DBD mutants introduced into the endogenous BRCA2 gene cause cytosolic mis-localization and disable DNA repair by HDR. ( A ) Subcellular fractionation of BRCA2 mutant cell lines, HeLa-W2626C and HeLa-D2723H. Two independent clones for each mutation were used. Nuclear and cytoplasmic fractions of BRCA2 and RAD51 were analysed by western blotting. MEK2 is a cytoplasmic marker. ( B ) mClover Lamin A HDR assay. Cells transfected with Lamin A-targeting sgRNA and mClover Lamin A donor constructs as shown in the schematic were analysed for mClover Lamin A-positive cells. Mean of HDR positive cells (%) ± s.e. from two repeats is shown. More than 100 cells per sample were analysed in each repeat. Statistical significance was tested by one-way ANOVA test, followed by Bonferroni's multiple comparison test.

Article Snippet: Primary antibodies used were α-BRCA2 (Ab-1, clone 2B, Merck Millipore OP95, 1:500; clone 5.23, Merck Millipore, 05-666, 1:2000), α-RAD51 (clone 14B4, GeneTex, GTX70230, 1:1000), α-GFP (clone JL-8, Clontech, 632381,1:1000), α-mCherry (α-dsRed, Clontech, 632496, 1:1000), α-Myc (clone 9E10, Santa Cruz, sc-40, 1:500), and α-MEK2 (BD Transduction, 610235, 1:1000).

Techniques: Fractionation, Mutagenesis, Clone Assay, Western Blot, Marker, Transfection, Construct, Comparison

Journal: Nucleic Acids Research

Article Title: Cancer-causing BRCA2 missense mutations disrupt an intracellular protein assembly mechanism to disable genome maintenance

doi: 10.1093/nar/gkab308

Figure Lengend Snippet: Pathogenic DBD mutants exhibit defective nuclear import. ( A ) Nuclear import assay using BRCA2 CT tagged with photoactivable GFP (paGFP). HeLa cells expressing wildtype or mutant forms of paGFP-BRCA2 CT were monitored by live imaging. As shown in the schematic, photoactivation of a cytosolic area (red square) was followed by measurement of fluorescence intensity in the designated area in the cytosol (red square) and the nucleus (blue square). Plots show the relative fluorescence intensity (mean ± s.e.), normalized to the maximum intensity in each cell. 8–10 cells per sample were analysed. Black arrows mark the timing of photoactivation. ( B ) HeLa cells expressing paGFP-BRCA2 CT W2626C with or without Myc-DSS1 were monitored by live imaging after cytosolic photoactivation and analysed as shown in (A). ( C ) Efficiency of DSS1 depletion by siRNA transfection. Relative level of DSS1 mRNA measured by RT-qPCR assay, a result from technical triplicate is shown. Statistical significance was assessed by paired t -test. ( D and E ) Nucleocytoplasmic distribution of the GFP-BRCA2 CT fragment after DSS1 depletion. HeLa cells were transfected with control or DSS1 siRNA 24 h before transfecting with the GFP-BRCA2 CT construct. Nucleocytoplasmic distribution was analysed as described in Figure . The graph (E) shows the GFP intensity ratio (nuclear/cytoplasmic, mean ± s.e.). More than 100 cells per sample were analysed. Statistical significance was assessed by unpaired t-test. A representative result from three independent experiments is shown. Representative microscopic images are shown in (D). Scale bar, 10 μm. ( F ) Subcellular fractionation of HeLa cells after DSS1 depletion. HeLa cells were transfected with control or DSS1 siRNA 48 h before fractionation. Nuclear and cytoplasmic fractions of BRCA2 and RAD51 were analysed by western blotting. The nuclear/cytoplasmic ratio of BRCA2 expression is shown. MEK2 is a cytoplasmic marker. Total cell extracts are also shown. A representative result from two independent experiments is shown.

Article Snippet: Primary antibodies used were α-BRCA2 (Ab-1, clone 2B, Merck Millipore OP95, 1:500; clone 5.23, Merck Millipore, 05-666, 1:2000), α-RAD51 (clone 14B4, GeneTex, GTX70230, 1:1000), α-GFP (clone JL-8, Clontech, 632381,1:1000), α-mCherry (α-dsRed, Clontech, 632496, 1:1000), α-Myc (clone 9E10, Santa Cruz, sc-40, 1:500), and α-MEK2 (BD Transduction, 610235, 1:1000).

Techniques: Expressing, Mutagenesis, Imaging, Fluorescence, Transfection, Quantitative RT-PCR, Control, Construct, Fractionation, Western Blot, Marker

Journal: Nucleic Acids Research

Article Title: Cancer-causing BRCA2 missense mutations disrupt an intracellular protein assembly mechanism to disable genome maintenance

doi: 10.1093/nar/gkab308

Figure Lengend Snippet: DSS1 expression restores the nuclear localization and HDR function of hypomorphic BRCA2 pathogenic mutants. ( A and B ) Nucleocytoplasmic distribution of the DBD mutants with DSS1 overexpression. HeLa cells expressing GFP-BRCA2 CT forms without (vector only) or with Myc-DSS1 were fixed with PFA and analysed by microscopy. Representative images are shown. Scale bar, 10μm. The graph (B) shows GFP intensity ratio (nuclear/cytoplasmic, mean ± s.e.) measured from the images. Myc-DSS1 expression increases the nuclear/cytoplasmic ratio of GFP-BRCA2 CT fragments to varying degrees: WT by 1.7 fold; W2626C and E2663V mutants by 3.5- to 5-fold; I2627F, R2659T, and G2748D mutants by 1.8- to 2.4-fold; T2722R, D2723H, and D2723G mutants, little change. More than 100 cells per sample were analysed. Statistical significance was assessed by one-way ANOVA test, followed by Bonferroni's multiple comparison test. A representative result from three independent experiments is shown. ( C ) mClover Lamin A HDR assay. HeLa cells harbouring BRCA2 mutations (Figure ) were transfected with vector or Myc-DSS1 plasmids in addition to mClover Lamin A HDR assay plasmids. Mean of HDR positive cells (%) ± s.e. from two repeats is shown. More than 100 cells per sample were analysed in each repeat. Statistical significance was assessed by one-way ANOVA test, followed by Bonferroni's multiple comparison test.

