wild type nqo1  (Addgene inc)

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Suppression of NAD(P)H-quinone oxidoreductase 1 enhanced the susceptibility of cholangiocarcinoma cells to chemotherapeutic agents

doi: 10.1186/1756-9966-33-11

Figure Lengend Snippet: Basal level of NQO1 mRNA, protein expression, and enzyme activity of CCA cells and NQO1 protein induction by chemotherapeutic agents (5-FU, Doxo, and Gem). (A) Basal NQO1 mRNA expression in CCA cell lines (KKU-100 and KKU-M214) and two other cell lines (Chang and MMNK1 cells) analyzed by qPCR. The bars represent relative mRNA expression of NQO1 normalized with β-actin as internal control. * p < 0.05 vs KKU-100 cells. (B) Basal NQO1 enzyme activity analyzed by enzymatic methods. * p < 0.05 vs KKU-100 cells. (C) Basal NQO1 protein expression analyzed by Western Blot analysis using β-actin as internal control. Representative images of NQO1 and β-actin are shown in the top panel of the figure. * p < 0.05 vs KKU-100 cells. (D) Effect of chemotherapeutic agents on NQO1 protein expression in KKU-100 cells. Cells were exposed to 5-FU (3 μM), Doxo (0.1 μM), and Gem (0.1 μM) for 24 hr. Data represent mean ± SEM, each from three separated experiments. * p < 0.05 vs the untreated control.

Article Snippet: A plasmid encoding human wild-type NQO1 in pCMV6-XL5 (4,707 bp) was purchased from Origene Technologies (#SC119599; Rockville, MD).

Techniques: Expressing, Activity Assay, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Suppression of NAD(P)H-quinone oxidoreductase 1 enhanced the susceptibility of cholangiocarcinoma cells to chemotherapeutic agents

doi: 10.1186/1756-9966-33-11

Figure Lengend Snippet: Knockdown of NQO1 by siRNA sensitized KKU-100 cells to chemotherapeutic agents. (A-B) Effect of NQO1 siRNA on mRNA and protein levels of NQO1 in KKU-100 cells. Cells were transfected with the pooled siRNA against NQO1 gene for 24 hr and 48 hr. Data represent mean ± SEM, each from three separated experiments. * p < 0.05 vs the non-targeting siRNA transfected cells. (C-E) Cytotoxicity of chemotherapeutic agents on NQO1 siRNA transfected KKU-100 cells. Forty-eight hour after transfection, cells were treated with varied concentration of chemotherapeutic agents; 5-FU, Doxo, and Gem for another 24 hr as described in the “Methods” section. The cytotoxicity was evaluated by SRB assay. Data represent mean ± SEM, each from three separated experiments. * p < 0.05 vs the non-targeting siRNA transfected cells.

Article Snippet: A plasmid encoding human wild-type NQO1 in pCMV6-XL5 (4,707 bp) was purchased from Origene Technologies (#SC119599; Rockville, MD).

Techniques: Transfection, Concentration Assay, Sulforhodamine B Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Suppression of NAD(P)H-quinone oxidoreductase 1 enhanced the susceptibility of cholangiocarcinoma cells to chemotherapeutic agents

doi: 10.1186/1756-9966-33-11

Figure Lengend Snippet: Altered expressions of proteins related to cell proliferation and apoptosis pathways. A-D , Expressions of proteins related to cell proliferation and apoptosis pathways. KKU-100 with NQO1 knocked down cells were exposed to chemotherapeutic agents; 5-FU (3 μM), Doxo (0.1 μM), and Gem (0.1 μM) for 24 hr. Whole cell lysates were prepared after indicated treatment and Western blot analysis was conducted using anti-p53 (A) , -p21 (B) , -cyclin D1 (C) , -Bax (D) and -β-actin antibodies. The relative bars that were normalized with β-actin as a loading control of each band is shown below the Western blot images. Data represent mean ± SEM, each from three separated experiments. * p < 0.05 vs the treated non-targeting knocked down cells. ** p < 0.05 vs the untreated non-targeting knocked down cells.

