Journal: Oncogene

Article Title: Human SNM1B is required for normal cellular response to both DNA interstrand crosslink-inducing agents and ionizing radiation.

doi: 10.1038/sj.onc.1207895

Figure Lengend Snippet: Figure 4 Depletion of hSNM1B by siRNA does not affect monoubiquitination of FANCD2. SV40-transformed fibroblasts (GM0637) were transfected with hSNM1B, FANCA or a negative control siRNA and assayed 66 h later for monoubiquitination of FANCD2 by immunoblot. Cells were either untreated or treated with MMC or IR as indicated

Article Snippet: Polyclonal Nibrin/p95 (NB100-143) and FANCD2 (NB 100- 182) antibodies were purchased from Novus-Biologicals (Littleton, CO, USA).

Techniques: Transformation Assay, Transfection, Negative Control, Western Blot

Journal: Oncogene

Article Title: Human SNM1B is required for normal cellular response to both DNA interstrand crosslink-inducing agents and ionizing radiation.

doi: 10.1038/sj.onc.1207895

Figure Lengend Snippet: Figure 3 Depletion of hSNM1B from HeLa cells increases sensitivity to ICL-inducing agents and to IR. (a) Clonogenic survival of HeLa cells transfected with hSNM1B siRNA, FANCD2 siRNA or control siRNA after treatment with increasing concentrations of MMC. (b) Survival of hSNM1B-depleted and control siRNA-transfected cells after treatment with cisplatin or (c) survival of HeLa cells transfected with hSNM1B siRNA, 53BP1 siRNA or control siRNA after treatment with increasing doses of IR. A fraction of HeLa cells treated with siRNAs for use in survival experiments was assayed for protein expression. Whole-cell lysates were immunoblotted for FANCD2 (d) or for 53BP1 (e). p95/nibrin expression was assayed as a control for specificity of RNA interference

Article Snippet: Polyclonal Nibrin/p95 (NB100-143) and FANCD2 (NB 100- 182) antibodies were purchased from Novus-Biologicals (Littleton, CO, USA).

Techniques: Transfection, Control, Expressing

Journal: Gynecologic oncology

Article Title: MTDH/AEG-1 downregulation using pristimerin-loaded nanoparticles inhibits Fanconi anemia proteins and increases sensitivity to platinum-based chemotherapy

doi: 10.1016/j.ygyno.2019.08.014

Figure Lengend Snippet: (A) Expression of FANCD2 and FANCI protein was detected and quantified by Western blotting in the brain (Br), liver (Li) and spleen (Sp) from wild type mice and two MTDH homozygous knockout mice (MTDH KO1 and MTDH KO2). Rad51 and β-actin were also analyzed by Western blotting as controls for other DNA repair proteins and loading. (B) Quantification of Western blots in (A). With the exception of Rad51, all data are relative to wide type brain. For Rad51 data are relative to MTDH−/− 2 spleen. (C, D) FANCD2, FANCI and MTDH were detected and quantified by Western blotting in parental Hec50 cells and MTDH CRISPR knockout Hec50 cells in the absence or presence of DNA damage via cisplatin for 24 h. Labels denote ubiquitinated (Ub) FANCI and the long (L) and short (S) isoforms of FANCD2. β-actin was used as a loading control. (E,F) FANCD2, FANCI and MTDH were detected and quantified by Western blotting in empty vector transfected Hec50 cells and MTDH overexpressed Hec50 cells. β-actin was used as a loading control. Each figure is representative of three independent experiments.

Article Snippet: Anti-FANCD2 (1:2000, #NB 200–182) and anti-FANCI (1:500, #ab74332) were obtained from Novus Biologicals (Centennial, CO, USA).

Techniques: Expressing, Western Blot, Knock-Out, CRISPR, Plasmid Preparation, Transfection

Journal: Gynecologic oncology

Article Title: MTDH/AEG-1 downregulation using pristimerin-loaded nanoparticles inhibits Fanconi anemia proteins and increases sensitivity to platinum-based chemotherapy

doi: 10.1016/j.ygyno.2019.08.014

Figure Lengend Snippet: (A) MTDH amplification portends worse prognosis for endometrial cancer. Kaplan Meier analysis of MTDH copy number gain (after removal of germline copy number variants) as a measure of MTDH amplification in TCGA dataset for endometrial cancer. Blue: low MTDH copy number; red: high MTDH copy number, indicative of gene amplification. (B) MTDH amplification positively correlates with expression of FANCD2 and FANCI in breast cancer. Heat maps for MTDH copy number variations and gene expression of MTDH, FANCI, and FANCD2 in TCGA dataset for breast cancer.

Article Snippet: Anti-FANCD2 (1:2000, #NB 200–182) and anti-FANCI (1:500, #ab74332) were obtained from Novus Biologicals (Centennial, CO, USA).