Article Snippet: Primary antibodies used were α-BRCA2 (Ab-1, clone 2B, Merck Millipore OP95, 1:500; clone 5.23, Merck Millipore, 05-666, 1:2000), α-RAD51 (clone 14B4, GeneTex, GTX70230, 1:1000), α-GFP (clone JL-8, Clontech, 632381,1:1000), α-mCherry (α-dsRed, Clontech, 632496, 1:1000), α-Myc (clone 9E10, Santa Cruz, sc-40, 1:500), and α-MEK2 (BD Transduction, 610235, 1:1000).

Techniques: Expressing, Over Expression, Plasmid Preparation, Microscopy, Comparison, Transfection

Journal: Nucleic Acids Research

Article Title: Cancer-causing BRCA2 missense mutations disrupt an intracellular protein assembly mechanism to disable genome maintenance

doi: 10.1093/nar/gkab308

Figure Lengend Snippet: Cancer-causing BRCA2 missense mutations disrupt an intracellular protein assembly mechanism to disable genome maintenance. Our results suggest a hypothetical model wherein the engagement of DSS1 by the DBD of BRCA2 antagonizes BRCA2 self-oligomerization and promotes protein folding. Assembly of a correctly folded BRCA2/DSS1 complex enables nuclear transport. Cancer-causing mutations in the BRCA2 DBD impair DSS1 binding, perturbing this protein assembly mechanism, and provoking the formation of intracellular BRCA2 oligomers that are excluded from the cell nucleus. The failure to assemble correctly folded mutant BRCA2/DSS1 complexes, and cytosolic mis-localization of the mutant BRCA2, disables HDR in cells harbouring the DBD mutations. DSS1 can antagonize the self-oligomerization of wild-type BRCA2, and of cancer-causing DBD mutants such as W2626C that retain some degree of DSS1 binding.

Article Snippet: Primary antibodies used were α-BRCA2 (Ab-1, clone 2B, Merck Millipore OP95, 1:500; clone 5.23, Merck Millipore, 05-666, 1:2000), α-RAD51 (clone 14B4, GeneTex, GTX70230, 1:1000), α-GFP (clone JL-8, Clontech, 632381,1:1000), α-mCherry (α-dsRed, Clontech, 632496, 1:1000), α-Myc (clone 9E10, Santa Cruz, sc-40, 1:500), and α-MEK2 (BD Transduction, 610235, 1:1000).

Techniques: Binding Assay, Mutagenesis

Journal: Nucleic Acids Research

Article Title: Cancer-causing BRCA2 missense mutations disrupt an intracellular protein assembly mechanism to disable genome maintenance

doi: 10.1093/nar/gkab308

Figure Lengend Snippet: Pathogenic DBD mutants introduced into the endogenous BRCA2 gene cause cytosolic mis-localization and disable DNA repair by HDR. ( A ) Subcellular fractionation of BRCA2 mutant cell lines, HeLa-W2626C and HeLa-D2723H. Two independent clones for each mutation were used. Nuclear and cytoplasmic fractions of BRCA2 and RAD51 were analysed by western blotting. MEK2 is a cytoplasmic marker. ( B ) mClover Lamin A HDR assay. Cells transfected with Lamin A-targeting sgRNA and mClover Lamin A donor constructs as shown in the schematic were analysed for mClover Lamin A-positive cells. Mean of HDR positive cells (%) ± s.e. from two repeats is shown. More than 100 cells per sample were analysed in each repeat. Statistical significance was tested by one-way ANOVA test, followed by Bonferroni's multiple comparison test.

Article Snippet: Primary antibodies used were α-BRCA2 (Ab-1, clone 2B, Merck Millipore OP95, 1:500; clone 5.23, Merck Millipore, 05-666, 1:2000), α-RAD51 (clone 14B4, GeneTex, GTX70230, 1:1000), α-GFP (clone JL-8, Clontech, 632381,1:1000), α-mCherry (α-dsRed, Clontech, 632496, 1:1000), α-Myc (clone 9E10, Santa Cruz, sc-40, 1:500), and α-MEK2 (BD Transduction, 610235, 1:1000).

Techniques: Fractionation, Mutagenesis, Clone Assay, Western Blot, Marker, Transfection, Construct, Comparison

Journal: Nucleic Acids Research

Article Title: Cancer-causing BRCA2 missense mutations disrupt an intracellular protein assembly mechanism to disable genome maintenance

doi: 10.1093/nar/gkab308

Figure Lengend Snippet: Pathogenic DBD mutants exhibit defective nuclear import. ( A ) Nuclear import assay using BRCA2 CT tagged with photoactivable GFP (paGFP). HeLa cells expressing wildtype or mutant forms of paGFP-BRCA2 CT were monitored by live imaging. As shown in the schematic, photoactivation of a cytosolic area (red square) was followed by measurement of fluorescence intensity in the designated area in the cytosol (red square) and the nucleus (blue square). Plots show the relative fluorescence intensity (mean ± s.e.), normalized to the maximum intensity in each cell. 8–10 cells per sample were analysed. Black arrows mark the timing of photoactivation. ( B ) HeLa cells expressing paGFP-BRCA2 CT W2626C with or without Myc-DSS1 were monitored by live imaging after cytosolic photoactivation and analysed as shown in (A). ( C ) Efficiency of DSS1 depletion by siRNA transfection. Relative level of DSS1 mRNA measured by RT-qPCR assay, a result from technical triplicate is shown. Statistical significance was assessed by paired t -test. ( D and E ) Nucleocytoplasmic distribution of the GFP-BRCA2 CT fragment after DSS1 depletion. HeLa cells were transfected with control or DSS1 siRNA 24 h before transfecting with the GFP-BRCA2 CT construct. Nucleocytoplasmic distribution was analysed as described in Figure . The graph (E) shows the GFP intensity ratio (nuclear/cytoplasmic, mean ± s.e.). More than 100 cells per sample were analysed. Statistical significance was assessed by unpaired t-test. A representative result from three independent experiments is shown. Representative microscopic images are shown in (D). Scale bar, 10 μm. ( F ) Subcellular fractionation of HeLa cells after DSS1 depletion. HeLa cells were transfected with control or DSS1 siRNA 48 h before fractionation. Nuclear and cytoplasmic fractions of BRCA2 and RAD51 were analysed by western blotting. The nuclear/cytoplasmic ratio of BRCA2 expression is shown. MEK2 is a cytoplasmic marker. Total cell extracts are also shown. A representative result from two independent experiments is shown.

