Journal: Human molecular genetics

Article Title: Loss of CHK1 function impedes DNA damage-induced FANCD2 monoubiquitination but normalizes the abnormal G2 arrest in Fanconi anemia.

doi: 10.1093/hmg/ddm340

Figure Lengend Snippet: Figure 3. CHK1 inhibition affects both the basal and the DNA damage-induced FANCD2 monoubiquitination. (A) HeLa cells transfected with siRNA targeting CHK1 or Luciferase (Luc) were treated with 20 J/m2 UVC or 10 mM 8-MOPþ10 kJ/m2 UVA 72 h after transfection and harvested at the indicated times. Phos- phorylation of several ATR targets was analyzed by western blotting with the indicated antibodies. (B) HeLa cells were transfected with siRNA targeting ATR, CHK1, both or Luciferase. Cells were lysed 72 h after transfection, and FANCD2 monoubiquitination and NBS1 phosphorylation were assessed by western blot. Asterisk indicates the remaining signal of CHK1 just below b-tubulin signal. (C) FANCD2 monoubiquitination was monitored by western blot in extracts from HeLa cells transfected and treated as in (A). (D) HeLa cells were pretreated with either 5 mM CHK1 inhibitor sb-218078 or solvent (DMSO) for 1 h and then exposed to 5 mM HU or 10 mM 8-MOPþ10 kJ/m2 UVA (8M). Cells were cultivated with or without CHK1 inhibitor until lysis 5 h later. FANCD2 ubiquitination was monitored by western blot. (E) HeLa cells were pretreated with either 300 nM CHK1 inhibitor UCN-01 or solvent (DMSO) for 1 h and then exposed to 2 mM HU or 20 J/m2 UVC. Cells were cultivated with or without CHK1 inhibitor until lysis 6 h later. FANCD2 ubiquitination and NBS1 phosphorylation were ana- lyzed by western blot. Where indicated, L/S represents the ratio of monoubiquitinated (L) to non-monoubiquitinated (S) FANCD2. Fold induction (FI) of the L/S ratio of the treated samples relative to the non-treated for each siRNA or drug is also indicated.

Article Snippet: The following primary antibodies were used and incubated in 5% non-fat milk PBS-T (or 5% BSA in TBS-T for antibodies from Cell Signaling) overnight at 48C: FANCD2, ATR, RAD1, HUS1, RAD17, CHK1, ACTIN, NBS1 (Santa Cruz Biotechnology), FANCD2, RAD9, RPA32, p343NBS1, SMC1, p966SMC1, H2AX (Abcam), RAD9 (Stratagene), p645RAD17, p345CHK1 (Cell Signaling), RPA70 (Oncogene), CLASPIN (Bethyl), NBS1 D ow nloaded from https://academ ic.oup.com /hm g/article/17/5/679/587141 by guest on 11 June 2024 (Calbiochem), ORC2 (BD Biosciences) and gH2AX (Upstate).

Techniques: Inhibition, Transfection, Luciferase, Western Blot, Phospho-proteomics, Solvent, Lysis, Ubiquitin Proteomics

Journal: Human molecular genetics

Article Title: Loss of CHK1 function impedes DNA damage-induced FANCD2 monoubiquitination but normalizes the abnormal G2 arrest in Fanconi anemia.

