Journal: Oncotarget

Article Title: FBW7 regulates DNA interstrand cross-link repair by modulating FAAP20 degradation

doi: 10.18632/oncotarget.9595

Figure Lengend Snippet: A. , B. Overexpression of GSK3β and FBW7 suppresses damage-induced FANCD2 monoubiquitination and foci formation. A. HeLa cells coexpressing HA-tagged GSK3β and FBW7 were treated with 1 μM MMC for 8 h and cell lysates analyzed by Western blotting. B. U2OS cells coexpressing HA-tagged GSK3β and FBW7 were treated with 100 ng/mL MMC for 16 h and subjected to anti-FANCD2 immunofluorescence. C. Quantification of cells in B. exhibiting more than 10 FANCD2 foci. Data shown are the mean ± SD from three independent experiments. * p < 0.01 compared with vector control. D. GSK3β and FBW7 overexpression facilitates the turnover of FANCA and FANCG. HeLa cells expressing HA-tagged GSK3β and FBW7 were treated with 50 μg/mL CHX for the indicated times and analyzed by Western blotting. E. Densitometry of FANCA and FANCG levels in D. quantitated by ImageJ. F. GSK3β and FBW7 overexpression sensitizes cells to a DNA interstrand cross-linking agent. U2OS cells expressing HA-GSK3β and HA-FBW7 were plated to 96 wells, treated with the indicated doses of MMC for 5 days, and cell viability was measured by luminescence assay. Data shown are the mean ± SD from three independent experiments. * p < 0.05 compared with control.

Article Snippet: Cells were incubated with an anti-FANCD2 primary antibody (1: 500, Novus Biologicals) in PBS/ 1 % BSA for 2 h at RT, washede three times in PBS, and incubated with 1:1000 Alexa Fluor ® 568 goat anti-mouse IgG secondary antibody (Molecular Probes) for 1 h at RT.

Techniques: Over Expression, Western Blot, Immunofluorescence, Plasmid Preparation, Control, Expressing, Luminescence Assay

Journal: Oncotarget

Article Title: FBW7 regulates DNA interstrand cross-link repair by modulating FAAP20 degradation

doi: 10.18632/oncotarget.9595

Figure Lengend Snippet: A. Depletion of FBW7 hypersensitizes cells to a DNA interstrand cross-linking agent. U2OS cells transfected with indicated siRNA for 48 h were plated to 96 wells, treated with the indicated doses of MMC for 5 days, and cell viability was measured by luminescence assay. FAAP20 depletion served as a positive control. Data shown are the mean ± SD from three independent experiments. * p < 0.05 compared with control. B. A schematic for the FAAP20 knockout strategy using CRISPR/Cas9. The 20-nucleotide sgRNA target loci in the exon 1 are marked in blue line along with a PAM sequence in red. The cleavage site for the Cas9 nuclease is shown by red triangle. The ATG start codon is marked in bold with arrow. C. U2OS wild-type (vector transfected) or FAAP20 knockout (KO) clones were treated with 100 ng/mL MMC for 16 h and analyzed by Western blotting. D. Western blot analyses of U2OS FAAP20 KO cells reconstituted with FAAP20 wild-type or SA mutant by retroviral transduction. E. Restoration of FANCD2 monoubiquitination by exogenous FAAP20 wild-type or SA mutant. FAAP20 KO cells stably expressing FAAP20 wild-type or SA mutant were treated with 100 ng/mL MMC for 16 h and analyzed by Western blotting. F. Accumulation of FANCA and FANCD2 monoubiquitin in the chromatin-enriched fraction in cells expressing the FAAP20 SA mutant. Indicated U2OS cells were treated with 1 μM MMC for 2 h, replenished with fresh medium to initiate the DNA repair process, and collected at the indicated times. Cells were fractionated, and chromatin-enriched fractions were analyzed by Western blotting. Asterisks denote nonspecific bands. G. The half-life of FANCA in the chromatin extends in the cells expressing the FAAP20 SA mutant. (Top) U2OS FAAP20 KO cells expressing FAAP20 wild-type or SA mutant were treated with 100 ng/mL MMC for 16 h, incubated with 50 μg/mL CHX for the indicated times and fractionated to isolate chromatin-enriched fractions. Cell lysates were analyzed by Western blotting. (Bottom) Quantification of the FANCA level normalized by ORC2. Error bars indicate SD from two independent experiments. * p < 0.05 compared with SA. H. U2OS cells serially transfected with siRNA and siRNA-resistant FAAP20 variants (siR*) were treated with indicated doses of MMC, and cell viability was measured by luminescence assay. Data shown are the mean ± SD from three independent experiments. * p < 0.05 (WT and SA) compared with control except 125 nM for SA ( p = 0.4940 not significant).