Article Snippet: Primary antibodies used were α-BRCA2 (Ab-1, clone 2B, Merck Millipore OP95, 1:500; clone 5.23, Merck Millipore, 05-666, 1:2000), α-RAD51 (clone 14B4, GeneTex, GTX70230, 1:1000), α-GFP (clone JL-8, Clontech, 632381,1:1000), α-mCherry (α-dsRed, Clontech, 632496, 1:1000), α-Myc (clone 9E10, Santa Cruz, sc-40, 1:500), and α-MEK2 (BD Transduction, 610235, 1:1000).

Techniques: Expressing, Mutagenesis, Imaging, Fluorescence, Transfection, Quantitative RT-PCR, Control, Construct, Fractionation, Western Blot, Marker

Journal: bioRxiv

Article Title: RAD54L regulates replication fork progression and nascent strand degradation in BRCA1/2-deficient cells

doi: 10.1101/2023.07.26.550704

Figure Lengend Snippet: ( A ) Schematic illustration of the iPOND assay. ( B ) Western blots of the input and iPOND samples probed for proteins, as indicated. NUCKS1 is a RAD54L interacting protein . Click rxn, Click reaction; the no Click condition represents cells pulsed with EdU and processed without biotin-azide in the Click reaction step. ( C ) Western blots of HeLa, RAD54L KO, and RAD54L KO cells ectopically expressing RAD54L-HA, as described earlier . ( D ) Schematic of the protocol for the DNA fiber assay to determine replication tract lengths. ( E ) Dot plot with medians of CldU+IdU tract lengths in parental cells and RAD54L (R54L) KOs in Hela, Hs578T, and MCF7 cells. RAD51AP1 KOs (AP1) and R54L KOs ectopically expressing R54L (R54L KO+R54L; ) are show for comparison purposes (n=3; at least 100 fiber tracts/experiment analyzed). Fiber tract lengths were analyzed by Kruskal-Wallis test followed by Dunn’s multiple comparisons test (ns, not significant; *, p<0.05; ****, p<0.0001).

Article Snippet: The following primary antibodies were used: α-RAD51AP1 ([ ]; 1:6,000), α-RAD54L (F-11; sc-374598; Santa Cruz Biotechnology; 1:500); α-RAD51 (Ab-1; EMD Millipore; 1:3,000), α-PARP1 (ab6079; Abcam; 1:1,000), α-α-Tubulin (DM1A; Santa Cruz Biotechnology; 1:1,000), α-Histone H3 (ab1791;Abcam; 1:10,000), α-HLTF (E9H5I; 45965; Cell Signaling; 1:6,000) α-FBH1 (sc-81563; 1:100), α-BRCA2 (OP95; EMD Millipore; 1:500), α-BRCA1 (MS110; ab16780; Abcam; 1:50), α-PCNA (sc-25280; Santa Cruz Biotechnology; 1:1,000), α-NUCKS1 ([ ]; 1:10,000).

Techniques: Western Blot, Expressing, Comparison

Journal: bioRxiv

Article Title: RAD54L regulates replication fork progression and nascent strand degradation in BRCA1/2-deficient cells

doi: 10.1101/2023.07.26.550704

Figure Lengend Snippet: ( A ) Schematic of the DNA fiber assay protocol used to assess replication restart. ( B ) Dot plot with medians of IdU tract lengths in untreated (NT) or HU treated Hs578T and RAD51AP1 (AP1) or RAD54L (R54L) KO cells. ( C ) Dot plot with medians of CldU tract lengths in untreated (NT) or HU treated Hs578T and RAD51AP1 (AP1) or RAD54L (R54L) KO cells. (n=3; at least 100 fiber tracts/experiment analyzed). ( D ) Schematic of the DNA fiber assay protocol used to evaluate replication progression during mild replication stress in HeLa cells and derivatives. ( E ) Dot plot with medians of IdU tract lengths in HeLa control cells, R54L KO+R54L, and two independently isolated R54L KO cell lines treated with or without 25 μM HU during the IdU pulse (n=3; at least 100 fiber tracts/experiment analyzed). ( F ) Dot plot with medians of IdU tract lengths in HeLa cells transfected with control (Ctrl), R54L, FBH1, or HLTF siRNA and treated with or without 25 μM HU during the IdU pulse (n=3; at least 100 fiber tracts/experiment analyzed). ( G ) Representative Western blots to show extent of protein knockdown for the experiments shown in F. Loading control: α-Tubulin. ( H ) Schematic of the DNA fiber assay protocol used to evaluate replication progression during mild replication stress in hTERT RPE-1 cells. ( I ) Dot plot with medians of IdU tract lengths in hTERT RPE-1 cells transfected with control (Ctrl) or R54L siRNA and treated with or without 25 μM HU during the IdU pulse (n=3; at least 100 fiber tracts/experiment analyzed). ( J ) Representative Western blots to show extent of protein knockdown for the experiments shown in I. Loading control: α-Tubulin. Fiber tract lengths were analyzed by Kruskal-Wallis test followed by Dunn’s multiple comparisons test (ns, not significant; ****, p<0.0001, or as indicated).