doi: 10.1093/hmg/ddm340

Figure Lengend Snippet: Figure 5. The ATR/CHK1 axis regulates MMC-induced G2 arrest in FANC-pathway-deficient cells. (A) MRC5 (WT) and FA-A PD220 fibroblasts (FA-A) were transfected in six-well plates with siRNA targeting Luciferase (Luc) or CHK1. Forty-eight hours later, cells were detached, re-suspended in fresh medium and seeded in 60 mm dishes. The day after, cells were treated with 100 ng/ml mitomycin C (MMC) for 2 h and then extensively washed with PBS. Cell cycle profiles were analyzed 24 h after MMC treatment by FACS using propidium iodide staining to visualized DNA content. The percentage of cells with a 4N DNA content (G2) is reported. Right panel, western blot showing the knock-down of CHK1 and subsequent ATR activation as revealed by NBS1 phosphorylation in MRC5 and FA-A fibroblasts. (B) Exponentially growing HSC536N (FA-C) and HSC536N-corrected (FA-CþFANCC) lymphoblasts were treated with 50 ng/ml MMC for 2 h, washed twice in PBS and incubated with 1 mM CHK1 inhibitor sb-218078 (CHK1i) or an equivalent volume of solvent (DMSO). Twenty-four hours later, cells were fixed, and cell cycle was analyzed as in (A). Right part, western blotting revealing CHK1 phosphorylation on S345 in FA-C or FA-CþFANCC lym- phoblasts 24 h after exposure to 50 ng/ml MMC for 2 h. (C) FA-A PD220 fibroblasts were transfected with siRNA against Luciferase, ATR, CHK1, CLASPIN or RAD17 as indicated, treated with 100 ng/ml MMC for 2 h and analyzed as in (A). Right panel, western blot revealing the levels of depleted proteins as well as CHK1 phosphorylation on S345. (D) Growth inhibition test on HSC536N (FA-C) and HSC536N-corrected (FA-CþFANCC) lymphoblasts treated with 5 ng/ml MMC, 1 mM caffeine and 500 nM sb-218078 alone or in combination for 4 days of culture as described in Material and Methods. Values represent the means+ standard variations from three independent experiments.

Article Snippet: The following primary antibodies were used and incubated in 5% non-fat milk PBS-T (or 5% BSA in TBS-T for antibodies from Cell Signaling) overnight at 48C: FANCD2, ATR, RAD1, HUS1, RAD17, CHK1, ACTIN, NBS1 (Santa Cruz Biotechnology), FANCD2, RAD9, RPA32, p343NBS1, SMC1, p966SMC1, H2AX (Abcam), RAD9 (Stratagene), p645RAD17, p345CHK1 (Cell Signaling), RPA70 (Oncogene), CLASPIN (Bethyl), NBS1 D ow nloaded from https://academ ic.oup.com /hm g/article/17/5/679/587141 by guest on 11 June 2024 (Calbiochem), ORC2 (BD Biosciences) and gH2AX (Upstate).

Techniques: Transfection, Luciferase, Staining, Western Blot, Knockdown, Activation Assay, Phospho-proteomics, Incubation, Solvent, Inhibition

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: 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: Human Molecular Genetics

Article Title: Functional cross talk between the Fanconi anemia and ATRX/DAXX histone chaperone pathways promotes replication fork recovery

doi: 10.1093/hmg/ddz250

Figure Lengend Snippet: FANCD2 interacts with ATRX and requires ATRX to maintain protein stability. Immunoprecipitation (IP) experiments were performed from nuclear extracts (NEs) of PD20 + 3xFLAG-FANCD2 (wild-type) cells. (A) FANCD2 co-IPs with ATRX. NEs were either untreated or treated with 2 mm HU for 24 h (lanes 1 and 2) and subjected to IP with rabbit IgG (lane 3) or an anti-ATRX antibody (lanes 4 and 5). NE and IP samples were analyzed for the presence of ATRX, FANCD2 and DAXX (positive co-IP control for ATRX). (B) ATRX co-IPs with FANCD2. NEs (lane 1) were subjected to IP with mouse IgG (lane 2) or an anti-FLAG antibody (lane 3). NE and IP samples were analyzed for the presence of ATRX, FANCD2 and CtIP (positive co-IP control for FANCD2). (C–E) ATRX protects FANCD2 protein stability. (C) WCEs were prepared from PD20 + D2 cells that had been treated with control siRNA (lanes 1 and 3) or ATRX-#1 siRNA (lanes 2 and 4) for the indicated time points and analyzed for the presence of ATRX and FANCD2. Ku-80, loading control. (D) WCEs were prepared from human hTERT-RPE1 cells that had been treated with control siRNA (lanes 1 and 3) or ATRX-#1 siRNA (lanes 2 and 4) for 72 h. At 67 h, cells were additionally supplemented with DMSO (lanes 1 and 2) or 10 μm MG132 (lanes 3 and 4). WCEs were then analyzed for ATRX and FANCD2. Ku-80, loading control. Immunoblot signals for FANCD2 were analyzed by densitometry and normalized against Ku80 signals using ImageJ. The relative FANCD2 protein level values are provided underneath each corresponding lane.

