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: International Journal of Clinical and Experimental Medicine

Article Title: p53 mediated apoptosis in osteosarcoma MG-63 cells by inhibition of FANCD2 gene expression

doi:

Figure Lengend Snippet: FANCD2 protein expression in MG-63 cells after RNAi. Lane 1: Control; Lane 2: siRNA-Control; Lane 3: siRNA-FANCD2 24 h; Lane 4: siRNA-FANCD2 48 h.

Article Snippet: Construction and transfection of the FANCD2 siRNA in MG-63 cells siRNA-FANCD2 and a control siRNA plasmid were designed and synthesized by Santa Cruz Biotechnology, Inc. (Texas, USA).

Techniques: Expressing, Control

Journal: International Journal of Clinical and Experimental Medicine

Article Title: p53 mediated apoptosis in osteosarcoma MG-63 cells by inhibition of FANCD2 gene expression

doi:

Figure Lengend Snippet: Absorbance of MG-63 cells after siRNA-FANCD2 interfere by CCK-8 assay ( x ̅ ±s, n = 6)

Article Snippet: Construction and transfection of the FANCD2 siRNA in MG-63 cells siRNA-FANCD2 and a control siRNA plasmid were designed and synthesized by Santa Cruz Biotechnology, Inc. (Texas, USA).

Techniques: CCK-8 Assay, Control

Journal: International Journal of Clinical and Experimental Medicine

Article Title: p53 mediated apoptosis in osteosarcoma MG-63 cells by inhibition of FANCD2 gene expression

doi:

Figure Lengend Snippet: Cell cycle distribution of MG-63 cells after siRNA-FANCD2 interfere ( x ̅ ±s, n = 4)

Article Snippet: Construction and transfection of the FANCD2 siRNA in MG-63 cells siRNA-FANCD2 and a control siRNA plasmid were designed and synthesized by Santa Cruz Biotechnology, Inc. (Texas, USA).

Techniques: Control

Journal: International Journal of Clinical and Experimental Medicine

Article Title: p53 mediated apoptosis in osteosarcoma MG-63 cells by inhibition of FANCD2 gene expression

doi:

Figure Lengend Snippet: Apoptotic percentages of MG-63 cells after FANCD2 siRNA interfere by Flow Cytometry ( x ̅ ±s, n = 4)

Article Snippet: Construction and transfection of the FANCD2 siRNA in MG-63 cells siRNA-FANCD2 and a control siRNA plasmid were designed and synthesized by Santa Cruz Biotechnology, Inc. (Texas, USA).

Techniques: Flow Cytometry, Control

Journal: International Journal of Clinical and Experimental Medicine

Article Title: p53 mediated apoptosis in osteosarcoma MG-63 cells by inhibition of FANCD2 gene expression

doi:

Figure Lengend Snippet: Relative expression of mRNAs in MG-63 cells after FANCD2 siRNA interfere ( x ̅ ±s, n = 3)

Article Snippet: Construction and transfection of the FANCD2 siRNA in MG-63 cells siRNA-FANCD2 and a control siRNA plasmid were designed and synthesized by Santa Cruz Biotechnology, Inc. (Texas, USA).

Techniques: Expressing, Control

Journal: International Journal of Clinical and Experimental Medicine

Article Title: p53 mediated apoptosis in osteosarcoma MG-63 cells by inhibition of FANCD2 gene expression

doi:

Figure Lengend Snippet: Western blotting picture of p53, phos-p53, p21, TP53INP1, cleaved caspase-9 and-3 protein expression after RNAi. Lane 1: Control; Lane 2: siRNA-Control; Lane 3: siRNA-FANCD2 24 h; Lane 4: siRNA-FANCD2 48 h.

Article Snippet: Construction and transfection of the FANCD2 siRNA in MG-63 cells siRNA-FANCD2 and a control siRNA plasmid were designed and synthesized by Santa Cruz Biotechnology, Inc. (Texas, USA).