Techniques: Amplification, Expressing

Journal: Gynecologic oncology

Article Title: MTDH/AEG-1 downregulation using pristimerin-loaded nanoparticles inhibits Fanconi anemia proteins and increases sensitivity to platinum-based chemotherapy

doi: 10.1016/j.ygyno.2019.08.014

Figure Lengend Snippet: (A) Schematic representation of full length MTDH and truncated MTDH constructs which contain different RNA binding domains. Circle denotes the region in MTDH that binds mRNAs (residues 145–260). (B) Immunoblot for flag-tagged MTDH fragments. Lysates were immunoprecipitated with anti-Flag, followed by Western blotting with anti-Flag antibody. (C) RNA immunoprecipatation of MTDH-associated mRNAs followed by RT-qPCR for FANCI and FANCD2. Data are presented as the fold enrichment of MTDH-bound FANCI and FANCD2 mRNA following immunoprecipitation with anti-Flag relative to IgG. Error bars represent the relative rate of enrichment of FANCD2 and FANCI from three independent experiments, P<0.01.

Article Snippet: Anti-FANCD2 (1:2000, #NB 200–182) and anti-FANCI (1:500, #ab74332) were obtained from Novus Biologicals (Centennial, CO, USA).

Techniques: Construct, RNA Binding Assay, Western Blot, Immunoprecipitation, Quantitative RT-PCR, Relative Rate

Journal: Gynecologic oncology

Article Title: MTDH/AEG-1 downregulation using pristimerin-loaded nanoparticles inhibits Fanconi anemia proteins and increases sensitivity to platinum-based chemotherapy

doi: 10.1016/j.ygyno.2019.08.014

Figure Lengend Snippet: (A) Viability of Hec50, MDA-MB-231 and KLE cells after treatment with pristimerin for 72 h was determined using WST-1 assay. (B-D) Viability of Hec50 (B), MDA-MB-231 (C) and KLE (D) cells after treatment with cisplatin alone or in combination with the indicated dose of pristimerin for 72 h was determined using WST-1 assay, *P<0.05, ***P<0.001, ****P<0.0001 by 2-way ANOVA. (E) Pristimerin decreased MTDH, FANCD2 and FANCI protein levels when used as a single drug (1µM) or in combination with cisplatin (5µM) in Hec50, MDA-MB-231 and KLE cell lines. CT: untreated control; C+P: cisplatin + pristimerin. (F) Comparison of the effect of pristimerin in solution(1µM) and loaded into nanoparticles (NP) (40µM) on expression of MTDH, FANCD2 and FANCI and induction of the apoptotic marker cleaved caspase 3 and the autophagy marker LC3 in Hec50, MDA-MB-231 and KLE cells. β-actin: loading control; CT: untreated. (G) Quantification of Western blots in (E). (H)Quantification of Western blots in (F) With the exception of cleaved caspase 3 and LC3B, all data are relative to control. For cleaved caspase 3 and LC3B data are relative to pristimerin in solution. All data are representative of three independent experiments.

Article Snippet: Anti-FANCD2 (1:2000, #NB 200–182) and anti-FANCI (1:500, #ab74332) were obtained from Novus Biologicals (Centennial, CO, USA).

Techniques: WST-1 Assay, Expressing, Marker, Western Blot

Journal: PLoS ONE

Article Title: Ouabain, a Cardiac Glycoside, Inhibits the Fanconi Anemia/BRCA Pathway Activated by DNA Interstrand Cross-Linking Agents

doi: 10.1371/journal.pone.0075905

Figure Lengend Snippet: (A) The data from chemical library screening and chemical structure of ouabain. U2OS cells were pre-treated with 5 µM of each compound for 1 h prior to incubation in 200 ng/ml MMC for 24 h. Representative data from plate containing ouabain compound are shown. Each dot in the scattergram represents FANCD2 foci area per cell after treatment with each compound tested. (B) Ouabain inhibits MMC-induced FANCD2 foci formation. U2OS cells were pretreated with the indicated concentration of ouabain or curcumin for 1 h and then treated with 200 ng/ml MMC for 24 h. After incubation, the cells were fixed and processed for FANCD2 immunofluorescence and the FANCD2 foci were analyzed with an IN Cell Analyzer. Representative graphs and images from three independent experiments are shown. Values represent the means ± SEM (Student’s t -test, ***, P < 0.001; *, P < 0.05). (C) U2OS cells treated as described in (B) were analyzed with an IN Cell Analyzer. % foci total area/ cell represents relative % to MMC-only treated samples. IC 50 was calculated using GraphPad Prism version 5.03. Graphs and values represent the means ± SEM from three independent experiments. (D) Ouabain inhibits MMC-induced FANCD2 monoubiquitination. Protein extracts fromU2OS cells treated as described in (B) were analyzed by Western blotting using antibodies against FANCD2 and β-actin. Values shown represent the ratio of FANCD2 (L)/FANCD2 (S). (E) Ouabain inhibits FANCD2 monoubiquitination after cisplatin treatment. Western blotting was performed with protein extracts from U2OS cells treated with 10 µM cisplatin.