Article Snippet: Cells were incubated with an anti-FANCD2 primary antibody (1: 500, Novus Biologicals) in PBS/ 1 % BSA for 2 h at RT, washede three times in PBS, and incubated with 1:1000 Alexa Fluor ® 568 goat anti-mouse IgG secondary antibody (Molecular Probes) for 1 h at RT.

Techniques: Transfection, Luminescence Assay, Positive Control, Control, Knock-Out, CRISPR, Sequencing, Plasmid Preparation, Clone Assay, Western Blot, Mutagenesis, Retroviral, Transduction, Stable Transfection, Expressing, Incubation

Journal: PLoS Pathogens

Article Title: High-risk human papillomavirus oncogenes disrupt the Fanconi anemia DNA repair pathway by impairing localization and de-ubiquitination of FancD2

doi: 10.1371/journal.ppat.1007442

Figure Lengend Snippet: (A) Cells were treated with increasing concentrations of cisplatin for 72 hr and % survival was measured using the Crystal Violet assay. LXSN, E6, E7, E6E7 expressing cells showed respective IC50 of 2.5, 0.74, 1.27 and 0.73 uM; 72 hr. (B-D) Cells were transfected with siControl or siFancD2 for 48hrs and plated for western blotting and cisplatin survival assay. (B) Western blot showing FancD2 knockdown in the cells harvested at the time of reading survival assay. (C) Survival curves of LXSN, E6, E7 and E6E7 cells transfected with siRNA (siFancD2 or siControl) and treated with indicated doses of cisplatin for 48 hr. (D) Survival curve of siControl cells treated with indicated doses of cisplatin for 48 hrs.

Article Snippet: Primary antibodies against p53 (Cell Signaling Technology, 9282), pRb (BD Pharmingen, 554136), FancD2 (Santa Cruz Biotechnology, FI17, sc-20022 or Abcam, ab2187), FancI (Santa Cruz Biotechnology, A-7, sc-271316), Phospho-Ser556 FancI and Phospho-Ser565-FancI (gifts from Ronald Cheung, Toshiyasu Taniguchi lab), pH2AX (Millipore, 05–636), Rad51 (Cosmo Bio Co. Ltd, 70–001), Palb2 (Bethyl Laboratories, A301-246A), Palb2 (a gift from Bing Xia, Rutgers Cancer Institute), Phospho-ATR (Ser428) (Cell Signaling Technology, 2853), Phospho-Chk1 (Ser345) (133D3) (Cell Signaling Technology, 2348), ATR (Cell Signaling Technology, 2790), CHK1 (2G1D5, Cell Signaling Technology, 2360), PCNA (PC10) (Cell Signaling Technology, 2586), Ubiquityl-PCNA (Lys164) (Cell Signaling Technology, 13439), UHRF1 (Santa Cruz Biotechnology, H-8, sc-373750), USP1 (C-term; a gift from Tony Huang, NYU School of Medicine), Cyclin A (in-house, Clurman lab), Beta actin (GeneTex, GTX110564 or Cell Signaling Technology, 5125), Vinculin (Sigma, V9131), and Histone H3 (Abcam, ab1791), GAPDH (14C10, Cell Signaling Technology, 2118)were used.

