Journal: Oncology reports

Article Title: Ammonium tetrathiomolybdate enhances the antitumor effect of cisplatin via the suppression of ATPase copper transporting beta in head and neck squamous cell carcinoma.

doi: 10.3892/or.2019.7367

Figure Lengend Snippet: Figure 1. Expression of ATP7B in head and neck normal tissues, head and neck cancers, and human oral squamous cell lines. (A) Immunohistochemistry results of ATP7B expression in (a) head and neck normal tissue and (b) HNC. (B) Scatterplot of the ATP7B‑positive areas in the head and neck normal tissues (n=11) and HNSCC (n=70). Error bars: Mean ± SD. There was a significantly increased expression of ATP7B in the HNSCC samples (P<0.0001). (C) The expression of ATP7B in the human oral squamous cell lines (HSC‑2, ‑3, ‑4) analyzed by western blotting. HNC, head and neck cancer; HNSCC, head and neck squamous cell carcinoma; ATP7B, ATPase copper transporting beta.

Article Snippet: ATP7B (anti-rabbit, polyclonal; cat. no. NB100-360) was purchased from Novus Biologicals.

Techniques: Expressing, Immunohistochemistry, Western Blot

Journal: Oncology reports

Article Title: Ammonium tetrathiomolybdate enhances the antitumor effect of cisplatin via the suppression of ATPase copper transporting beta in head and neck squamous cell carcinoma.

doi: 10.3892/or.2019.7367

Figure Lengend Snippet: Figure 2. Effect of TM on ATP7B expression and the synergistic antitumor effect of cisplatin in HNSCC cell proliferation in vitro via cisplatin accumulation. HSC3 cells were plated in triplicate and treated with 5 µM TM for 24 h. (A) The expression of ATP7B in control HSC‑3 cells and TM‑treated HSC‑3 cells was analyzed based on the image blot density revealed by western blotting. (B) Immunofluorescence analysis of ATP7B and DAPI in the cells. Upper images, ATP7B (green); middle images, DAPI (blue); lower images, merged. Sections were incubated with rabbit anti‑ATP7B (1:100), then with Alexa Fluor 488 anti‑rabbit IgG (1:1,000) and encapsulated with DAPI. (C) The cisplatin concentration in HSC‑3 cells was evaluated after 6‑h cisplatin treatment with or without 24‑h TM pre‑incubation. (D) Antitumor and synergistic antitumor effect of TM against HSC‑3 HNSCC cells. HSC‑3 cells were treated with cisplatin with or without TM for 24 h. Live cells were counted with trypan blue reagent. Data represent the relative ratio (the control is indicated as 1.0). *P<0.05 cisplatin + TM group vs. the cisplatin treated group. TM, tetrathiomolybdate; ATP7B, ATPase copper transporting beta; HNSCC, head and neck squamous cell carcinoma.

Article Snippet: ATP7B (anti-rabbit, polyclonal; cat. no. NB100-360) was purchased from Novus Biologicals.

Techniques: Expressing, In Vitro, Control, Western Blot, Immunofluorescence, Incubation, Concentration Assay

Journal: Oncology reports

Article Title: Ammonium tetrathiomolybdate enhances the antitumor effect of cisplatin via the suppression of ATPase copper transporting beta in head and neck squamous cell carcinoma.

doi: 10.3892/or.2019.7367

Figure Lengend Snippet: Figure 6. Immunofluorescence analysis of the tumor in the bone marrow in the mouse model of bone invasion. (A, green: High magnification) KI‑67, (B, green: High magnification) ATP7B and DAPI stained with blue (n=6/group). ATP7B, ATPase copper transporting beta.

Article Snippet: ATP7B (anti-rabbit, polyclonal; cat. no. NB100-360) was purchased from Novus Biologicals.