Article Snippet: The following primary antibodies were used: α-RAD51AP1 ([ ]; 1:6,000), α-RAD54L (F-11; sc-374598; Santa Cruz Biotechnology; 1:500); α-RAD51 (Ab-1; EMD Millipore; 1:3,000), α-PARP1 (ab6079; Abcam; 1:1,000), α-α-Tubulin (DM1A; Santa Cruz Biotechnology; 1:1,000), α-Histone H3 (ab1791;Abcam; 1:10,000), α-HLTF (E9H5I; 45965; Cell Signaling; 1:6,000) α-FBH1 (sc-81563; 1:100), α-BRCA2 (OP95; EMD Millipore; 1:500), α-BRCA1 (MS110; ab16780; Abcam; 1:50), α-PCNA (sc-25280; Santa Cruz Biotechnology; 1:1,000), α-NUCKS1 ([ ]; 1:10,000).

Techniques: Control, Isolation, Transfection, Western Blot, Knockdown

Journal: bioRxiv

Article Title: RAD54L regulates replication fork progression and nascent strand degradation in BRCA1/2-deficient cells

doi: 10.1101/2023.07.26.550704

Figure Lengend Snippet: ( A ) Schematic of the DNA fiber assay protocol to assess fork degradation after HU. ( B ) Representative Western blots to show extent of BRCA2 knockdown in HeLa cells and derivatives for the experiments shown in C. Loading control: PARP1. ( C ) Dot plot with medians of IdU/CldU tract length ratios in HeLa, R54L KO, and R54L KO+R54L cells transfected with Ctrl or BRCA2 siRNA and treated with or without HU (n=3; at least 100 fiber tracts/experiment analyzed). ( D ) Representative Western blots to show extent of RAD54L knockdown in DLD1 cells (BRCA2 KO) and DLD1 cells expressing BRCA2 for the experiments shown in E and F. and derivatives for the experiments shown in C. Loading control: α-Tubulin. ( E ) Dot plot with medians of IdU/CldU tract length ratios in DLD1 and DLD1+BRCA2 cells transfected with Ctrl or RAD54L siRNA and treated with or without HU (n=3; at least 100 fiber tracts/experiment analyzed). ( F ) Dot plot with medians after neutral Comet assay in DLD1 and DLD1+BRCA2 cells transfected with Ctrl or RAD54L siRNA and treated with or without HU (n=3; at least 100 Comet tails/experiment analyzed). IdU/CldU ratios and length of Comet signals were analyzed by Kruskal-Wallis test followed by Dunn’s multiple comparisons test (ns, not significant; *, p<0.05; **, p<0.01, or as indicated).

Article Snippet: The following primary antibodies were used: α-RAD51AP1 ([ ]; 1:6,000), α-RAD54L (F-11; sc-374598; Santa Cruz Biotechnology; 1:500); α-RAD51 (Ab-1; EMD Millipore; 1:3,000), α-PARP1 (ab6079; Abcam; 1:1,000), α-α-Tubulin (DM1A; Santa Cruz Biotechnology; 1:1,000), α-Histone H3 (ab1791;Abcam; 1:10,000), α-HLTF (E9H5I; 45965; Cell Signaling; 1:6,000) α-FBH1 (sc-81563; 1:100), α-BRCA2 (OP95; EMD Millipore; 1:500), α-BRCA1 (MS110; ab16780; Abcam; 1:50), α-PCNA (sc-25280; Santa Cruz Biotechnology; 1:1,000), α-NUCKS1 ([ ]; 1:10,000).

Techniques: Western Blot, Knockdown, Control, Transfection, Expressing, Neutral Comet Assay

Journal: bioRxiv

Article Title: RAD54L regulates replication fork progression and nascent strand degradation in BRCA1/2-deficient cells

doi: 10.1101/2023.07.26.550704

Figure Lengend Snippet: ( A ) ClustalW sequence alignment of the N-terminal domains of RAD54L from human (Homo sapiens), chimpanzee (Pan troglodytes), mouse (Mus musculus), and bovine (Bos taurus). Basic residues are shown in blue, and acidic residues are shown in red. The pink box indicates basic residues mutated to alanines, and the green box indicates CDK2 phosphorylation consensus sequence and mutated serine to glutamate, as previously described . ( B ) Representative Western blots to show expression of wild type RAD54L (lane 1) and RAD54L mutants (lanes 3-6) in HeLa RAD54L KO cells. RAD54L-S49E expressing lines are clonal isolates resistant to puromycin; RAD54L-4A/S49E expressing cells are puromycin-resistant cell populations. Loading control: α-Tubulin. ( C ) Schematic of the DNA fiber assay protocol used in D. ( D ) Dot plot with medians of IdU tract lengths in HeLa RAD54L KO cells expressing wild type RAD54L (WT) or RAD54L mutants (S49E and 4A/S49E) treated with or without 25 μM HU during the IdU pulse (n=3; at least 100 fiber tracts/experiment analyzed). Fiber tract lengths were analyzed by Kruskal-Wallis test followed by Dunn’s multiple comparisons test (ns, not significant; (*, p<0.05; **, p<0.01; ****, p<0.0001). ( E ) Model to show that RAD54L contributes to fork reversal in human cells.

Article Snippet: The following primary antibodies were used: α-RAD51AP1 ([ ]; 1:6,000), α-RAD54L (F-11; sc-374598; Santa Cruz Biotechnology; 1:500); α-RAD51 (Ab-1; EMD Millipore; 1:3,000), α-PARP1 (ab6079; Abcam; 1:1,000), α-α-Tubulin (DM1A; Santa Cruz Biotechnology; 1:1,000), α-Histone H3 (ab1791;Abcam; 1:10,000), α-HLTF (E9H5I; 45965; Cell Signaling; 1:6,000) α-FBH1 (sc-81563; 1:100), α-BRCA2 (OP95; EMD Millipore; 1:500), α-BRCA1 (MS110; ab16780; Abcam; 1:50), α-PCNA (sc-25280; Santa Cruz Biotechnology; 1:1,000), α-NUCKS1 ([ ]; 1:10,000).

