Journal: PLoS ONE

Article Title: Mycoplasma fermentans Inhibits the Activity of Cellular DNA Topoisomerase I by Activation of PARP1 and Alters the Efficacy of Its Anti-Cancer Inhibitor

doi: 10.1371/journal.pone.0072377

Figure Lengend Snippet: Infection of cells with live M. fermentans inhibits the DNA relaxation activity of Topo I. MCF7 (A,B) and U251 (C,D) cancer cells were infected with live M. fermentans ( M.f ) for various intervals at MOI of 10 3 CFU/cell. Total nuclear protein (12.5 ng) was added to a specific reaction mixture for Topo I. Reaction products were analyzed by agarose gel electrophoresis. ( A,C ) A representative picture (n = 4–5) of Topo I DNA-relaxation activity. ( B,D ) Quantification analysis of Topo I activity. Symbols: R and S are the relaxed and supercoiled form of the pUC19 DNA, respectively; Topo- no protein added to the reaction mixture. t -test: * p

Article Snippet: Supercoiled DNA plasmid pUC19 was purchased from MBI Fermentas (Hanover, MD, USA).

Techniques: Infection, Activity Assay, Agarose Gel Electrophoresis

Journal: PLoS ONE

Article Title: Mycoplasma fermentans Inhibits the Activity of Cellular DNA Topoisomerase I by Activation of PARP1 and Alters the Efficacy of Its Anti-Cancer Inhibitor

doi: 10.1371/journal.pone.0072377

Figure Lengend Snippet: Sonicated M. fermentans Protein inhibits the DNA relaxation activity of Topo I. MCF7 (A–C) and U251 (D–F) cancer cells were treated for 24 hrs with various concentrations of M. fermentans proteins. Total nuclear proteins (12.5 ng) were added to a specific reaction mixture for Topo I. Reaction products were analyzed by agarose gel electrophoresis. ( A,D ) A representative picture (n = 4–5) of Topo I DNA relaxation activity. ( B,E ) Quantification analysis of Topo I activity. ( C,F ) Topo I protein level examined by Western blot analysis. Symbols: R and S are the relaxed and supercoiled form of the pUC19 DNA, respectively, Topo- no protein added to the reaction mixture. t -test: * p

Article Snippet: Supercoiled DNA plasmid pUC19 was purchased from MBI Fermentas (Hanover, MD, USA).

Techniques: Sonication, Activity Assay, Agarose Gel Electrophoresis, Western Blot

Journal: PLoS ONE

Article Title: Mycoplasma fermentans Inhibits the Activity of Cellular DNA Topoisomerase I by Activation of PARP1 and Alters the Efficacy of Its Anti-Cancer Inhibitor

doi: 10.1371/journal.pone.0072377

Figure Lengend Snippet: PARP inhibitor prevented the Mycoplasma-induced inhibitory effect on Topo I activity. MCF7 cells were pre-incubated with 3-aminobenzamide (3AB) for 1 hr at various concentrations followed by M. fermentans infection (MOI of 10 3 CFU/cell) for an additional 6 hrs. Total nuclear protein (12.5 ng) was added to a specific reaction mixture for Topo I. Reaction products were analyzed by agarose gel electrophoresis (A) and quantification of Topo I activity was performed (B). Symbols: R and S are the relaxed and supercoiled form of the pUC19 DNA, respectively, Topo- no protein added to the reaction mixture t -test: * p

Article Snippet: Supercoiled DNA plasmid pUC19 was purchased from MBI Fermentas (Hanover, MD, USA).

Techniques: Activity Assay, Incubation, Infection, Agarose Gel Electrophoresis

Journal: PLoS ONE

Article Title: Mycoplasma fermentans Inhibits the Activity of Cellular DNA Topoisomerase I by Activation of PARP1 and Alters the Efficacy of Its Anti-Cancer Inhibitor

doi: 10.1371/journal.pone.0072377

Figure Lengend Snippet: M. fermentans diminished the CPT inhibition effect on the DNA relaxation activity of Topo I. MCF7 cells were infected with M. fermentans ( M.f ) for 6 hrs followed by CPT (30 µM) treatments for additional 1.5 hrs. Total nuclear protein (12.5 ng) was added to a specific reaction mixture for Topo I. Reaction products were analyzed by agarose gel electrophoresis. ( A ) A representative picture n = 5, of Topo I DNA-relaxation activity. ( B ) Quantification analysis of Topo I activity. ( C ) Topo I protein level examined by Western blot analysis. Symbols: R and S are the relaxed and supercoiled form of the pUC19 DNA, respectively, Topo- no protein added to the reaction mixture t -test: * p

Article Snippet: Supercoiled DNA plasmid pUC19 was purchased from MBI Fermentas (Hanover, MD, USA).

Techniques: Cycling Probe Technology, Inhibition, Activity Assay, Infection, Agarose Gel Electrophoresis, Western Blot

Journal: PLoS ONE

Article Title: Mycoplasma fermentans Inhibits the Activity of Cellular DNA Topoisomerase I by Activation of PARP1 and Alters the Efficacy of Its Anti-Cancer Inhibitor

doi: 10.1371/journal.pone.0072377

Figure Lengend Snippet: The Effect of MEK inhibitor on Mycoplasma-induced inhibitory effect on Topo I activity and on ERK 1/2 Phosphorylation in MCF7 cells. MCF7 cells were pre-incubated with MEK inhibitors (PD) for 1 hour at a concentration of 25 µM followed by M. fermentans infection (MOI of 10 3 CFU/cell) for an additional 6 hrs. Total nuclear protein (12.5 ng) was added to a specific reaction mixture for Topo I. Reaction products were analyzed by agarose gel electrophoresis (A) and Topo I activity was quantified (B). Phosphorylated ERK 1/2 protein level from MCF7-extract was examined by Western blot analysis (C). Symbols: R and S are the relaxed and supercoiled form of the pUC19 DNA, respectively, Topo- no protein added to the reaction mixture t -test: *** p

Article Snippet: Supercoiled DNA plasmid pUC19 was purchased from MBI Fermentas (Hanover, MD, USA).

Techniques: Activity Assay, Incubation, Concentration Assay, Infection, Agarose Gel Electrophoresis, Western Blot