Article Snippet: After blocking in 5% milk, the membranes were incubated with the following primary antibodies: FANCD2 (1:1000), ATRX (1:2000), NBS1 (1:1500), Ku80 (1:5000) and tubulin (1:5000), A horseradish peroxidase-conjugated rabbit secondary antibody (Jackson Laboratories) or a mouse secondary antibody (Bio-Rad) was used at a dilution of 1:10000 or 1:3000, respectively.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Control, Western Blot

Journal: Human Molecular Genetics

Article Title: Functional cross talk between the Fanconi anemia and ATRX/DAXX histone chaperone pathways promotes replication fork recovery

doi: 10.1093/hmg/ddz250

Figure Lengend Snippet: FANCD2 and ATRX can support normal cell proliferation independently, but cooperate to promote cellular HU resistance. (A) Analysis of ATRX and FANCD2 protein expressions in HCT116 WT, FANCD2−/−, ATRX−/0 and AD2DKO cells. WCEs were prepared in the absence or presence of HU and analyzed for FANCD2 and ATRX. Tubulin, loading control. (B) The absence of ATRX and FANCD2 affects cell proliferation synergistically. Proliferation rates of WT, FANCD2−/−, ATRX−/0 and AD2DKO cells were measured following plating at Days 1, 3 and 5 via an MTS assay. (C) ATRX and FANCD2 act in concert to promote HU resistance. WT, FANCD2−/−, ATRX−/0 and AD2DKO cells were untreated or treated with 50, 100 or 150 μm HU and assayed for their colony-forming ability after 10 days.

Article Snippet: After blocking in 5% milk, the membranes were incubated with the following primary antibodies: FANCD2 (1:1000), ATRX (1:2000), NBS1 (1:1500), Ku80 (1:5000) and tubulin (1:5000), A horseradish peroxidase-conjugated rabbit secondary antibody (Jackson Laboratories) or a mouse secondary antibody (Bio-Rad) was used at a dilution of 1:10000 or 1:3000, respectively.

Techniques: Control, MTS Assay

Journal: Human Molecular Genetics

Article Title: Functional cross talk between the Fanconi anemia and ATRX/DAXX histone chaperone pathways promotes replication fork recovery

doi: 10.1093/hmg/ddz250

Figure Lengend Snippet: ATRX acts in concert with FANCD2 and the MRN exonuclease activity to mediate replication fork restart and suppression of new origin firing. (A) Schematic of the replication restart protocol with representative images of DNA fibers. Red tracks: DigU; green tracks: BioU. (B) ATRX and FANCD2 cooperate to mediate replication fork restart. Replication fork restart efficiencies were compared between HCT116 WT, FANCD2−/−, ATRX−/0 and AD2DKO cells. Replication restart efficiency was measured as the number of restarted forks after HU-mediated fork stalling (DigU → BioU tracts), in contrast with the total number of DigU-labeled tracts (DigU plus DigU → BioU). (C) ATRX and FANCD2 act in concert to suppress new origin firing during replication blockade. The fraction of new sites of replication originating during the 40 min recovery period after HU treatment was compared between WT, FANCD2−/−, ATRX−/0 and AD2DKO cells. Fractions were measured as the number of green-only (BioU) tracts in contrast with the total number of spreading replication tracts (BioU plus DigU→BioU). [N.B. The same DNA fiber assay analysis described in Fig. 3B and C for analyzing replication fork restart and new origin firing following HU treatment was used throughout this study.] (D) ATRX and FANCD2 cooperate during replication fork restart in an MRN exonuclease-dependent manner. Replication fork restart efficiencies were compared between in HCT116 WT, FANCD2−/−, ATRX−/0 and AD2DKO cells that had additionally been either untreated or supplemented with 50 μm mirin. (E) ATRX and FANCD2 cooperate with the MRN exonuclease to suppress new origin firing during replication blockade. The number of new replication sites originating during BioU labeling after HU treatment was compared between WT, FANCD2−/−, ATRX−/0 and AD2DKO cells that had additionally been either untreated or treated with 50 μm mirin.

Article Snippet: After blocking in 5% milk, the membranes were incubated with the following primary antibodies: FANCD2 (1:1000), ATRX (1:2000), NBS1 (1:1500), Ku80 (1:5000) and tubulin (1:5000), A horseradish peroxidase-conjugated rabbit secondary antibody (Jackson Laboratories) or a mouse secondary antibody (Bio-Rad) was used at a dilution of 1:10000 or 1:3000, respectively.