Techniques: Sequencing, Phospho-proteomics, Western Blot, Expressing, Control

Journal: Nucleic acids research

Article Title: Disparate requirements for RAD54L in replication fork reversal.

doi: 10.1093/nar/gkae828

Figure Lengend Snippet: Figure 2. Loss of RAD54L does not hinder replication restart, leads to faster replication during mild replication stress, and partially restores fork restraint in cells with FBH1 knockdown. ( A ) Schematic of the DNA fiber assay protocol used to assess replication restart. ( B ) Dot plot with medians of IdU fork speeds in untreated (NT) or HU-treated parental Hs578T, RAD51AP1 KO, and RAD54L KO cells ( n = 3; 65–113 fiber tracts / experiment analyzed). ( C ) Fractions of stalled and restarted forks after 4 mM HU in the Hs578T cells and deriv ativ es sho wn in (B). ( D ) Dot plot with medians of CldU fork speeds in untreated (NT) or HU-treated Hs578T cells and deriv ativ es ( n = 3; 65–80 fiber tracts / experiment analyzed). ( E ) Schematic of the DNA fiber assay protocol used to e v aluate the progression of replication forks during mild replication stress in (F). ( F ) Dot plot with medians of IdU fork speeds in parental HeLa cells, RAD54L KO cells expressing RAD54L KO, and two independently isolated RAD54L KO cell lines treated with or without 25 μM HU during the IdU pulse ( n = 3; 82–220 fiber tracts / experiment analyzed). ( G ) Schematic of the DNA fiber assay protocol used to evaluate the progression of replication forks during mild replication stress in (H), (I) and (J). ( H ) Dot plot with medians of IdU fork speeds in hTERT RPE-1 cells transfected with Ctrl or RAD54L siRNA and treated with or without 25 μM HU during the IdU pulse ( n = 3; 76–293 fiber tracts / experiment analyzed). ( I ) Dot plot with medians of IdU fork speeds in HeLa cells transfected with control (Ctrl), RAD54L, FBH1 or HLTF siRNA and treated with or without 25 μM HU during the IdU pulse ( n = 3; 92–139 fiber tracts / experiment analyzed). ( J ) Dot plot with medians of IdU fork speeds in HeLa and RAD54L KO cells transfected with Ctrl, FBH1, HLTF or SMARCAL1 siRNA ( n = 3; 122–420 fiber tracts / experiment analyzed). All data were analyzed by Kruskal–Wallis test followed by Dunn’s multiple comparisons test (ns, not significant; ∗P < 0.05; ∗∗∗∗P < 0.0 0 01; or as indicated).

Article Snippet: The following primary antibodies were used: α-RAD51AP1 (( 41 ); 1:6000), α-RAD54L (F11; sc-374598; Santa Cruz Biotechnology; 1:500); α-RAD51 (Ab-1; EMD Millipore; 1:3000), α-PARP1 (ab6079; Abcam; 1:1000), α- α-Tubulin (DM1A; Santa Cruz Biotechnology; 1:1000), α-Histone H3 (ab1791; Abcam; 1:10 000), α-HLTF (E9H5I; 45965; Cell Signaling; 1:6000) α-FBH1 (sc-81563; 1:500), α-BRCA2 (OP95; EMD Millipore; 1:500), α-BRCA1 (MS110; ab16780; Abcam; 1:50), α-PCNA (sc-25280; Santa Cruz Biotechnology; 1:1000), α-NUCKS1 (( 42 ); 1:10000), αMSH2 (ab52266; Abcam; 1:5000), α-53BP1 (A300-272A; Bethyl Laboratories; 1:10 000).

Techniques: Knockdown, Expressing, Isolation, Transfection, Control

Journal: Nucleic acids research

Article Title: Disparate requirements for RAD54L in replication fork reversal.

doi: 10.1093/nar/gkae828

Figure Lengend Snippet: Figure 6. RAD54L’s branch migration activity is specifically required for the FBH1 pathway of RAD51-mediated fork reversal. ( A ) Schematic of the DNA fiber assay protocol used in (C). ( B ) Representative Western blots to show extent of FBH1 and HLTF knockdown in RAD54L KO cells (lanes 4–6) and RAD54L KO cells expressing wild type RAD54L (lanes 1–3) or mutant RAD54L-4A / S49E (lanes 7–12). RAD54L-4A / S49E-1 dignifies a clonal isolate; RAD54L-4A / S49E #2 dignifies a purom y cin-resistant cell population. Loading control: α-tubulin. ( C ) Dot plot with medians of IdU fork speeds in HU-treated RAD54L KO cells and RAD54L KO cells expressing wild type RAD54L or RAD54L-4A / S49E and transfected with Ctrl, FBH1 or HLTF siRNA ( n = 3; 85–224 fiber tracts / experiment analyzed; n = 1 for RAD54L-4A / S49E #2). ( D ) Schematic of the DNA fiber assay protocol used in (F). ( E ) R epresentativ e w estern blots to sho w e xtent of BR CA2 knockdo wn in RAD54L KO cells (lanes 3–4) and RAD54L KO cells expressing wild type RAD54L (lanes 1–2) or mutant RAD54L-4A / S49E (lanes 5–8) and RAD54L-K189R (lanes 9–10). RAD54L-4A / S49E-1 dignifies a clonal isolate; RAD54L-4A / S49E #2 and RAD54L-K189R #1 dignifies purom y cin-resistant cell populations. Loading control: MSH2. ( F ) Dot plot with medians of IdU / CldU tract length ratios in HU-treated RAD54L KO cells and RAD54L KO cells expressing wild type RAD54L or mutant RAD54L-4A / S49E and RAD54L-K189R transfected with Ctrl or BRCA2 siRNA ( n = 3; 46–104 fiber tracts / experiment analyzed; n = 1 for RAD54L-K189R #1). Data were analyzed by Kruskal–Wallis test followed by Dunn’s multiple comparisons test (ns, not significant; ∗∗∗∗P < 0.0 0 01). ( G ) Model to explain the role of RAD54L in the FBH1 pathway of RAD51-mediated fork reversal. RAD54L may be recruited through its interaction with RAD51 on extended parental ssDNA. FBH1 is recruited to stalled replication forks by PCNA (( 78 ); PCNA is not shown here). Unwinding of lagging strand DNA through FBH1 ( 72 ) may initiate nascent strand annealing. RAD54L may then oligomerize on a 4-way junction and drive reversal through BM. The dependency of FBH1 on RAD54L may be indicative of coordinated recruitment and / or post-translational modification orchestrating sequential coaction. The number of subunits in the RAD54L BM oligomer remain to be determined. Schematic created with Biorender.com.