Techniques: Western Blot, Expressing, Control

Journal: Apoptosis : an international journal on programmed cell death

Article Title: Crosstalk of ferroptosis regulators and tumor immunity in pancreatic adenocarcinoma: novel perspective to mRNA vaccines and personalized immunotherapy.

doi: 10.1007/s10495-023-01868-8

Figure Lengend Snippet: Fig. 6 Correlation analysis between FANCD2 expression and immune cell infiltration in PAAD. A–F The correlation of FANCD2 expression and immune infiltration cells. *P < 0.05, **P < 0.01, and ***P < 0.001

Article Snippet: Regents as following: FANCD2: Santa Cruz Biotechnology Cat#sc-20022, RRID:AB_2278211, dilution 1:10.

Techniques: Expressing

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: Alcoholism, clinical and experimental research

Article Title: Alcohol metabolism in human cells causes DNA damage and activates the Fanconi anemia-breast cancer susceptibility (FA-BRCA) DNA damage response network.

doi: 10.1111/j.1530-0277.2011.01563.x

Figure Lengend Snippet: Fig. 3. Ethanol exposure of HeLa-ADH1B cells stimulates FANCD2 monoubiquitination. (A) Cells were exposed to 20 mM ethanol or 2 mM hydroxyurea (HU) and whole cell extracts probed with anti-FANCD2. The positions of the long (L) and short (S) forms of FANCD2 are indicated. The ratio of the L and S forms of FANCD2 was determined using the NIH ImageJ program and results shown at the right. (B) Cells were exposed to media alone, 20 mM ethanol, or 20 mM ethanol in the presence of different concentrations of 4-methyl pyrazole (4-MP), and extracts were analyzed for FANCD2 monoubiquitination. Quantification is shown at the right.

Article Snippet: Blots were probed with antibodies against FANCD2 (Novus Biologicals, Littleton, CO), BRCA1, or BRCA1 pSer 1524 (both from Bethyl Laboratories, Montgomery, TX).

Techniques:

Journal: Alcoholism, clinical and experimental research

Article Title: Alcohol metabolism in human cells causes DNA damage and activates the Fanconi anemia-breast cancer susceptibility (FA-BRCA) DNA damage response network.

doi: 10.1111/j.1530-0277.2011.01563.x

Figure Lengend Snippet: Fig. 5. FANCD2 monoubiquitination and BRCA1 phosphorylation following ethanol exposure in HeLa-ADH1B and HeLa-ADH1B-ALDH2 cells. Increased FANCD2 monoubiquitination is seen in both HeLa-ADH1B cells and HeLa-ADH1B-ALDH2 cells after ethanol exposure, whereas ethanol increased BRCA1 phosphorylation at Ser 1524 only in HeLa-ADH1B cells.

Article Snippet: Blots were probed with antibodies against FANCD2 (Novus Biologicals, Littleton, CO), BRCA1, or BRCA1 pSer 1524 (both from Bethyl Laboratories, Montgomery, TX).

Techniques: Phospho-proteomics

Journal: bioRxiv

Article Title: RNA transcripts suppress G-quadruplex structures through G-loop formation

doi: 10.1101/2023.03.09.531892

Figure Lengend Snippet: a , pG4 BOT and pPolyT were incubated in NPE pre-treated with buffer or RNaseA, and products were analysed by ChIP-qPCR with the FANCD2 antibody and a primer pair for the G4 locus. The relative values compared to the highest signal among the conditions were plotted. b , Mock- and FANCA-depleted NPEs were analysed by Western blot with the FANCA antibody. The asterisk represents a non-specific band. c , pG4 BOT was incubated in the NPEs as described in b , and products were analysed by ChIP-qPCR as in a with XPF (i) and SLX4 (ii) antibodies using primers for the G4 locus (schematic, right). d , Mock-, XPF-, MUS81-, SLX1-, and XPA-depleted NPEs supplemented with buffer, or where indicated with the XPF-ERCC1 complex or SLX4, were analysed by Western blot with XPF, MUS81, SLX1, SLX4, and XPA antibodies (bottom). The asterisks represent non-specific bands. Schematic representation of SLX4 and its interacting nucleases is depicted (top). e , pG4 TOP was incubated in the NPEs as described in d , and products were digested with AflIII, end-labelled, separated by denaturing PAGE alongside a sequencing ladder, and visualized by autoradiography. Incised fragments (−1 to −8) are indicated with a bracket. f , Mock- and DHX36-FANCJ-depleted NPEs supplemented with buffer or wild-type DHX36 and FANCJ were analysed by Western blot with DHX36 and FANCJ antibodies. g, pG4 TOP was incubated in the NPEs as described in f, and products were analysed by denaturing PAGE as in e . h, pG4 BOT was incubated in the NPEs as described in f , and products were analysed by DRIP- qPCR using primers for the G4 locus. Relative values compared to input signals were plotted. i, pPolyT and pG4 BOT were incubated in NPE, and products were analysed by ChIP-qPCR with DHX36 (i) and FANCJ (ii) antibodies using primers for the G4 locus (schematic, right). Where indicated, NPE was pre-treated with RNase A. The relative values compared to the highest signal among the conditions were plotted.