Techniques: Activity Assay, Labeling

Journal: Human Molecular Genetics

Article Title: Functional cross talk between the Fanconi anemia and ATRX/DAXX histone chaperone pathways promotes replication fork recovery

doi: 10.1093/hmg/ddz250

Figure Lengend Snippet: FANCD2 and ATRX function to recruit CtIP to stalled replication forks and cooperate to promote efficient HR repair. (A and B) ATRX and FANCD2 promote CtIP foci formation at HU-stalled replication forks. HCT116 WT, FANCD2−/−, ATRX−/0 and AD2DKO cells were untreated or treated with 2 mm HU for 20 h and cellular nuclei were analyzed for the presence of CtIP foci. Nuclei with > 5 foci were considered positive for CtIP foci. (A) Representative images; (B) graphic depiction of the calculated CtIP foci numbers. (C–E) ATRX and FANCD2 cooperate to promote HR-mediated DNA DSB repair in two different cell lines. (C) A plasmid-based GFP-HDR reporter assay was used to determine HDR efficiencies in HCT116 WT, FANCD2−/−, ATRX−/0 and AD2DKO cells. In this assay, I-SceI restriction enzyme digestion creates a DSB in the HR reporter plasmid (DR-GFP). HDR repair of the DSB restores GFP expression. The repair efficiency was determined by dual GFP and mCherry-positive cells divided by the mCherry-positive cells. Results were averaged from a minimum of three replicates and normalized to the average repair efficiency of the WT cells. (D) Upper panel: HEK293T cells harboring a chromosomally integrated GFP-HDR reporter system were treated with control siRNA (lane 1), siATRX (lane 2) siFANCD2 (lane 3) or siATRX/siFANCD2 (lane 4) and analyzed for the presence of ATRX and FANCD2 by WB. Actin, loading control. Lower panel: The HEK 293T cells were treated with either control siRNA (lane 1) or siRAD51 (right lane 2), and analyzed for the presence of RAD51 by WB. (E) Following siRNA treatment of the HEK293T cells (see D), HDR was initiated by induction of DSB following transient I-SceI expression. The repair efficiency was measured by dual GFP and dsRED-positive cells divided by the dsRED-positive cells. Results were averaged from three independent experiments and normalized to the average repair efficiency of the WT cells. siRAD51-treated cells were used as a control.

Article Snippet: After blocking in 5% milk, the membranes were incubated with the following primary antibodies: FANCD2 (1:1000), ATRX (1:2000), NBS1 (1:1500), Ku80 (1:5000) and tubulin (1:5000), A horseradish peroxidase-conjugated rabbit secondary antibody (Jackson Laboratories) or a mouse secondary antibody (Bio-Rad) was used at a dilution of 1:10000 or 1:3000, respectively.

Techniques: Plasmid Preparation, Reporter Assay, Expressing, Control

Journal: Human Molecular Genetics

Article Title: Functional cross talk between the Fanconi anemia and ATRX/DAXX histone chaperone pathways promotes replication fork recovery

doi: 10.1093/hmg/ddz250

Figure Lengend Snippet: FANCD2 cooperates with DAXX to mediate replication fork restart. (A) Efficiency of siRNA-mediated DAXX knockdown. WCEs were prepared from human FANCD2-proficient cells (PD20 + D2, WT; lanes 1 and 2), or FANCD2-deficient cells (PD20, lanes 3 and 4) that had been untreated or treated with DAXX siRNA (siDAXX) and analyzed by WB for FANCD2 and DAXX. Ku80, loading control. (B) FANCD2 and DAXX cooperate to mediate replication fork restart. Replication fork restart efficiencies were compared between WT, FANCD2-, DAXX- or FANCD2/DAXX double-deficient cells. (C) FANCD2 and DAXX act in concert to suppress new origin firing during replication blockade. The number of new replication sites originating during BioU labeling after HU treatment was compared between WT, FANCD2-, DAXX- or FANCD2/DAXX double-deficient cells.

Article Snippet: After blocking in 5% milk, the membranes were incubated with the following primary antibodies: FANCD2 (1:1000), ATRX (1:2000), NBS1 (1:1500), Ku80 (1:5000) and tubulin (1:5000), A horseradish peroxidase-conjugated rabbit secondary antibody (Jackson Laboratories) or a mouse secondary antibody (Bio-Rad) was used at a dilution of 1:10000 or 1:3000, respectively.

