Journal: eLife

Article Title: LIN37-DREAM prevents DNA end resection and homologous recombination at DNA double-strand breaks in quiescent cells

doi: 10.7554/eLife.68466

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-FANCD2 (Rabbit monoclonal) , R and D Systems , MAB93691 , WB (1:1000).

Techniques: Recombinant, Plasmid Preparation, Genome Wide, CRISPR, Clone Assay, Flow Cytometry, Staining, Sequencing, Software, Microscopy

Journal: Molecular Cancer

Article Title: Non-specific chemical inhibition of the Fanconi anemia pathway sensitizes cancer cells to cisplatin

doi: 10.1186/1476-4598-11-26

Figure Lengend Snippet: Effects of 26 chemicals that inhibit the FA pathway on HR efficiency, IR-induced foci formation of FANCD2, RAD51 and BRCA1, and IR-induced FANCD2 monoubiquitination. (See Additional file : Figures S2, Additional file : Figure S4, and Additional file : Figure S5 for details) Color-coded representation of HR efficiency in DR-GFP assay, the proportion of cells with IR-induced foci of the indicated proteins, and the proportion of FANCD2 monoubiquitinated form on Western blot (8 hours after 10 Gy), compared to untreated controls. U2OS-DR-GFP cells were used for all the experiments. An asterisk (*) indicates significant decrease compared to controls (p ≤0.05, paired t test, n = 3 to 7) in DR-GFP and foci formation experiments. N.D. = not determined.

Article Snippet: Antibodies against BRCA1 (D-9, Santa Cruz, 1/100), γH2AX (JBW301, Upstate, 1/1000), FANCD2 (NB 100–182, Novus Biologicals, 1/1000) and RAD51 (PC130, Calbiochem, 1/1000 or H-92, Santa Cruz, 1/200) were used.

Techniques: Western Blot

Journal: Communications Biology

Article Title: Fanconi anemia associated protein 20 (FAAP20) plays an essential role in homology-directed repair of DNA double-strand breaks

doi: 10.1038/s42003-023-05252-9

Figure Lengend Snippet: a Measure of HR repair events at an I-SceI DSB in 282-U2OS reporter cell lines with the indicated knockdown conditions performed prior to DSB induction. Events are reported as % of siCtrl. BRCA2 knockdown is shown as a “HR factor control.” Representative dot plots are shown for effect. b Measure of HR events in 282-U2OS cells with FANCD2 siRNA knockdown compared with FAAP20 siRNA knockdown c Measure of HR events in 282-U2OS cells with indicated protein siRNA knockdown in FANCA knock out background generated by CRISPR-Cas9. Comparison with 282-U2OS WT cells shows non-redundant relationship with FANCA during HR for FAAP20 and FANCD2. d Schematic of Δ7 RMR-U2OS reporter cell experiments to measure SSTR using short regions of homology. A non-homologous insert is removed from an interrupted GFP cassette through expression of CRISPR and dual sgRNAs that target the 5’ and 3’ recognition sites surrounding the sequence. GFP is restored through a targeted insertion at the break site using an exogenous single-strand oligonucleotide repair template with varying degrees of RNA content. For all RMR experiments, cells are first gated on mCherry due to a constitutively active mCherry construct present within the CRISPR expression plasmids, then as a % GFP within the mCherry (+) cell population. They are then reported as a % of a control condition (siCtrl or vector) that has been normalized to 1. e RMR using 100% DNA template with the indicated knockdown conditions. f RMR using a DNA/RNA hybrid repair template (H2) with 7 bp of RNA spanning the targeted insertion region needed to restore GFP. Indicated knockdown conditions are shown. All graphs in this figure show bars as mean values and error bars as SD. All statistical tests were two-tailed Student’s t test where * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; n = 3 for all experiments.

Article Snippet: Gels were run 1–1.5 h at 150 V then transferred to nitrocellulose membranes, blocked for 1 h with 5% milk, then probed with the following antibodies: FANCA (Bethyl-1:1000), FAAP20 (Sigma, Weidong Wang-1:250), FANCG (Santa Cruz-1:50), Actin (Santa Cruz-1:2000), H2B (Cell Signaling Technologies-1:1000), BRCA2 (Sigma, OHSU-1:500), RAD51 (BioAcademia-1:500), HSP90 (Santa Cruz-1:2000), FANCD2 (Proteintech-1:1000).

Techniques: Knockdown, Control, Knock-Out, Generated, CRISPR, Comparison, Expressing, Sequencing, Construct, Plasmid Preparation, Two Tailed Test

Journal: Molecular Cancer Research

Article Title: Constitutive Activation of Caspase-3 and Poly ADP Ribose Polymerase Cleavage in Fanconi Anemia Cells

doi: 10.1158/1541-7786.mcr-09-0373

Figure Lengend Snippet: FIGURE 1. PARP cleavage and upregulation in FA cells. A. Immunoblot showing PARP cleavage in FANCD2-deficient (PD20), FANCD2-proficient (PD20cor), or nonmonoubiquitinable mutant (K561R) cells. B. Immunoblot showing PARP processing in PD20 and PD20-corrected cells treated (+) or untreated (−) with PARP inhibitor 3AB. C. PARP cleavage is increased in primary fibroblasts transfected with FANCD2 siRNA for the corresponding time intervals. Arrows, mobility shift representing PARP phosphorylation. Note the reduction of PARP signal in the sample treated with λ-phosphatase or in the siRNA FANCD2–depleted cells pretreated with PKC inhibitor. D. Immunoblot of the above FANCD2 knockdown samples immunoprecipitated with PARP antibody followed by probing with phosphor-PARP or total PARP antibodies. E. Immunoblots showing PARP cleavage in different FA subgroups treated or untreated with the PARP inhibitor, 3AB.

Article Snippet: Proteins were then transferred to a polyvinylidene difluoride membrane and were probed with an anti-PARP, cleaved anti–caspase-3, cleaved anti–caspase-8, anti–glyceraldehyde-3-phosphate dehydrogenase, antivinclulin (all from AbCAM), anti-PAR antibody (a rabbit polyclonal IgG from AbNOVA or SantaCruz), or with an anti-FANCD2 antibody (monoclonal mouse IgG, Novus Biologicals) followed by a horseradish peroxidase–conjugated secondary antibody (Amersham Biosciences).

Techniques: Western Blot, Mutagenesis, Transfection, Mobility Shift, Phospho-proteomics, Knockdown, Immunoprecipitation