Techniques: Crystal Violet Assay, Expressing, Transfection, Western Blot, Clonogenic Cell Survival Assay, Knockdown

Journal: PLoS Pathogens

Article Title: High-risk human papillomavirus oncogenes disrupt the Fanconi anemia DNA repair pathway by impairing localization and de-ubiquitination of FancD2

doi: 10.1371/journal.ppat.1007442

Figure Lengend Snippet: (A) Immunoblot showing FancD2/ FancI expression and monoubiquitination status in transduced HFK cells which were either untreated or treated with 3 uM cisplatin for 24 hr. Ub refers to the monoubiquitinated forms of FancD2 and FancI, and non-Ub refers to the non-ubiquitinated forms. Ratios of monoubiquitinated to non-ubiquitinated FancD2 (D2 Ub: Non-Ub) and total FancD2 (Ub + Non-Ub) levels are indicated beneath the corresponding lanes. (B) Immunoblot of soluble and chromatin-bound fractions prepared from transduced HFK cells that were either untreated or treated with 3 uM cisplatin for 24 hr. Vinculin and Histone H3 act as loading controls respectively for soluble and chromatin-bound fractions. (C) Immunoblot of whole cell lysates showing levels of phosphorylated S556 FancI, total FancI, Ub-PCNA, non-Ub PCNA, and UHFR1. Vinculin acts as a loading control.

Article Snippet: Primary antibodies against p53 (Cell Signaling Technology, 9282), pRb (BD Pharmingen, 554136), FancD2 (Santa Cruz Biotechnology, FI17, sc-20022 or Abcam, ab2187), FancI (Santa Cruz Biotechnology, A-7, sc-271316), Phospho-Ser556 FancI and Phospho-Ser565-FancI (gifts from Ronald Cheung, Toshiyasu Taniguchi lab), pH2AX (Millipore, 05–636), Rad51 (Cosmo Bio Co. Ltd, 70–001), Palb2 (Bethyl Laboratories, A301-246A), Palb2 (a gift from Bing Xia, Rutgers Cancer Institute), Phospho-ATR (Ser428) (Cell Signaling Technology, 2853), Phospho-Chk1 (Ser345) (133D3) (Cell Signaling Technology, 2348), ATR (Cell Signaling Technology, 2790), CHK1 (2G1D5, Cell Signaling Technology, 2360), PCNA (PC10) (Cell Signaling Technology, 2586), Ubiquityl-PCNA (Lys164) (Cell Signaling Technology, 13439), UHRF1 (Santa Cruz Biotechnology, H-8, sc-373750), USP1 (C-term; a gift from Tony Huang, NYU School of Medicine), Cyclin A (in-house, Clurman lab), Beta actin (GeneTex, GTX110564 or Cell Signaling Technology, 5125), Vinculin (Sigma, V9131), and Histone H3 (Abcam, ab1791), GAPDH (14C10, Cell Signaling Technology, 2118)were used.

Techniques: Western Blot, Expressing, Control

Journal: PLoS Pathogens

Article Title: High-risk human papillomavirus oncogenes disrupt the Fanconi anemia DNA repair pathway by impairing localization and de-ubiquitination of FancD2

doi: 10.1371/journal.ppat.1007442

Figure Lengend Snippet: (A) HFK cells were treated with cisplatin (3 uM) for 24 hr and immunostained with FancD2 (red), pH2AX (green) and DAPI (blue). Representative images are shown. (B) Cells with >5 foci were counted, and the percentage of positive cells is plotted (n = 3, mean ± SEM). (C) Quantification of percentage of FancD2 foci co-localization with pH2AX with or without cisplatin treatment. * (p-value ≤ 0.05) and ** (p-value ≤ 0.01) denote a statistically significant difference from the similarly treated LXSN control cells. Error bars represent standard error of the mean. Quantification was based on data observed from ≥ 15 nuclei from three independent experiments. (D) U2OS-DR cells (transduced with LXSN or E6/E7) were transfected with I-SceI expression plasmid for 24 hr before fixation. Cells were immunostained with pH2AX, FancD2, and DAPI (blue). Cells with a single large pH2AX focus (red) were examined for the colocalization with FancD2 (green). Representative images are shown. (E) Quantification of the frequency of colocalization of FancD2 with pH2AX foci in U2OS-DR cells. Data represent mean ± SEM and was based on observations from ≥ 50 cells from at least three independent experiments. (F-H) U2OS-DR cells transduced with the indicated constructs were transfected with the siControl or siFancD2. They were transfected with I-SceI expression plasmid and then fixed after 24 hr of transfection and stained with Rad51 and pH2AX antibodies. (F) Cell lysates were subjected to western blotting to confirm depletion of FancD2. (G) Cells with a single large pH2AX focus (red) were inspected for the colocalization with Rad51 (green). Representative images are shown. (H) Quantification of the frequency of colocalization of Rad51 with pH2AX foci. Data represent mean ± SEM and was based on observations from ≥ 25 cells from at least three independent experiments. * and ** indicate significance respectively at p<0.05 and p<0.01 (compared to LXSN) whereas n.s. indicates non-significant.