Article Snippet: The following primary antibodies were used: α-RAD51AP1 (( 41 ); 1:6000), α-RAD54L (F11; sc-374598; Santa Cruz Biotechnology; 1:500); α-RAD51 (Ab-1; EMD Millipore; 1:3000), α-PARP1 (ab6079; Abcam; 1:1000), α- α-Tubulin (DM1A; Santa Cruz Biotechnology; 1:1000), α-Histone H3 (ab1791; Abcam; 1:10 000), α-HLTF (E9H5I; 45965; Cell Signaling; 1:6000) α-FBH1 (sc-81563; 1:500), α-BRCA2 (OP95; EMD Millipore; 1:500), α-BRCA1 (MS110; ab16780; Abcam; 1:50), α-PCNA (sc-25280; Santa Cruz Biotechnology; 1:1000), α-NUCKS1 (( 42 ); 1:10000), αMSH2 (ab52266; Abcam; 1:5000), α-53BP1 (A300-272A; Bethyl Laboratories; 1:10 000).

Techniques: Migration, Activity Assay, Western Blot, Knockdown, Expressing, Mutagenesis, Control, Transfection, Modification

Journal: bioRxiv

Article Title: RAD54L regulates replication fork progression and nascent strand degradation in BRCA1/2-deficient cells

doi: 10.1101/2023.07.26.550704

Figure Lengend Snippet: ( A ) Schematic of the DNA fiber assay protocol used to assess replication restart. ( B ) Dot plot with medians of IdU tract lengths in untreated (NT) or HU treated Hs578T and RAD51AP1 (AP1) or RAD54L (R54L) KO cells. ( C ) Dot plot with medians of CldU tract lengths in untreated (NT) or HU treated Hs578T and RAD51AP1 (AP1) or RAD54L (R54L) KO cells. (n=3; at least 100 fiber tracts/experiment analyzed). ( D ) Schematic of the DNA fiber assay protocol used to evaluate replication progression during mild replication stress in HeLa cells and derivatives. ( E ) Dot plot with medians of IdU tract lengths in HeLa control cells, R54L KO+R54L, and two independently isolated R54L KO cell lines treated with or without 25 μM HU during the IdU pulse (n=3; at least 100 fiber tracts/experiment analyzed). ( F ) Dot plot with medians of IdU tract lengths in HeLa cells transfected with control (Ctrl), R54L, FBH1, or HLTF siRNA and treated with or without 25 μM HU during the IdU pulse (n=3; at least 100 fiber tracts/experiment analyzed). ( G ) Representative Western blots to show extent of protein knockdown for the experiments shown in F. Loading control: α-Tubulin. ( H ) Schematic of the DNA fiber assay protocol used to evaluate replication progression during mild replication stress in hTERT RPE-1 cells. ( I ) Dot plot with medians of IdU tract lengths in hTERT RPE-1 cells transfected with control (Ctrl) or R54L siRNA and treated with or without 25 μM HU during the IdU pulse (n=3; at least 100 fiber tracts/experiment analyzed). ( J ) Representative Western blots to show extent of protein knockdown for the experiments shown in I. Loading control: α-Tubulin. Fiber tract lengths were analyzed by Kruskal-Wallis test followed by Dunn’s multiple comparisons test (ns, not significant; ****, p<0.0001, or as indicated).

Article Snippet: The following primary antibodies were used: α-RAD51AP1 ([ ]; 1:6,000), α-RAD54L (F-11; sc-374598; Santa Cruz Biotechnology; 1:500); α-RAD51 (Ab-1; EMD Millipore; 1:3,000), α-PARP1 (ab6079; Abcam; 1:1,000), α-α-Tubulin (DM1A; Santa Cruz Biotechnology; 1:1,000), α-Histone H3 (ab1791;Abcam; 1:10,000), α-HLTF (E9H5I; 45965; Cell Signaling; 1:6,000) α-FBH1 (sc-81563; 1:100), α-BRCA2 (OP95; EMD Millipore; 1:500), α-BRCA1 (MS110; ab16780; Abcam; 1:50), α-PCNA (sc-25280; Santa Cruz Biotechnology; 1:1,000), α-NUCKS1 ([ ]; 1:10,000).

Techniques: Control, Isolation, Transfection, Western Blot, Knockdown

Journal: bioRxiv

Article Title: RAD54L regulates replication fork progression and nascent strand degradation in BRCA1/2-deficient cells

doi: 10.1101/2023.07.26.550704

Figure Lengend Snippet: ( A ) Schematic of the DNA fiber assay protocol used to assess replication restart. ( B ) Dot plot with medians of IdU tract lengths in untreated (NT) or HU treated Hs578T and RAD51AP1 (AP1) or RAD54L (R54L) KO cells. ( C ) Dot plot with medians of CldU tract lengths in untreated (NT) or HU treated Hs578T and RAD51AP1 (AP1) or RAD54L (R54L) KO cells. (n=3; at least 100 fiber tracts/experiment analyzed). ( D ) Schematic of the DNA fiber assay protocol used to evaluate replication progression during mild replication stress in HeLa cells and derivatives. ( E ) Dot plot with medians of IdU tract lengths in HeLa control cells, R54L KO+R54L, and two independently isolated R54L KO cell lines treated with or without 25 μM HU during the IdU pulse (n=3; at least 100 fiber tracts/experiment analyzed). ( F ) Dot plot with medians of IdU tract lengths in HeLa cells transfected with control (Ctrl), R54L, FBH1, or HLTF siRNA and treated with or without 25 μM HU during the IdU pulse (n=3; at least 100 fiber tracts/experiment analyzed). ( G ) Representative Western blots to show extent of protein knockdown for the experiments shown in F. Loading control: α-Tubulin. ( H ) Schematic of the DNA fiber assay protocol used to evaluate replication progression during mild replication stress in hTERT RPE-1 cells. ( I ) Dot plot with medians of IdU tract lengths in hTERT RPE-1 cells transfected with control (Ctrl) or R54L siRNA and treated with or without 25 μM HU during the IdU pulse (n=3; at least 100 fiber tracts/experiment analyzed). ( J ) Representative Western blots to show extent of protein knockdown for the experiments shown in I. Loading control: α-Tubulin. Fiber tract lengths were analyzed by Kruskal-Wallis test followed by Dunn’s multiple comparisons test (ns, not significant; ****, p<0.0001, or as indicated).