Techniques: Knockdown, Control, Labeling

Journal: Human Molecular Genetics

Article Title: Functional cross talk between the Fanconi anemia and ATRX/DAXX histone chaperone pathways promotes replication fork recovery

doi: 10.1093/hmg/ddz250

Figure Lengend Snippet: FANCD2’s histone H3 chaperone activity is required to mediate replication fork restart. (A) FANCD2 protein expression in different cell clones. WCEs were prepared from HCT116 WT and D2−/− cells, as well as from several complemented D2−/− + D2WT (#3, 18, 20) and D2−/− + D2L231R (#5, 7, 8) clones, and analyzed for FANCD2 protein levels by WB. Tubulin, loading control. (B) Chromatin-binding efficiencies of D2L231R versus D2WT. HCT116-derived D2−/− + D2WT (clone #3), D2−/− + D2L231R (clone #8) and FANCL−/− cells were either untreated or HU-treated as indicated. Chromatin fractions were prepared from the cells and analyzed for the presence of FANCD2 by WB. TFIIH loading control. Immunoblot signals for chromatin-bound FANCD2WT (lanes 1 and 2) and FANCD2L231R (lanes 3 and 4) were analyzed by densitometry and normalized against TFIIH signals using ImageJ. Relative chromatin-bound FANCD2 levels: lane 1, 1.0; lane 2, 1.0; lane 3, 1.7; lane 4, 1.2. (C) Replication fork restart requires the FANCD2 histone chaperone activity. Replication fork restart efficiencies were compared between WT, FANCD2−/−, FANCD2−/− + D2WT, D2−/− + D2L231R and L−/− cells. (D) The FANCD2 histone chaperone activity is required to suppress new origin firing during replication blockade. The number of new replication sites originating during BioU labeling after HU treatment was compared between WT, FANCD2−/−, FANCD2−/− + D2WT, D2−/− + D2L231R and L−/− cells.

Article Snippet: After blocking in 5% milk, the membranes were incubated with the following primary antibodies: FANCD2 (1:1000), ATRX (1:2000), NBS1 (1:1500), Ku80 (1:5000) and tubulin (1:5000), A horseradish peroxidase-conjugated rabbit secondary antibody (Jackson Laboratories) or a mouse secondary antibody (Bio-Rad) was used at a dilution of 1:10000 or 1:3000, respectively.

Techniques: Activity Assay, Expressing, Clone Assay, Control, Binding Assay, Derivative Assay, Western Blot, Labeling

Journal: G3: Genes|Genomes|Genetics

Article Title: Conserved function of Drosophila Fancd2 monoubiquitination in response to double-strand DNA breaks

doi: 10.1093/g3journal/jkac129

Figure Lengend Snippet: Fancd2 genetic reagents and their impact on viability with and without X-irradiation. a) Diagram of the protein level changes on fancd2 for each indicated mutant. The horizontal line represents the fancd2 protein. Box = point mutant, stop = truncated protein. b) The frequency of eclosed homozygotes of the indicated genotypes, from crosses with heterozygous animals. From multiple independent replicates, a total of at least 60 animals were examined per genotype. Control genotype was derived from a cross between the strain w 1118 and Ly/TM3 , and for the experiment TM3 (non-Ly) animals were crossed to each other. * = significantly different from control, P < 0.01, Chi-squared test. c) The frequency of eclosed homozygotes of the indicated genotypes, as for panel (b), from crosses heterozygous animals where F1 progeny were irradiated (15 Gy X-ray). From multiple independent replicates, a total of at least 100 animals were irradiated per genotype. Combined number of surviving heterozygotes in each condition from all replicates (from left to right on graph): 68, 33, 7, 11, 8, 38. A “0” indicates no homozygotes survived to adulthood in any replicate. * = significantly different from control, P < 0.01, Chi-squared test. d) The relative frequency of homozygous animal survival for each indicated genotype. Data for each genotype represent the ratio of the value for each genotype in panel (c) divided by the same value in panel (b). e) Schematics of GFP-fancd2 transgenic constructs with WT monoubiquitination reside and the K595R point mutation. f) Quantification of the % survival of flies that eclose as adults in fancd2 K595R mutants with and without GFP-fancd2 following 20 Gy X-ray larval irradiation.

Article Snippet: GFP-fancd2 and GFP-fancd2 K595R were synthesized and cloned into the pBID vector (Addgene) by Twist Biosciences (twistbioscience.com).