Article Snippet: Primary antibodies against p53 (Cell Signaling Technology, 9282), pRb (BD Pharmingen, 554136), FancD2 (Santa Cruz Biotechnology, FI17, sc-20022 or Abcam, ab2187), FancI (Santa Cruz Biotechnology, A-7, sc-271316), Phospho-Ser556 FancI and Phospho-Ser565-FancI (gifts from Ronald Cheung, Toshiyasu Taniguchi lab), pH2AX (Millipore, 05–636), Rad51 (Cosmo Bio Co. Ltd, 70–001), Palb2 (Bethyl Laboratories, A301-246A), Palb2 (a gift from Bing Xia, Rutgers Cancer Institute), Phospho-ATR (Ser428) (Cell Signaling Technology, 2853), Phospho-Chk1 (Ser345) (133D3) (Cell Signaling Technology, 2348), ATR (Cell Signaling Technology, 2790), CHK1 (2G1D5, Cell Signaling Technology, 2360), PCNA (PC10) (Cell Signaling Technology, 2586), Ubiquityl-PCNA (Lys164) (Cell Signaling Technology, 13439), UHRF1 (Santa Cruz Biotechnology, H-8, sc-373750), USP1 (C-term; a gift from Tony Huang, NYU School of Medicine), Cyclin A (in-house, Clurman lab), Beta actin (GeneTex, GTX110564 or Cell Signaling Technology, 5125), Vinculin (Sigma, V9131), and Histone H3 (Abcam, ab1791), GAPDH (14C10, Cell Signaling Technology, 2118)were used.

Techniques: Control, Transduction, Transfection, Expressing, Plasmid Preparation, Construct, Staining, Western Blot

Journal: PLoS Pathogens

Article Title: High-risk human papillomavirus oncogenes disrupt the Fanconi anemia DNA repair pathway by impairing localization and de-ubiquitination of FancD2

doi: 10.1371/journal.ppat.1007442

Figure Lengend Snippet: (A) Outline of an experiment to evaluate FancD2 monoubiquitination/de-ubiquitination pattern. Transduced HFK cells were untreated or treated with cisplatin (1.5 uM for 24 hr) or exposed to 10 mJ/cm 2 UVB and allowed to repair. Whole-cell lysates were prepared for immunoblot at various time points during the experiments (represented by Δ). (B-C) Immunoblots of HFKs subjected to the experiment outlined in , following cisplatin withdrawal (B) or recovery after UVB exposure (C). Ratios of monoubiquitinated to non-ubiquitinated FancD2 (D2 Ub: non-Ub) are indicated beneath the corresponding lanes. FancD2 Ub:non-Ub ratio in cells following cisplatin withdrawal and UVB exposure was plotted alongside Figure 5B and 5C. ** (p< 0.01) denotes a statistically significant difference from 0 hr cisplatin withdrawal. ‘ns’ denotes non-significant differences. (D) USP1 immunoblot in cells untreated and treated with cisplatin. (E) Immunoblot of soluble and chromatin-bound fractions prepared from transduced HFK cells subjected to the experiment outlined in . Vinculin and Histone H3 act as loading controls respectively for soluble and chromatin-bound fractions. (F) Immunoblot showing levels of p-FancI-S565 and total FancI in cisplatin-treated or untreated cells. (G) Immunoblot for p-ATR, pCHK1, FancD2 and p-FancI-S565 following cisplatin withdrawal for 18 and 24 hrs. Actin or vinculin act as a loading control for immunoblots (B-G). (H) Proposed mechanisms for delayed de-ubiquitination of FancD2 in E6 cells.