Techniques: Immunofluorescence, Staining

Journal: Oncology reports

Article Title: Ammonium tetrathiomolybdate enhances the antitumor effect of cisplatin via the suppression of ATPase copper transporting beta in head and neck squamous cell carcinoma.

doi: 10.3892/or.2019.7367

Figure Lengend Snippet: Figure 7. The role of ATP7B in cisplatin‑resistant cancer cells. (A) The expression of ATP7B in the parental and cisplatin‑resistant A431 cancer cells (A431/CDDP‑R) was evaluated by a western blot analysis. (B) A431 and A431/CDDP‑R cells were incubated with cisplatin with or without TM. Cleaved caspase‑3 was evaluated with western blotting. TM, tetrathiomolybdate; ATP7B, ATPase copper transporting beta; CDDP, cis‑dichloro‑diamine‑plat- inum‑resistant.

Article Snippet: ATP7B (anti-rabbit, polyclonal; cat. no. NB100-360) was purchased from Novus Biologicals.

Techniques: Expressing, Western Blot, Incubation

Journal: PLoS ONE

Article Title: Novel ATPase Cu 2+ Transporting Beta Polypeptide Mutations in Chinese Families with Wilson's Disease

doi: 10.1371/journal.pone.0066526

Figure Lengend Snippet: Clinical data from the 20 patients with the ATP7B gene mutations.

Article Snippet: The blots were blocked for 1 h in 5% milk in TBST and incubated at 4°C overnight with rabbit anti- ATP7B polyclonal antibody (Novus Biologicals, Littleton, CO, USA), rabbit anti- minichromosome maintenance protein 7 (MCM7) monoclonal antibody (Millipore Biologicals, Billerica, MA, USA), rabbit anti- sterol regulatory element binding protein 1 (SREBP1) polyclonal antibody (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA), mouse anti- B-cell CLL/lymphoma 2 (BCL2) polyclonal antibody (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA), mouse anti- BCL2-associated X protein (BAX) polyclonal antibody (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA) and mouse anti-β-actin polyclonal antibody (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA).

Techniques:

Journal: PLoS ONE

Article Title: Novel ATPase Cu 2+ Transporting Beta Polypeptide Mutations in Chinese Families with Wilson's Disease

doi: 10.1371/journal.pone.0066526

Figure Lengend Snippet: Statistical differences between ATP7B siRNA transfected groups and AllStars Negative Control siRNA groups are indicated as * ( P <0.01), or untreated group are indicated as # ( P <0.01).

Article Snippet: The blots were blocked for 1 h in 5% milk in TBST and incubated at 4°C overnight with rabbit anti- ATP7B polyclonal antibody (Novus Biologicals, Littleton, CO, USA), rabbit anti- minichromosome maintenance protein 7 (MCM7) monoclonal antibody (Millipore Biologicals, Billerica, MA, USA), rabbit anti- sterol regulatory element binding protein 1 (SREBP1) polyclonal antibody (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA), mouse anti- B-cell CLL/lymphoma 2 (BCL2) polyclonal antibody (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA), mouse anti- BCL2-associated X protein (BAX) polyclonal antibody (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA) and mouse anti-β-actin polyclonal antibody (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA).

Techniques: Transfection, Negative Control

Journal: PLoS ONE

Article Title: Novel ATPase Cu 2+ Transporting Beta Polypeptide Mutations in Chinese Families with Wilson's Disease

doi: 10.1371/journal.pone.0066526

Figure Lengend Snippet: (A) Western blot analysis of ATP7B , BCL2, BAX, SREBP1 and MCM7 in HepG2 cells following treatment with targeted siRNA and AllStars Negative Control siRNA for 24, 48, and 72 h. β-actin was shown below as loading control. Protein levels of (B) ATP7B , (C) BCL2, (D) BAX, (E) SREBP1 and (F) MCM7 were quantified by densitometry and expression is shown as arbitrary units. Statistical differences for expression of the proteins between ATP7B siRNA transfected groups and AllStars Negative Control siRNA groups are indicated as * ( P <0.05), or ATP7B siRNA untreated group are indicated as # ( P <0.05).