Article Snippet: Antibodies against xl BRCA2 , xl DHX36 , xl FANCD2 , xl FANCJ , xl MUS81 , hs RAD51 , xl SLX1 , xl SLX4 , xl XPA , xl XPF and hs RNA Polymerase II subunit A (Bethyl) were previously described.

Techniques: Incubation, ChIP-qPCR, Western Blot, Sequencing, Autoradiography

Journal: Apoptosis : an international journal on programmed cell death

Article Title: Crosstalk of ferroptosis regulators and tumor immunity in pancreatic adenocarcinoma: novel perspective to mRNA vaccines and personalized immunotherapy.

doi: 10.1007/s10495-023-01868-8

Figure Lengend Snippet: Fig. 6 Correlation analysis between FANCD2 expression and immune cell infiltration in PAAD. A–F The correlation of FANCD2 expression and immune infiltration cells. *P < 0.05, **P < 0.01, and ***P < 0.001

Article Snippet: The information of antibodies was included: FANCD2 (1:1000, Proteintech, China), GAPDH (1:3000, Proteintech, China).

Techniques: Expressing

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: Cell death & disease

Article Title: FANCD2 maintains replication fork stability during misincorporation of the DNA demethylation products 5-hydroxymethyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxyuridine.

doi: 10.1038/s41419-022-04952-0

Figure Lengend Snippet: Fig. 1 5hmdC exposure induces genome instability in Fancd2−/−cells. A Cell proliferation assay of wild type, Fancd2−/−exposed to the indicated dose of 5dC, 5mdC, 5hmdC, 5fdC and 5cadC for 3 days (n = 4, mean ± s.d.). Cell proliferation assay of a lymphoblast FANCA-deficient patient-derived HSC72, NV012 cell lines and FANCD2−KO eHAP CRISPR clones exposed to the indicated doses of 5hmdC for 3 days (n = 4, mean ± s.d.). B Top left, representative PAR (red) immunofluorescence images of wild type and Fancd2−/−cells exposed to 5hmdC (10 μM) for 16 h. DAPI (blue) stains nuclear DNA. Top right, plot depicting PAR mean intensity signal per nucleus (n = 3, Mann–Whitney test; central line represents median value). Bottom left, representative γ-H2AX (green) immunofluorescence images of wild type and Fancd2−/−cells exposed to 5hmdC (10 μM) for 16 h. DAPI (blue) stains nuclear DNA. Bottom right, plot depicting γ-H2AX foci per nucleus (n = 3, Student’s t test; central line represents mean ± s.d.). C Left, representative images of chromosome aberration test (red arrowhead) from wild type and Fancd2−/−cells following 5hmdC treatment (10 μM) for 40 h. Right, bar plot of breakdown of the different types of chromosomal aberrations (n = 150 of each of 3 biological replicates, Student’s t test; bar represents mean ± s.e.m.). D Western blot of wild type and Fancd2−/−MEFs extracts to detect γ- H2AX, ser345-CHK1, total CHK1, and PCNA (loading control) after exposure to 5dC, 5mdC, 5hmdC, 5fdC or 5cadC (10 μM) for 16 h. E Bar plot depicting frequency of G1, S or G2 populations of wild type and Fancd2−/−cells exposed to 5dC, 5mdC and 5hmdC (100 μM) for 30 min and subsequently analyzed after 48 h in culture (n = 3, Student’s t test; bar represents mean ± s.d.). F Left, plot depicting γ-H2AX foci per nucleus of wild type and Fancd2−/−cells exposed to AZD7762 (2.5 nM), 5hmdC (10 μM) or combination of both for 16 h (n = 3, Mann–Whitney test; central line represents mean value). Right, as in Left but AZD7762 was substituted by UCN-01 (2.5 nM). G Bar plot of breakdown of the different types of chromosomal aberrations from wild type and Fancd2−/−cells treated with 5hmdC (10 μM), AZD7762 (2.5 nM), UCN-01 (2.5 nM) or combinations for 48 h. AZD7762 or UCN-01 were added 24 h before harvesting the cells (n = 100 of each of 2 biological replicates, Student’s t test; bar represents mean ± s.d).

