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: 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, Data-independent acquisition, Plasmid Preparation, Sequencing, Software