Techniques: Irradiation, Mutagenesis, Control, Derivative Assay, Transgenic Assay, Construct

Journal: G3: Genes|Genomes|Genetics

Article Title: Conserved function of Drosophila Fancd2 monoubiquitination in response to double-strand DNA breaks

doi: 10.1093/g3journal/jkac129

Figure Lengend Snippet: Papillar cell survival after DSB induction in fancd2 mutants. a) Representative image of an adult rectum of control animals +/− I-Cre. Papillar cells (pseudo-colored), green; DNA (DAPI), magenta. (DNA). Scale bars = 50 μm. b) Representative image of an adult rectum of fancd2 K595R (line # 4-3) animals +/− hs-I-CreI . Labeling as in (a). c) Representative image of an adult rectum of fancd2 A623E/D644E (line #5- 2) animals +/− hs-I-CreI . Labeling as in (a). d) Representative images of adult rectums of fancd2 deletion animals +/− hs-I-CreI. Labeling as in (a). e) Representative images of adult rectums of fancd2 deletion animals +/− hs-I-CreI + GFP-fancd2 . f) Quantification of adult papillar cell number in fancd2 513ΔC animals +/− hs - I-CreI with and without GFP-fancd2 . Red dashed line = expected number of papillar cells in a WT adult. Each condition has at least 2 biological replicates. Each data point represents a single animal ( N ). Statistical test: ordinary 1-way ANOVA. See Materials and methods for statistical notations.

Article Snippet: GFP-fancd2 and GFP-fancd2 K595R were synthesized and cloned into the pBID vector (Addgene) by Twist Biosciences (twistbioscience.com).

Techniques: Control, Labeling

Journal: G3: Genes|Genomes|Genetics

Article Title: Conserved function of Drosophila Fancd2 monoubiquitination in response to double-strand DNA breaks

doi: 10.1093/g3journal/jkac129

Figure Lengend Snippet: GFP-Fancd2 localization in tissues. a) Representative GFP-Fancd2 expression in the adult midgut. Enterocytes are outlined with white hatched lines. DAPI, magenta, Fancd2-GFP, green. Scale bars = 10 μm. b) Representative GFP-Fancd2 expression in the wandering third instar (L3) brain. Neuroblasts and ganglion mother cells are outlined with white hatched lines. All other labeling as in (a). c) Representative GFP-Fancd2 expression in the L3 rectum. Papillar cells are outlined with white hatched lines. All other labeling as in (a). d) Representative GFP-Fancd2 (WT and K595R) expression in the mitotic stage pupal rectum ( Fox et al. 2010 ) after IR, as well as WT GFP-Fancd2 no IR control. Papillar cells are outlined with white hatched lines. GFP-Fancd2, gray. Yellow arrows = Fancd2 + foci. Hatched box = area magnified 10× in the corresponding inset below each panel. White arrowhead = micronucleus. Scale bars = 10 μm.

Article Snippet: GFP-fancd2 and GFP-fancd2 K595R were synthesized and cloned into the pBID vector (Addgene) by Twist Biosciences (twistbioscience.com).

Techniques: Expressing, Labeling, Control

Journal: G3: Genes|Genomes|Genetics

Article Title: Conserved function of Drosophila Fancd2 monoubiquitination in response to double-strand DNA breaks

doi: 10.1093/g3journal/jkac129

Figure Lengend Snippet: Fancd2 Lysine 595 is required for Mre11 foci removal during mitosis. a) Mre11 and gH2Av recruitment to fancd2 K595R papillar cells +/− hs - I-CreI . DNA (DAPI), magenta; Mre11, green; gH2Av, red. Yellow arrows = Mre11 + gH2Av + foci. Hatched box = area magnified 10× in the corresponding inset below each panel. White arrowhead = micronucleus. Hatched outline = nuclei. Scale bars = 10 μm. NEBD = Nuclear Envelope Break Down. b) Quantification of Mre11+ gH2Av+ foci recruitment in fancd2 K595R papillar cells + hs - I-CreI pre- and post- the first pupal mitotic division of the pupal stage (first mitotic stage). Focus index= average number of foci/cell in papillar cells of a given animal. Each data point represents a single animal ( N ). Statistical test: Ordinary 1-way ANOVA. See Materials and methods for statistical notations.

Article Snippet: GFP-fancd2 and GFP-fancd2 K595R were synthesized and cloned into the pBID vector (Addgene) by Twist Biosciences (twistbioscience.com).

Techniques: