Journal: bioRxiv

Article Title: Break-induced replication drives large-scale genomic amplifications in cancer cells

doi: 10.1101/2024.08.27.609980

Figure Lengend Snippet: a , b , Histogram ( b ) showing the impact of IR (9 Gy) on the induction of genomic amplifications in control U2OS cells or in U2OS cells depleted of CtIP or EXO1 by siRNA (immunoblot shown in ( a )). c , Quantitation of the number of 53BP1 foci per cell in control (pX330) cells or U2OS deleted of SHILD1, SHILD2, or SHILD3. Representative images are shown in the . The results represent the average of a minimum of 100 nuclei in each of three independent experiments for each condition ± S.D. ns: non-significant. d , Histogram showing the percentage of cells with genomic amplifications following the exposure of control (pX330) U2OS cells or individual independent clones of U2OS cells lacking SHILD1, SHILD2, or SHILD3 to 9 Gy. Genomic amplifications were monitored 72 h post IR. Representative FACS profiles shown in . e , Induction of genomic amplifications by IR requires RAD51. Representative FACS (PI) profiles of control U2OS cells or U2OS cells depleted of RAD51 by two different siRNAs and exposed to 9 Gy. Genomic amplifications were monitored 72 h post IR. f , Dose-dependent suppression of strand invasion following incubation of U2OS cells with the RAD51 recombinase inhibitor B02. Representative FACS profiles shown in . g-i , Induction of genomic amplifications by IR requires the break-induced replication factors RAD52, POLD3 and POLD4. Representative FACS (PI) profiles of control U2OS cells or U2OS cells depleted of RAD52 ( g ), POLD3 ( h ) or POLD4 ( i ) by two different siRNAs each and exposed to 9 Gy. Genomic amplifications were monitored 72 h post IR. j , k , Histogram ( k ) showing the percentage of U2OS cells undergoing genomic amplifications when analysed at 72 h following the exposure of U2OS synchronized at various stages of the cell cycle to 9 Gy. Cells were irradiated at Early (E), Mid (M) or late (L) S-S-phase of the cell cycle following the synchronization by DTB and release (depicted in the schematic in ( j ; top) or in early (E) or Mid (M) G1 phase of the cell cycle following the synchronization in prometaphase by nocodazole and release (depicted in the schematic in ( j ; bottom)). Genomic amplifications in all cases was monitored 72 h following the exposure to IR. The synchronization PI-FACS profiles and representative rereplication PI-FACS profiles are shown in Extended data Fig. 10. h , Data in all the histograms represent the average of three independent experiments ± S.D. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Primary antibodies recognizing the following proteins were used: ATM (1:1000, ab78; Abcam), tubulin (1:1000, sc-53646; Santa Cruz), SET8 (1:1000, C18B7; Cell Signaling), mono-methyl Histone H4 (K20) (1:500, #9724; Cell Signaling), di-methyl Histone H4 (K20) (1:500, #9759; Cell Signaling), Tri-methyl Histone H4 (K20) (1:500, #5737; Cell Signaling), DNA-PKcs (1:1000, ab44815; Abcam), 53BP1 (1:5000, N100-304; Novus Biologicals) POLD3 (1:1000, H00010714-M01; Abnova), POLQ (1:1000, H00010721-M09; Abnova), RIF1 (1:1000, A300-569A-M; Bethyl Laboratories), RNF8 (1:1000, sc-2711462; Santa Cruz), RNF168 (1:1000, ABE367; Millipore), CtIP (1:1000, sc-271339, Santa Cruz), EXO1 (1:1000, ab95012; Abcam), XRCC4 (1:1000, sc-271087; Santa Cruz), XLF (1:1000, A300-730A-M; Bethyl Laboratories), NBS1 (1:1000, ab32074; Abcam), MDC1 (1:25,000, ab11171; Abcam), LIG1 (1:1000, 18051-1-AP; Proteintech), and LIG4 (1:1000, HPA001334; Sigma Aldrich).

Techniques: Control, Western Blot, Quantitation Assay, Clone Assay, Incubation, Irradiation

Journal: Nucleic acids research

Article Title: Human HELQ regulates DNA end resection at DNA double-strand breaks and stalled replication forks.