Article Snippet: A plasmid encoding human wild-type NQO1 in pCMV6-XL5 (4,707 bp) was purchased from Origene Technologies (#SC119599; Rockville, MD).

Techniques: Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Suppression of NAD(P)H-quinone oxidoreductase 1 enhanced the susceptibility of cholangiocarcinoma cells to chemotherapeutic agents

doi: 10.1186/1756-9966-33-11

Figure Lengend Snippet: Effects of NQO1 over-expression on the susceptibility of KKU-M214 cells to chemotherapeutic agents (5-FU, Doxo, and Gem). A-B , Effect of NQO1 over-expression on mRNA and protein levels of NQO1 in KKU-M214 cells. The pCMV6-XL5-NQO1 (wild type NQO1) or pCMV6-XL5 (control vector) was transfected to KKU-M214 for 24 hr. The whole cells were collected for NQO1 enzyme activity assay (A) and Western blot analysis (B) . The data represent mean ± SEM, each from three experiments. * p < 0.05 vs the control vector transfected cells. (C-E) Cytotoxicity of chemotherapeutic agents on NQO1 over-expressed KKU-M214 cells. Twenty-four hour after transfection, cells were incubated with chemotherapeutic agents for additional 24 hr (Doxo) and 48 hr (5-FU and Gem). The cytotoxicity was evaluated by SRB assay. Data represent mean ± SEM, each from three separated experiments. * p < 0.05 vs the control vector transfected cells.

Article Snippet: A plasmid encoding human wild-type NQO1 in pCMV6-XL5 (4,707 bp) was purchased from Origene Technologies (#SC119599; Rockville, MD).

Techniques: Over Expression, Plasmid Preparation, Transfection, Enzyme Activity Assay, Western Blot, Incubation, Sulforhodamine B Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Suppression of NAD(P)H-quinone oxidoreductase 1 enhanced the susceptibility of cholangiocarcinoma cells to chemotherapeutic agents

doi: 10.1186/1756-9966-33-11

Figure Lengend Snippet: NQO1 over-expression attenuates the p53 pathway in KKU-M214 cells. A-D , Western blots of p53 (A) , p21 (B) , cyclin D1 (C) , and Bax (D) protein in KKU-M214-NQO1 over-expressed cells after treatment with 5-FU 3 μM (48 hr), Doxo 0.1 μM (24 hr), and Gem 0.1 μM (48 hr). The relative bars that were normalized with β-actin of each band are shown below the Western blot images. * p < 0.05 vs the treated control vector transfected cells. ** p < 0.05 vs the untreated control vector transfected cells.

Article Snippet: A plasmid encoding human wild-type NQO1 in pCMV6-XL5 (4,707 bp) was purchased from Origene Technologies (#SC119599; Rockville, MD).

Techniques: Over Expression, Western Blot, Plasmid Preparation, Transfection

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Suppression of NAD(P)H-quinone oxidoreductase 1 enhanced the susceptibility of cholangiocarcinoma cells to chemotherapeutic agents

doi: 10.1186/1756-9966-33-11

Figure Lengend Snippet: Double knockdown of NQO1 and p53 by siRNA altered KKU-100 cells to chemotherapeutic agents. (A) Effect of co-transfected NQO1 and p53 siRNA in KKU-100 cells. Cells were transfected with the pooled siRNA against NQO1 and p53 for 24 hr. The bars represent relative expression of NQO1 and p53 normalized with β-actin as internal control. (B-D) After co-transfection, cells were treated with varied concentrations of chemotherapeutic agents; 5-FU, Doxo, and Gem for another 24 hr as described in the “Methods” section. The cytotoxicity was evaluated by SRB assay. Data represent mean ± SEM, each from three separated experiments. * p < 0.05 vs the non-targeting knocked down cells and # p < 0.05 vs NQO1 knocked down cells.

Article Snippet: A plasmid encoding human wild-type NQO1 in pCMV6-XL5 (4,707 bp) was purchased from Origene Technologies (#SC119599; Rockville, MD).

Techniques: Transfection, Expressing, Cotransfection, Sulforhodamine B Assay