Article Snippet: The blots were blocked for 1 h in 5% milk in TBST and incubated at 4°C overnight with rabbit anti- ATP7B polyclonal antibody (Novus Biologicals, Littleton, CO, USA), rabbit anti- minichromosome maintenance protein 7 (MCM7) monoclonal antibody (Millipore Biologicals, Billerica, MA, USA), rabbit anti- sterol regulatory element binding protein 1 (SREBP1) polyclonal antibody (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA), mouse anti- B-cell CLL/lymphoma 2 (BCL2) polyclonal antibody (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA), mouse anti- BCL2-associated X protein (BAX) polyclonal antibody (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA) and mouse anti-β-actin polyclonal antibody (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA).

Techniques: Western Blot, Negative Control, Control, Expressing, Transfection

Journal: PLoS ONE

Article Title: Novel ATPase Cu 2+ Transporting Beta Polypeptide Mutations in Chinese Families with Wilson's Disease

doi: 10.1371/journal.pone.0066526

Figure Lengend Snippet: HepG2 cells were stained with 1 μM of Hoechst 33342. Cells were treated with siRNA for 24 h and followed by copper sulfate treatment for 24 h. (A) Treated with 5 nM AllStars Negative Control siRNA. (B) Treated with 5 nM ATP7B siRNA. (C) Treated with 5 nM AllStars Negative Control siRNA+ 50 μM copper sulfate . (D) Treated with 5 nM ATP7B siRNA+ 50 μM copper sulfate. (E) Treated with 5 nM AllStars Negative Control siRNA + 200 μM copper sulfate. (F) Treated with 5 nM ATP7B siRNA+ 200 μM copper sulfate.

Article Snippet: The blots were blocked for 1 h in 5% milk in TBST and incubated at 4°C overnight with rabbit anti- ATP7B polyclonal antibody (Novus Biologicals, Littleton, CO, USA), rabbit anti- minichromosome maintenance protein 7 (MCM7) monoclonal antibody (Millipore Biologicals, Billerica, MA, USA), rabbit anti- sterol regulatory element binding protein 1 (SREBP1) polyclonal antibody (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA), mouse anti- B-cell CLL/lymphoma 2 (BCL2) polyclonal antibody (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA), mouse anti- BCL2-associated X protein (BAX) polyclonal antibody (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA) and mouse anti-β-actin polyclonal antibody (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA).

Techniques: Staining, Negative Control

Journal:

Article Title: Therapeutic Targeting of ATP7B in Ovarian Carcinoma

doi: 10.1158/1078-0432.CCR-08-2306

Figure Lengend Snippet: A, Western blot analysis of ATP7A and ATP7B in A2780-CP20 cells following treatment with targeted and control siRNA for 24, 48, and 72 h. β-Actin was used as loading control. Protein levels were quantified by densitometry and expression is shown as arbitrary units. B, effect of ATP7A and ATP7B silencing on cisplatin sensitivity in ovarian cancer cell lines. A2780-PAR and A2780-CP20 cells were transfected with ATP7A or ATP7B or control siRNA. After 24 h, cells were treated with cisplatin (0.01-32 μmol/L). Following 72 h of cisplatin exposure, MTT assay was done to determine the effects on cell viability. Columns, mean values for the IC50 of three independent experiments; bars, SE. *, P < 0.05; **, P < 0.01, compared with cisplatin alone.

Article Snippet: The blots were blocked for 1 h in 5% milk powder in TBST [10 mmol/L Tris (pH 8), 150 mmol/L NaCl, 0.05% Tween 20] and incubated at 4°C overnight with anti-ATP7A and anti-ATP7B antibodies (Novus Biologicals) at dilutions of 1:1,000 and 1:500, respectively.