Article Snippet: The antibodies used were against ser139-H2AX (Millipore, 05-636), PAR (Millipore, MABE1016), FANCD2 (Novus, NB100-316), ser345-CHK1 (Cell Signaling 2348), CHK1 (sc-8408), ERCC1 (sc-8408), PCNA (sc-56), Lamin A/C (sc-376248), BrdU (AbSerotec, OBT0030), BrdU (BD Bioscience, 347580), αtubulin (Sigma, T9026), ser4/ser8 RPA32 (Bethyl, A300-245A).

Techniques: Proliferation Assay, Derivative Assay, CRISPR, Clone Assay, MANN-WHITNEY, Western Blot, Control

Journal: Cell death & disease

Article Title: FANCD2 maintains replication fork stability during misincorporation of the DNA demethylation products 5-hydroxymethyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxyuridine.

doi: 10.1038/s41419-022-04952-0

Figure Lengend Snippet: Fig. 2 5hmdC-induced DNA damage associates to S-phase and impairs replication fork progression. A HPLC-MS/MS quantitation of endogenous and exogenous 5hmdC levels in genomic DNA samples of wild type and Fancd2−/−cells exposed to D3-labeled 5hmdC (0, 10 and 20 μM) for 16 h. Wild type cells showed a 0.49 vs 4.8 exogenous vs endogenous 5hmdC per 104 dG upon 10 μM 5hmdC-D3 exposure; 1.2 5hmdC-D3 vs 4.5 5hmdC per 104 dG for 20 μM exposure). Fancd2−/−cells showed a 3.2 5hmdC-D3 vs 5.8 5hmdC per 104 dG upon 10 μM 5hmdC-D3 exposure; 2.82 5hmdC-D3 vs 4.91 5hmdC per 104 dG upon 20 μM 5hmdC-D3 exposure. Plots represent mean values from 3 biological replicates. B Left, representative immunofluorescence of wild type and Fancd2−/−MEFs showing PAR nuclear staining and EdU positive (EdU+) cells after exposure to 5hmdC (10 μM) for 16 h (n = 3). Right, bar plot showing percentage of PAR+ cells in EdU negative (EdU−) or EdU+ cell populations (n = 250 of each of 3 independent biological replicates, Student’s t test; bars represents mean ± s.d.). C Western blot of iPOND-captured and input protein extracts from wild type and Fancd2−/−cells treated with 5hmdC (160 μM) or HU (1 mM) for 3 h to detect γ- H2AX, PCNA and RAD51. D Top left, scheme of BrdU incorporation assay. Bottom left, representative immunofluorescence images of wild type and Fancd2−/−MEFs showing BrdU + (Red) or BrdU- cells and nuclear DNA (DAPI, blue). Right, percentage of BrdU+ or BrdU−cells after HU (1 mM) or 5hmdC (40 μM) for 16 h (n = 200 of each of 3 biological replicates, Student’s t test; Bar represents mean ± s.d.). E Top, plot depicting PAR mean intensity signal per nucleus of wild type and Fancd2−/−cells exposed to 10, 40, 80 or 160 μM 5hmdC for 1 h (n = 3, Mann–Whitney test; central line represents median value). Bottom, plot depicting γ-H2AX foci per nucleus of wild type and Fancd2−/−cells exposed to 10, 40, 80 or 160 μM 5hmdC for 1 h (n = 3, Student’s t test; central line represents mean ± s.d.). F Top left, scheme of the DNA fiber assay. Bottom left, representative images of DNA fibers from wild type and Fancd2−/−MEFs after 10, 40, 80 or 160 μM 5hmdC exposure. Right, plot representing fork speed (Kbp min−1) of wild type and Fancd2−/−MEFs exposed to 10, 40, 80 or 160 μM 5hmdC (n = 150 of each of 3 biological replicates, Mann–Whitney test; central line represents median value).