Article Snippet: Primary antibodies against p53 (Cell Signaling Technology, 9282), pRb (BD Pharmingen, 554136), FancD2 (Santa Cruz Biotechnology, FI17, sc-20022 or Abcam, ab2187), FancI (Santa Cruz Biotechnology, A-7, sc-271316), Phospho-Ser556 FancI and Phospho-Ser565-FancI (gifts from Ronald Cheung, Toshiyasu Taniguchi lab), pH2AX (Millipore, 05–636), Rad51 (Cosmo Bio Co. Ltd, 70–001), Palb2 (Bethyl Laboratories, A301-246A), Palb2 (a gift from Bing Xia, Rutgers Cancer Institute), Phospho-ATR (Ser428) (Cell Signaling Technology, 2853), Phospho-Chk1 (Ser345) (133D3) (Cell Signaling Technology, 2348), ATR (Cell Signaling Technology, 2790), CHK1 (2G1D5, Cell Signaling Technology, 2360), PCNA (PC10) (Cell Signaling Technology, 2586), Ubiquityl-PCNA (Lys164) (Cell Signaling Technology, 13439), UHRF1 (Santa Cruz Biotechnology, H-8, sc-373750), USP1 (C-term; a gift from Tony Huang, NYU School of Medicine), Cyclin A (in-house, Clurman lab), Beta actin (GeneTex, GTX110564 or Cell Signaling Technology, 5125), Vinculin (Sigma, V9131), and Histone H3 (Abcam, ab1791), GAPDH (14C10, Cell Signaling Technology, 2118)were used.

Techniques: Ubiquitin Proteomics, Western Blot, Control

Journal: PLoS Pathogens

Article Title: High-risk human papillomavirus oncogenes disrupt the Fanconi anemia DNA repair pathway by impairing localization and de-ubiquitination of FancD2

doi: 10.1371/journal.ppat.1007442

Figure Lengend Snippet: (A) Outline of synchronization assay in HFK cells using double-thymidine block. Cells were synchronized by double-thymine block and released at various time points. Immunoblot for FancD2, FancI, cyclin A and vinculin of LXSN (B) and E6 cells (C). Cell-cycle phases at each time point were determined by flow cytometry of DNA content (D). Asynchronous (Asyn.) cells, which have not undergone thymidine block, were included for comparison.

Article Snippet: Primary antibodies against p53 (Cell Signaling Technology, 9282), pRb (BD Pharmingen, 554136), FancD2 (Santa Cruz Biotechnology, FI17, sc-20022 or Abcam, ab2187), FancI (Santa Cruz Biotechnology, A-7, sc-271316), Phospho-Ser556 FancI and Phospho-Ser565-FancI (gifts from Ronald Cheung, Toshiyasu Taniguchi lab), pH2AX (Millipore, 05–636), Rad51 (Cosmo Bio Co. Ltd, 70–001), Palb2 (Bethyl Laboratories, A301-246A), Palb2 (a gift from Bing Xia, Rutgers Cancer Institute), Phospho-ATR (Ser428) (Cell Signaling Technology, 2853), Phospho-Chk1 (Ser345) (133D3) (Cell Signaling Technology, 2348), ATR (Cell Signaling Technology, 2790), CHK1 (2G1D5, Cell Signaling Technology, 2360), PCNA (PC10) (Cell Signaling Technology, 2586), Ubiquityl-PCNA (Lys164) (Cell Signaling Technology, 13439), UHRF1 (Santa Cruz Biotechnology, H-8, sc-373750), USP1 (C-term; a gift from Tony Huang, NYU School of Medicine), Cyclin A (in-house, Clurman lab), Beta actin (GeneTex, GTX110564 or Cell Signaling Technology, 5125), Vinculin (Sigma, V9131), and Histone H3 (Abcam, ab1791), GAPDH (14C10, Cell Signaling Technology, 2118)were used.

