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: 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