Article Snippet: The following primary antibodies were used: α-RAD51AP1 ([ ]; 1:6,000), α-RAD54L (F-11; sc-374598; Santa Cruz Biotechnology; 1:500); α-RAD51 (Ab-1; EMD Millipore; 1:3,000), α-PARP1 (ab6079; Abcam; 1:1,000), α-α-Tubulin (DM1A; Santa Cruz Biotechnology; 1:1,000), α-Histone H3 (ab1791;Abcam; 1:10,000), α-HLTF (E9H5I; 45965; Cell Signaling; 1:6,000) α-FBH1 (sc-81563; 1:100), α-BRCA2 (OP95; EMD Millipore; 1:500), α-BRCA1 (MS110; ab16780; Abcam; 1:50), α-PCNA (sc-25280; Santa Cruz Biotechnology; 1:1,000), α-NUCKS1 ([ ]; 1:10,000).

Techniques: Control, Isolation, Transfection, Western Blot, Knockdown

Journal: bioRxiv

Article Title: RAD54L regulates replication fork progression and nascent strand degradation in BRCA1/2-deficient cells

doi: 10.1101/2023.07.26.550704

Figure Lengend Snippet: ( A ) Schematic of the DNA fiber assay protocol to assess fork degradation after HU. ( B ) Representative Western blots to show extent of BRCA2 knockdown in HeLa cells and derivatives for the experiments shown in C. Loading control: PARP1. ( C ) Dot plot with medians of IdU/CldU tract length ratios in HeLa, R54L KO, and R54L KO+R54L cells transfected with Ctrl or BRCA2 siRNA and treated with or without HU (n=3; at least 100 fiber tracts/experiment analyzed). ( D ) Representative Western blots to show extent of RAD54L knockdown in DLD1 cells (BRCA2 KO) and DLD1 cells expressing BRCA2 for the experiments shown in E and F. and derivatives for the experiments shown in C. Loading control: α-Tubulin. ( E ) Dot plot with medians of IdU/CldU tract length ratios in DLD1 and DLD1+BRCA2 cells transfected with Ctrl or RAD54L siRNA and treated with or without HU (n=3; at least 100 fiber tracts/experiment analyzed). ( F ) Dot plot with medians after neutral Comet assay in DLD1 and DLD1+BRCA2 cells transfected with Ctrl or RAD54L siRNA and treated with or without HU (n=3; at least 100 Comet tails/experiment analyzed). IdU/CldU ratios and length of Comet signals were analyzed by Kruskal-Wallis test followed by Dunn’s multiple comparisons test (ns, not significant; *, p<0.05; **, p<0.01, or as indicated).

Article Snippet: The following primary antibodies were used: α-RAD51AP1 ([ ]; 1:6,000), α-RAD54L (F-11; sc-374598; Santa Cruz Biotechnology; 1:500); α-RAD51 (Ab-1; EMD Millipore; 1:3,000), α-PARP1 (ab6079; Abcam; 1:1,000), α-α-Tubulin (DM1A; Santa Cruz Biotechnology; 1:1,000), α-Histone H3 (ab1791;Abcam; 1:10,000), α-HLTF (E9H5I; 45965; Cell Signaling; 1:6,000) α-FBH1 (sc-81563; 1:100), α-BRCA2 (OP95; EMD Millipore; 1:500), α-BRCA1 (MS110; ab16780; Abcam; 1:50), α-PCNA (sc-25280; Santa Cruz Biotechnology; 1:1,000), α-NUCKS1 ([ ]; 1:10,000).

Techniques: Western Blot, Knockdown, Control, Transfection, Expressing, Neutral Comet Assay

Journal: bioRxiv

Article Title: RAD54L regulates replication fork progression and nascent strand degradation in BRCA1/2-deficient cells

doi: 10.1101/2023.07.26.550704

Figure Lengend Snippet: ( A ) ClustalW sequence alignment of the N-terminal domains of RAD54L from human (Homo sapiens), chimpanzee (Pan troglodytes), mouse (Mus musculus), and bovine (Bos taurus). Basic residues are shown in blue, and acidic residues are shown in red. The pink box indicates basic residues mutated to alanines, and the green box indicates CDK2 phosphorylation consensus sequence and mutated serine to glutamate, as previously described . ( B ) Representative Western blots to show expression of wild type RAD54L (lane 1) and RAD54L mutants (lanes 3-6) in HeLa RAD54L KO cells. RAD54L-S49E expressing lines are clonal isolates resistant to puromycin; RAD54L-4A/S49E expressing cells are puromycin-resistant cell populations. Loading control: α-Tubulin. ( C ) Schematic of the DNA fiber assay protocol used in D. ( D ) Dot plot with medians of IdU tract lengths in HeLa RAD54L KO cells expressing wild type RAD54L (WT) or RAD54L mutants (S49E and 4A/S49E) treated with or without 25 μM HU during the IdU pulse (n=3; at least 100 fiber tracts/experiment analyzed). Fiber tract lengths were analyzed by Kruskal-Wallis test followed by Dunn’s multiple comparisons test (ns, not significant; (*, p<0.05; **, p<0.01; ****, p<0.0001). ( E ) Model to show that RAD54L contributes to fork reversal in human cells.