Techniques: Western Blot, Control, Expressing, Transfection, MTT Assay

Journal:

Article Title: Therapeutic Targeting of ATP7B in Ovarian Carcinoma

doi: 10.1158/1078-0432.CCR-08-2306

Figure Lengend Snippet: Effect of cisplatin on intracellular localization of ATP7B. A, A2780-CP20 cells were immunostained with the anti-ATP7B and anti–syntaxin-6 antibodies. Overlay images showing colocalization (white) of ATP7B (green) and syntaxin-6 (pink). B, HepG2 and A2780-CP20 cells were treated with copper chelator BCS (50 μmol/L) to deplete cells of copper and then either kept in BCS or treated with CuCl2 or cisplatin. To verify the lack of ATP7B trafficking in response to cisplatin in A2780-CP20 cells, we evaluated colocalization between ATP7B and syntaxin using multiple images and Zeiss software package. Trafficking in response to copper in HepG2 was used as a positive control. Arrows indicate direction and length of the densitometry scan.

Article Snippet: The blots were blocked for 1 h in 5% milk powder in TBST [10 mmol/L Tris (pH 8), 150 mmol/L NaCl, 0.05% Tween 20] and incubated at 4°C overnight with anti-ATP7A and anti-ATP7B antibodies (Novus Biologicals) at dilutions of 1:1,000 and 1:500, respectively.

Techniques: Software, Positive Control

Journal:

Article Title: Therapeutic Targeting of ATP7B in Ovarian Carcinoma

doi: 10.1158/1078-0432.CCR-08-2306

Figure Lengend Snippet: Effect of ATP7B gene silencing on DNA adduct formation in A2780-sensitive and A2780-resistant cells. A, the platinum content was determined using flame atomic absorption spectrometer after incubating A2780-PAR and A2780-CP20 cells with cisplatin for 4 h followed by digestion with 1 mol/L benzethonium hydroxide and acidification with 1 N HCl. Columns, mean values from three independent experiments; bars, SE. *, P < 0.05, compared with untreated or control siRNA–treated cells. B, cisplatin binds to the NH2-terminal domain of ATP7B (N-ATP7B). The recombinant NH2-terminal domain of ATP7B was incubated with increasing concentrations of copper or cisplatin (copper and cisplatin were added to the N-ATP7B in increasing molar ratios up to 60-fold excess over protein). Metal-coordinating cysteines in N-ATP7B were labeled with cysteine-directed probe CPM. Top, copper/cisplatin protects against labeling with the CPM without affecting total amount of protein; bottom, fluorescence intensity for average of three replicates, defining 100% as the fluorescence/protein ratio where no ligand was used before labeling. The densitometry of fluorescent gels indicates that this protection is partial and hence not all metal-binding sites in the N-ATP7B bind cisplatin. C, overexpression of N-ATP7B increases cisplatin resistance in platinum-sensitive cells. A2780-PAR cells were transfected with empty vector pTriEx-cDNA or pTriEx-N-ATP7B cDNA (WND cDNA) using Lipofectamine 2000. After 48 h, cells were incubated with cisplatin (2 μmol/L) for 72 h at 37°C, and MTT assay was done to determine the difference in the IC50 levels. Columns, mean values for the IC50 of three independent experiments; bars, SE. *, P < 0.03, compared with A2780-PAR cells. D, Western blot analysis of overexpression of NH2-terminal domain of ATP7B in A2780-PAR cells. Cells were transfected with pTriEx-cDNA or pTriEx-N-ATP7B cDNA as mentioned above and proteins were separated by SDS gel electrophoresis. β-Actin was used as loading control.

Article Snippet: The blots were blocked for 1 h in 5% milk powder in TBST [10 mmol/L Tris (pH 8), 150 mmol/L NaCl, 0.05% Tween 20] and incubated at 4°C overnight with anti-ATP7A and anti-ATP7B antibodies (Novus Biologicals) at dilutions of 1:1,000 and 1:500, respectively.