doi: 10.1093/nar/gkad940

Figure Lengend Snippet: Figure 7. HELQ functions in parallel to CtIP to preserve fork integrity. ( A ) Fork degradation assays were performed in HELQ KO #1 cells expressing the indicated shRNAs. ( B ) HU (2 mM, 4 h) induced RPA2 phosphorylation assa y s w ere perf ormed in wild type (WT) and HELQ KO #1 cells expressing shCtIP or vector (Ctrl). ( C ) HCT116 cells (WT) and HCT116 CtIP KO cells (CtIP KO) expressing shHELQ or vector (Ctrl) were treated with 2 mM HU for 4 h and subjected to metaphase spread assay. The chromosomal aberrations per chromosome spread are plotted. ( D ) The HU (2 mM, 4 h) induced RAD51-nascent DNA (EdU) interaction w as analyz ed b y PLA in U2OS cells (WT) and HELQ KO #1 cells expressing shCtIP or v ector (Ctrl). R epresentativ e images and quantification of the a v erage number of PLA foci per nucleus are shown. Scale bar = 5 μm. ( E ) WT and HELQ KO #1 cells expressing shCtIP or vector (Ctrl) were treated with the indicated concentration of HU for 48 h, then a cell viability assay was performed. ( F ) The viability of WT and HELQ KO #1 cells expressing shCtIP or vector (Ctrl) was analyzed by colony formation assay. Representative images (left) and quantifications of the relative survival (right) relative to vector (Ctrl) infected wild type U2OS cells (WT), which was arbitrarily set to 100%, are shown. In panels A, C, D, E and F, the data represent the means ± SD from at least three independent experiments. * P < 0.05, *** P < 0.001, **** P < 0.0001, n.s.: not significant. Mann–Whitney test was used in A, C and D; Student’s t -test was used in E and F.

Article Snippet: Antibodies used in this study included the following: FLAG (F1804, Sigma-Aldrich), HELQ (PA5-65181, Sigma-Aldrich and 19436s, Cell Signaling Technology), Biotin (200-002-211, Jackson Immunoresearch and A150-109A, Bethyl), γH2AX (05–636, Millipore), CtIP (A300-488A, Bethyl), RPA2 S4 / 8p (A300-245A, Bethyl), RPA2 (A300-244A, Bethyl), BRCA1 (9010s, Cell Signaling Technology), BRCA2 (10741S, Cell Signaling Technology), MRE11 (ab208020, Abcam), RAD51 (ab133534, Abcam), BrdU (ab6326, Abcam and 347580, BD), MUS81(ab247136, Abcam), BLM (ab2179, Abcam), DNA2 (18727-1-AP , Proteintech), EXO1 (16253-1-AP , Proteintech), HLTF (Ab17984, Abcam), SMARCAL1 (sc-376377, Santa Cruze), ZRANB3 (A303-033A, Bethyl).

Techniques: Expressing, Phospho-proteomics, Plasmid Preparation, Concentration Assay, Viability Assay, Colony Assay, Infection, MANN-WHITNEY

Journal: Cancer cell

Article Title: BRD4 inhibition is synthetic lethal with PARP inhibitors through the induction of homologous recombination deficiency

doi: 10.1016/j.ccell.2018.01.019

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: CtIP (D76F7) Rabbit mAb antibody , Cell Signaling Technology , #9201S, RRID:AB_10828593.

Techniques: Western Blot, Immunofluorescence, Virus, Recombinant, Staining, Lysis, Extraction, SYBR Green Assay, Transfection, Plasmid Preparation, In Vitro, In Vivo, Clinical Proteomics, Mutagenesis, Reverse Transcription, Chromatin Immunoprecipitation, Fractionation, Cell Culture, Gene Expression, Inhibition, Software

Journal: PLoS genetics

Article Title: CtIP Mutations Cause Seckel and Jawad Syndromes.

doi: 10.1371/journal.pgen.1002310

Figure Lengend Snippet: Figure 2. SCKL2 cells express a shorter version of CtIP and are mildly defective in RPA-coated ssDNA formation and ATR activation. (A) Protein samples from cells expressing the indicated CtIP variant after 1 h treatment with etoposide were separated by SDS-PAGE and detected with the indicated antibodies. (B) CtIP was immunoprecipitated from protein extracts from the indicated cell lines treated with etoposide using an anti-CtIP antibody (Sigma) and blotted using a different anti-CtIP (Novus Biological Ltd.). (C) Relative abundance of full-length CtIP and CtIPSCKL2 in different cell lines. (D) Immunoprecipitated CtIP from B were blotted with an antibody that recognized ATM and ATR phosphorylation sites (anti- phospho S/TQ). (E) Relative abundance of Chk1 phosphorylated on Ser-345 with respect to total Chk1. Ratios were normalized to wild-type cells after 10 mM etoposide, taken as 100%. (F) Cells treated for 1 h with 10 mM etoposide were immunostained with RPA and cH2AX antibodies. The percentages of total cells positive for cH2AX foci and proportions of cH2AX positive cells also positive for RPA foci are plotted. The averages and standard deviations of three independent experiments are shown. (G) As in (F), but in cells not treated with etoposide. doi:10.1371/journal.pgen.1002310.g002

Article Snippet: Immunoprecipitation was performed by using a CtIP antibody from Novus.

Techniques: Activation Assay, Expressing, Variant Assay, SDS Page, Immunoprecipitation, Phospho-proteomics