Techniques: Blocking Assay, Western Blot, Flow Cytometry, Comparison

Journal: PLoS Pathogens

Article Title: High-risk human papillomavirus oncogenes disrupt the Fanconi anemia DNA repair pathway by impairing localization and de-ubiquitination of FancD2

doi: 10.1371/journal.ppat.1007442

Figure Lengend Snippet: (A) Immunoblot for p53 (upper panel) and RT-PCR analysis of 16E6 and GAPDH expression (lower panel) in HFK cells transduced with LXSN, E6 and E6 mutant (8S/9A/10T). (B-C) Immunoblot showing FancD2 expression and monoubiquitination status in cells which were either untreated or treated with cisplatin (B) or nutlin (C) at 1.5 uM for 24 hr. (D-E) Cells were untreated or treated with cisplatin (E) or exposed to 10 mJ/cm 2 UVB (D) and processed similarly as in .

Article Snippet: Primary antibodies against p53 (Cell Signaling Technology, 9282), pRb (BD Pharmingen, 554136), FancD2 (Santa Cruz Biotechnology, FI17, sc-20022 or Abcam, ab2187), FancI (Santa Cruz Biotechnology, A-7, sc-271316), Phospho-Ser556 FancI and Phospho-Ser565-FancI (gifts from Ronald Cheung, Toshiyasu Taniguchi lab), pH2AX (Millipore, 05–636), Rad51 (Cosmo Bio Co. Ltd, 70–001), Palb2 (Bethyl Laboratories, A301-246A), Palb2 (a gift from Bing Xia, Rutgers Cancer Institute), Phospho-ATR (Ser428) (Cell Signaling Technology, 2853), Phospho-Chk1 (Ser345) (133D3) (Cell Signaling Technology, 2348), ATR (Cell Signaling Technology, 2790), CHK1 (2G1D5, Cell Signaling Technology, 2360), PCNA (PC10) (Cell Signaling Technology, 2586), Ubiquityl-PCNA (Lys164) (Cell Signaling Technology, 13439), UHRF1 (Santa Cruz Biotechnology, H-8, sc-373750), USP1 (C-term; a gift from Tony Huang, NYU School of Medicine), Cyclin A (in-house, Clurman lab), Beta actin (GeneTex, GTX110564 or Cell Signaling Technology, 5125), Vinculin (Sigma, V9131), and Histone H3 (Abcam, ab1791), GAPDH (14C10, Cell Signaling Technology, 2118)were used.

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Transduction, Mutagenesis

Journal: Cell Death & Disease

Article Title: Resistance to DNA-damaging treatment in non-small cell lung cancer tumor-initiating cells involves reduced DNA-PK/ATM activation and diminished cell cycle arrest

doi: 10.1038/cddis.2012.211

Figure Lengend Snippet: DDR/DNA repair proteins show suboptimal activation in TICS compared with bulk cells. ( a ) Western blot analysis of H125 bulk cells and TICs, 1, 4 and 24 h after IR, and A549, H1299 and H23 2 h after IR with 8 Gy. Phosphorylated and total forms of DNA-PK, ATM and phosphorylated forms of the ATM substrates H2AX, KAP1 and Chk2 were analyzed. α -Tubulin or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading controls. ( b ) Western blot analysis of H125 bulk cells and TICs, 4 h after bleomycin (2.5 μg/ml) or 24 h after cisplatin (1 or 10 μ M) exposure. Phosphorylated and total forms of DNA-PK, ATM, phosphorylated forms of the ATM substrates H2AX, KAP1, SMC1, Chk2 as well as the levels of FANCD2 and Rad51 were analyzed. A representative blot of at least two independent experiments is shown. β -Tubulin or GAPDH was used as loading controls. ( c ) Western blot analysis of H125 and A549 bulk cells and TICs, 24 h after 8 Gy IR, or 1 or 10 μ M continuous cisplatin exposure. FANCD2 and cdc25A was assayed. A representative blot of two independent experiments is shown. GAPDH was used as loading control. ( d ) Western blot analysis of PARP full-length and cleavage fragment 24 h after 10 μ M of continuous cisplatin treatment, with or without 1 h pretreatment with DNA-PKcs (NU7026) or ATM (KU55933) inhibitors (both 10 μ M). A representative blot of two independent experiments is shown. GAPDH was used as a loading control