Article Snippet: The following primary antibodies were used: α-RAD51AP1 ([ ]; 1:6,000), α-RAD54L (F-11; sc-374598; Santa Cruz Biotechnology; 1:500); α-RAD51 (Ab-1; EMD Millipore; 1:3,000), α-PARP1 (ab6079; Abcam; 1:1,000), α-α-Tubulin (DM1A; Santa Cruz Biotechnology; 1:1,000), α-Histone H3 (ab1791;Abcam; 1:10,000), α-HLTF (E9H5I; 45965; Cell Signaling; 1:6,000) α-FBH1 (sc-81563; 1:100), α-BRCA2 (OP95; EMD Millipore; 1:500), α-BRCA1 (MS110; ab16780; Abcam; 1:50), α-PCNA (sc-25280; Santa Cruz Biotechnology; 1:1,000), α-NUCKS1 ([ ]; 1:10,000).

Techniques: Sequencing, Phospho-proteomics, Western Blot, Expressing, Control

Journal: Nucleic Acids Research

Article Title: Disparate requirements for RAD54L in replication fork reversal

doi: 10.1093/nar/gkae828

Figure Lengend Snippet: Loss of RAD54L prevents fork degradation in both BRCA2- and 53BP1-deficient cells. ( A ) Schematic of the DNA fiber assay protocol. This experimental scheme was followed to assess fork degradation in (C), (D) and (F) and double-strand break formation in (E). ( B ) Representative Western blots to show extent of BRCA2 knockdown in HeLa cells and derivatives for the experiments shown in (C). Loading control: PARP1. ( C ) Dot plot with medians of IdU/CldU tract length ratios in HeLa, RAD54L KO, and RAD54L KO + RAD54L cells transfected with Ctrl ( – ) or BRCA2 siRNA and treated with or without HU ( n = 3; 42–117 fiber tracts/experiment analyzed). ( D ) Dot plot with medians of IdU/CldU tract length ratios in DLD1 and DLD1 + BRCA2 cells transfected with Ctrl ( – ) or RAD54L siRNA and treated with or without HU ( n = 3; 38–89 fiber tracts/experiment analyzed). ( E ) Dot plot with medians after neutral Comet assay in DLD1 and DLD1 + BRCA2 cells transfected with Ctrl ( – ) or RAD54L siRNA and treated with or without HU ( n = 3; 74–206 Comet tails/experiment analyzed). ( F ) Dot plot with medians of IdU/CldU tract length ratios in HeLa and RAD54L KO cells transfected with Ctrl ( – ) or 53BP1 siRNA and treated with or without HU ( n = 3; 60–82 fiber tracts/experiment analyzed). IdU/CldU ratios and lengths of Comet tails were analyzed by Kruskal–Wallis test followed by Dunn's multiple comparisons test (ns, not significant; ∗ P < 0.05; ∗∗ P < 0.01; or as indicated). NT: not treated.

Article Snippet: The following primary antibodies were used: α-RAD51AP1 (( ); 1:6000), α-RAD54L (F-11; sc-374598; Santa Cruz Biotechnology; 1:500); α-RAD51 (Ab-1; EMD Millipore; 1:3000), α-PARP1 (ab6079; Abcam; 1:1000), α-α-Tubulin (DM1A; Santa Cruz Biotechnology; 1:1000), α-Histone H3 (ab1791; Abcam; 1:10 000), α-HLTF (E9H5I; 45965; Cell Signaling; 1:6000) α-FBH1 (sc-81563; 1:500), α-BRCA2 (OP95; EMD Millipore; 1:500), α-BRCA1 (MS110; ab16780; Abcam; 1:50), α-PCNA (sc-25280; Santa Cruz Biotechnology; 1:1000), α-NUCKS1 (( ); 1:10000), α-MSH2 (ab52266; Abcam; 1:5000), α-53BP1 (A300-272A; Bethyl Laboratories; 1:10 000).

Techniques: Western Blot, Knockdown, Control, Transfection, Neutral Comet Assay

Journal: Cell

Article Title: A Class of Environmental and Endogenous Toxins Induces BRCA2 Haploinsufficiency and Genome Instability

doi: 10.1016/j.cell.2017.05.010

Figure Lengend Snippet:

Article Snippet: Membranes were incubated with primary antibodies (α-BRCA2 (Merck Millipore, ab-1, 1:500), α-BRCA1 (Santa Cruz, C-20, 1:200), α-PALB2 (Bethyl A301-246A, 1:1000), α-RAD51 (GeneTex, 14B4, 1:500), α-p-ATM (S1981) (Abcam, ab81292, 1:1000), α-ATM (Sigma, A1106, 1:500), α-p-ATR (T1989) (GeneTex, GTX128145, 1:500), α-ATR (Bethyl, A300-137A, 1:10000), α-LC3B (Cell Signaling, D11, 1:1000), α-FLAG (Sigma, M2, 1:500), α-polyubiquitin (Enzo, FK2, 1:1000), α-Hsp90 (Cell Signaling, C45G5, 1:1000), α-β-actin (Sigma, A5441, 1:10000), α-XRCC3 (Oncogene, PC691, 1:5000), α-FANCI (Abcam, ab15344, 1:2000), α-Ku80 (Santa Cruz, sc-1485, 1:500), α-Ku70 (Abcam, ab3114, 1:500), α-XRCC4 (Abcam, ab145, 1:2000), α-mCherry (Novus Biologicals, NBP1-96752, 1:2000), α-Histone H3 (Cell Signaling, #9715, 1:1000, α-c-Myc (Santa Cruz, sc-40, 1:500), α-PLK1 (ThermoFisher, 331700, 1:1000), α-Aurora B (Abcam, ab2254, 1:1000), α-TXNL1 (Abcam, ab188328, 1:2000), α-JAK1 (Santa Cruz, sc-376996, 1:500)) overnight at 4°C followed by washing in 0.1%Tween/TBS.

Techniques: Immunofluorescence, Western Blot, Recombinant, Giemsa Stain, Blocking Assay, Nucleic Acid Hybridization, Transfection, Protease Inhibitor, Imaging, Fractionation, Cell Culture, Plasmid Preparation, Sequencing, Software