Article Snippet: The antibodies used were against ser139-H2AX (Millipore, 05-636), PAR (Millipore, MABE1016), FANCD2 (Novus, NB100-316), ser345-CHK1 (Cell Signaling 2348), CHK1 (sc-8408), ERCC1 (sc-8408), PCNA (sc-56), Lamin A/C (sc-376248), BrdU (AbSerotec, OBT0030), BrdU (BD Bioscience, 347580), αtubulin (Sigma, T9026), ser4/ser8 RPA32 (Bethyl, A300-245A).

Techniques: Tandem Mass Spectroscopy, Quantitation Assay, Labeling, Staining, Western Blot, BrdU Incorporation Assay, MANN-WHITNEY

Journal: Cell death & disease

Article Title: FANCD2 maintains replication fork stability during misincorporation of the DNA demethylation products 5-hydroxymethyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxyuridine.

doi: 10.1038/s41419-022-04952-0

Figure Lengend Snippet: Fig. 4 5hmdC derived 5hmdU is responsible for the DNA damage and genomic instability in Fancd2−/−cells. A Plot representing the quantitation by HPLC-MS/MS of exogenous 5hmdU-D3 level in genomic DNA samples from wild type and Fancd2−/−cells exposed to isotopically labeled 5hmdC-D3 (0,10 and 20 μM) for 16 h. B Top, plot depicting PAR mean intensity signal per nucleus of wild type and Fancd2−/− cells exposed to 5hmdU (2.5 μM) for 16 h (n = 3, Mann–Whitney test; central line represents median value). Bottom, plot depicting γ-H2AX foci obtained from immunofluorescence images from wild type or Fancd2−/−cells exposed to 5hmdU (2.5 μM) for 16 h (n = 3, Student’s t test; central line represents mean ± s.d.). C Top, representative images of chromosome aberrations (red arrowhead) test from wild type and Fancd2−/−cells following 5hmdU treatment (1.25 and 2.5 μM) for 40 h. Bottom, bar plot of breakdown of the different types of chromosomal aberrations (n = 150 of each of 3 biological replicates, Student’s t test; bar represents mean ± s.d.). D Cell prolifera- tion assay of wild type and Fancd2−/−MEFs exposed to the indicated doses of 5hmdU for 3 days (n = 4, mean ± s.d.). E Cell proliferation assays of MDA-MB-231 (BRCA1 proficient), MDA-MB-436 (BRCA1 deficient), BRCA2+/+ and BRCA2−/−DLD-1 cell exposed to the indicated doses of 5hmdC or 5hmdU for 7 days (n = 4, mean ± s.d).

Article Snippet: The antibodies used were against ser139-H2AX (Millipore, 05-636), PAR (Millipore, MABE1016), FANCD2 (Novus, NB100-316), ser345-CHK1 (Cell Signaling 2348), CHK1 (sc-8408), ERCC1 (sc-8408), PCNA (sc-56), Lamin A/C (sc-376248), BrdU (AbSerotec, OBT0030), BrdU (BD Bioscience, 347580), αtubulin (Sigma, T9026), ser4/ser8 RPA32 (Bethyl, A300-245A).

Techniques: Derivative Assay, Quantitation Assay, Tandem Mass Spectroscopy, Labeling, MANN-WHITNEY