Article Snippet: The following primary antibodies were used: γ H2AX and pDNA-PKcs (pSer139; ab2893 and pSer2056; ab18192, respectively; Abcam), total DNA-PKcs (MS-423P0; Thermo Fisher Scientific, Fremont, CA, USA), pATM (pSer1981; 05-740; Upstate/Millipore, Solna, Sweden), total ATM and FANCD2 (1549-1 and 2986-1, respectively; Epitomics, Burlingame, CA, USA), pKAP1 and pSMC1 (pSer824; A300-767A and pSer966; A300-050A, respectively; Bethyl Laboratories, Montgomery, TX, USA).

Techniques: Activation Assay, Western Blot, Control

Journal:

Article Title: A Rad50-dependent pathway of DNA repair is deficient in Fanconi anemia fibroblasts

doi: 10.1093/nar/gkh649

Figure Lengend Snippet: Co-introduction of anti-DNA ligase IV antibody reduces DNA end-joining frequency in normal diploid fibroblasts but does not affect FA cells. (A) End-joining frequency of cohesive-ended DNA was determined in HT1080 cells (black bars) and normal HDFs (white bars) in the presence of no antibody (N), in the presence of anti-DNA ligase IV antibody (L), in the presence of anti-Fancd2 antibody (D), and in the presence of both anti-DNA ligase IV and anti-Fancd2 antibodies (L + D). In all cases, antibody treatment significantly reduced plasmid end-joining levels compared to those observed in cells not treated with antibody, P < 0.0001, χ2-test. (B) End-joining frequency of cohesive-ended DNA (black bars) and blunt-ended DNA (white bars) was determined in patient-derived FA-C cells in the absence of antibody (−) and in the presence of anti-DNA ligase IV antibody (+).

Article Snippet: Mouse polyclonal anti-DNA ligase III, rabbit polyclonal anti-Fancd2, rabbit polyclonal anti-Rad50, rabbit polyclonal anti-Mre11 and rabbit polyclonal anti-Nbs1 antibodies were obtained from Novus Biologicals, Inc. (Littleton, CO).

Techniques: Plasmid Preparation, Derivative Assay

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: The following antibodies were used for western blot analysis: 53BP1 (Bethyl Laboratories, A300-272A, 1:3000), LIN37 (Santa Cruz Biotechnology, sc-515686, 1:200), BLM (Bethyl Laboratories, A300-572A, 1:2000), BRCA1 for mouse (R and D Systems, gift from Dr. Andre Nussenzweig, NCI, 1:1000) , BRCA1 for human (Millipore Sigma, 07-434, 1:1000), RAD51 (Millipore Sigma, ABE257, 1:2000), BARD1 (Thermo Fisher Scientific, PA5-85707, 1:1000), CtIP (gift from Dr. Richard Baer, [Columbia University, New York], 1:1000), MRE11 (Novus Biologicals, NB100-142, 1:2000), RIF1 (Abcam, ab13422, 1:500), SHLD1/C20orf196 (Thermo Fisher Scientific, PA5-559280, 1:200), GAPDH (Sigma, G8795, 1:10,000), KAP1 (Genetex, GTX102226, 1:2000), FANCD2 (R and D Systems, MAB93691, 1:1000), BRCA2 for human (Proteintech, 19791-1-AP, 1:500), Rb1 (Thermo Fisher Scientific, LF-MA0173, 1:1000), Phospho-Rb (Ser780) (Cell Signaling Technology, 8180T, 1:1000), Phospho-Rb (Ser807/811) (Cell Signaling Technology, 8516T, 1:1000), PCNA (Bethyl Laboratories, A300-276A, 1:3000), CDK4: (Novus Biologicals, NBP1-31308, 1:1000), CDK4 (phosphor Thr 172) (GeneTex, GTX00778, 1:1000), and RPA (Cell Signaling Technology, 2208S, 1:1000).

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