Techniques: Control, Recombinant, Incubation, Labeling, Fluorescence, Binding Assay, Over Expression, Transfection, Plasmid Preparation, MTT Assay, Western Blot, SDS-Gel, Electrophoresis

Journal:

Article Title: Therapeutic Targeting of ATP7B in Ovarian Carcinoma

doi: 10.1158/1078-0432.CCR-08-2306

Figure Lengend Snippet: Effect of ATP7B gene silencing on ovarian carcinoma. A, Western blot of lysates from orthotopic tumor samples collected at 0, 1, 2, 3, and 4 d after a single administration of ATP7B siRNA or control siRNA incorporated in DOPC. Protein levels were quantified by densitometry and expression is shown as arbitrary units. B, therapeutic efficacy of siRNA-mediated ATP7B down-regulation. Nude mice were injected i.p. with 1.0 × 106 A2780-CP20 or 3.0 × 106 RMG2 cells and randomly allocated to one of the following groups: empty liposome, control siRNA-DOPC, control siRNA-DOPC + cisplatin, ATP7B siRNA-DOPC, and ATP7B siRNA-DOPC + cisplatin. Treatments were started 1 wk after tumor cell injection and siRNA/liposomes were administered twice weekly at a dose of 150 μg/kg body weight. All of the animals were sacrificed when animals in any group appeared moribund (A2780-CP20, after 3 wk; RMG2, 5 wk starting therapy) and necropsy was done and mouse weight, tumor weight, and location were recorded. Statistical analysis for tumor weights was done by Student's t test. *, P < 0.05, compared with empty liposome or control siRNA-DOPC; **, P < 0.001, compared with control siRNA-DOPC + cisplatin or ATP7B siRNA-DOPC.

Article Snippet: The blots were blocked for 1 h in 5% milk powder in TBST [10 mmol/L Tris (pH 8), 150 mmol/L NaCl, 0.05% Tween 20] and incubated at 4°C overnight with anti-ATP7A and anti-ATP7B antibodies (Novus Biologicals) at dilutions of 1:1,000 and 1:500, respectively.

Techniques: Western Blot, Control, Expressing, Drug discovery, Injection, Liposomes

Journal:

Article Title: Therapeutic Targeting of ATP7B in Ovarian Carcinoma

doi: 10.1158/1078-0432.CCR-08-2306

Figure Lengend Snippet: Characteristics of tumors after ATP7B siRNA-DOPC treatment with or without chemotherapy

Article Snippet: The blots were blocked for 1 h in 5% milk powder in TBST [10 mmol/L Tris (pH 8), 150 mmol/L NaCl, 0.05% Tween 20] and incubated at 4°C overnight with anti-ATP7A and anti-ATP7B antibodies (Novus Biologicals) at dilutions of 1:1,000 and 1:500, respectively.

Techniques: Control

Journal:

Article Title: Therapeutic Targeting of ATP7B in Ovarian Carcinoma

doi: 10.1158/1078-0432.CCR-08-2306

Figure Lengend Snippet: ATP7B gene silencing leads to reduced proliferation, increased apoptosis, and reduced MVD of A2780-CP20 tumors. Immunohistochemical staining for PCNA (A), TUNEL assay (B), and MVD (C) was conducted to assess cell proliferation, apoptosis, and MVD in A2780-CP20 tumors collected at completion of ATP7B siRNA-DOPC therapy. Original magnification, ×100. Quantification of effects is shown graphically on the right. Bars, 95% confidence intervals. Treatment arms were compared by Student's t test. *, P < 0.05; **, P < 0.001, compared with the empty liposome or control siRNA-DOPC groups. D, ATP7B silencing decreased VEGF levels in tumors. ELISA of A2780-CP20 tumors harvested at the completion of ATP7B therapy with or without cisplatin. Experiment was done twice. *, P < 0.05; **, P < 0.001, compared with empty liposome group.

Article Snippet: The blots were blocked for 1 h in 5% milk powder in TBST [10 mmol/L Tris (pH 8), 150 mmol/L NaCl, 0.05% Tween 20] and incubated at 4°C overnight with anti-ATP7A and anti-ATP7B antibodies (Novus Biologicals) at dilutions of 1:1,000 and 1:500, respectively.

Techniques: Immunohistochemical staining, Staining, TUNEL Assay, Control, Enzyme-linked Immunosorbent Assay