Article Snippet: Antibodies against FANCD2 (NB 100-182) and FANDI (A300-212A) were purchased from NOVUS (Littleton, CO) and Bethyl Laboratories (Montgomery, TX), respectively.

Techniques: Library Screening, Incubation, Concentration Assay, Immunofluorescence, Western Blot

Journal: PLoS ONE

Article Title: Ouabain, a Cardiac Glycoside, Inhibits the Fanconi Anemia/BRCA Pathway Activated by DNA Interstrand Cross-Linking Agents

doi: 10.1371/journal.pone.0075905

Figure Lengend Snippet: (A) U2OS cells were pretreated with the indicated concentration of digitoxin or digoxin for 1 h and then incubated in 200 ng/ml MMC for 24 h. After incubation, the cells were fixed and processed for FANCD2 immunofluorescence and the FANCD2 foci were analyzed with IN Cell Analyzer. Representative graphs from three independent experiments are shown. Values represent the means ± SEM.(B) (C) Protein extracts from U2OS cells pretreated with 5 µM curcumin, 100 nM ouabain, 100 nM digitoxin and 100 nM digoxin and incubated in 200 ng/ml MMC for 24 h as described in (A) were analyzed by Western blotting using antibodies against (B) FANCD2, (C) FANCI, and β-actin. L/S values shown represent the ratio of FANCD2 (L)/FANCD2 (S) or FANCI (L)/FANCI (S).

Article Snippet: Antibodies against FANCD2 (NB 100-182) and FANDI (A300-212A) were purchased from NOVUS (Littleton, CO) and Bethyl Laboratories (Montgomery, TX), respectively.

Techniques: Concentration Assay, Incubation, Immunofluorescence, Western Blot

Journal: PLoS ONE

Article Title: Ouabain, a Cardiac Glycoside, Inhibits the Fanconi Anemia/BRCA Pathway Activated by DNA Interstrand Cross-Linking Agents

doi: 10.1371/journal.pone.0075905

Figure Lengend Snippet: (A) Protein extracts from U2OS cells treated with 100 nM ouabain for the indicated times were analyzed by Western blotting using antibodies against phosphorylated p38, p38, phosphorylated ERK, ERK, phosphorylated JNK, and JNK. (B) U2OS cells were pretreated with the indicated concentration of MAPK inhibitors for 30 min and then with 100 nM ouabain for 30 min and incubated in 200 ng/ml MMC for 24 h. The cell lysates were subjected to Western blot analysis using antibodies to FANCD2 and β-actin. PD, PD 98059 (ERK inhibitor); SP, SP 600215 (JNK inhibitor); SB, SB 203580 (p38 inhibitor).

Article Snippet: Antibodies against FANCD2 (NB 100-182) and FANDI (A300-212A) were purchased from NOVUS (Littleton, CO) and Bethyl Laboratories (Montgomery, TX), respectively.

Techniques: Western Blot, Concentration Assay, Incubation

Journal: The EMBO Journal

Article Title: Dysregulation of DNA polymerase ? recruitment to replication forks results in genomic instability

doi: 10.1038/emboj.2011.457

Figure Lengend Snippet: Increased micronuclei formation caused by USP1 depletion is Polκ dependent. (A, B) U2OS cells were transfected with the indicated siRNAs and assayed for micronucleation (n=600). Western blots were performed with cells from the same transfection to demonstrate efficiency of siRNA knockdown and probed with the indicated antibodies. (C) U2OS cells were transfected with the indicated siRNAs and assayed for micronucleation and western blot analysis. Hydroxyurea (HU) treatment was used as a control for DNA damage-induced monoubiquitination of FANCI and FANCD2 to validate the functional knockdown of the FA pathway. (D) Patient-derived FANCA-deficient (GM6914) fibroblasts retrovirally complemented with either vector or wild-type FANCA cDNA were treated with the indicated siRNAs and analysed by western blot or assayed for micronucleation. Graph displays percentage (%) of cells with micronuclei. Error bars represent standard deviation of experiment done in triplicate (n=300). Single asterisk represents P-value <0.05, double asterisks represent P-value<0.01.

Article Snippet: Cells were blocked with PBS containing 1% BSA, 0.2% Triton X-100, and incubated with anti-FANCD2 (1:400, NB 100-182 Novus) for 2 h. GFP TLS Pol nuclear foci formation was detected after methanol fixation and stained with anti-GFP (B-2, Santa Cruz Biotechnology) for 2 h. Micronuclei were measured in cells treated with cytochalasin-B (2 μg ml −1 ) (Sigma) for 24 h before fixation with methanol.

Techniques: Transfection, Western Blot, Functional Assay, Derivative Assay, Plasmid Preparation, Standard Deviation