Journal: Arthritis Research & Therapy

Article Title: Decreased catalytic function with altered sumoylation of DNA topoisomerase I in the nuclei of scleroderma fibroblasts

doi: 10.1186/ar3435

Figure Lengend Snippet: Measurement of catalytic function of topo I in cultured fibroblasts . A serial dilution of topo I in the nuclear extracts obtained from SSc and control fibroblasts was used to relax 0.25 μg supercoiled DNA. A . The supercoiled DNA band is gradually diminished following increased amounts of topo I in the nuclear extracts in the relaxing assays. The efficiency of SSc topo I in relaxing the supercoiled DNA appeared to be less than that of control topo I in each concentration of nuclear extracts. B . Comparison of 11 paired SSc and control fibroblasts for mean values of intensity of supercoiled DNA bands after relaxing assay with different concentrations of topo I in the nuclear extracts. Each P- value of comparison at different dilution points is listed in the figure. Comparison of average band intensity of remaining supercoiled DNA in each of six dilutions between all SSc and all control fibroblasts showed a significant P- value ( P = 0.0041) (Student's t -test). A = standard supercoiled DNA band; B = standard relaxed DNA bands; the numbers (1/32, 1/16, 1/8, 1/4, 1/2 and 1) indicate serial dilutions of topo I in nuclear extracts used for relaxing supercoiled DNA. The error bars indicate standard deviation (SD).

Article Snippet: Briefly, supercoiled DNA substrate (0.25 μg) (TopoGen, Inc.) was reacted with nuclear proteins containing topo I at serial dilutions.

Techniques: Cell Culture, Serial Dilution, Control, Concentration Assay, Comparison, Standard Deviation

Journal: Arthritis Research & Therapy

Article Title: Decreased catalytic function with altered sumoylation of DNA topoisomerase I in the nuclei of scleroderma fibroblasts

doi: 10.1186/ar3435

Figure Lengend Snippet: Catalytic function of topo I in cultured SSc fibroblasts with and without SUMO1 siRNA transfection . A serial dilution of the nuclear extract containing topo I obtained from SSc fibroblasts was used to relax 0.25 μg supercoiled DNA. In this figure, the supercoiled DNA band was completely transformed to relaxed DNA at dilutions of one half and one in the fibroblasts without siRNA transfection or non-target siRNA transfection. In contrast, this change was observed between the one-eighth and one-fourth dilutions in the fibroblasts with SUMO1 transfection, which indicates a higher efficiency of catalytic function of topo I after SUMO1 inhibition in the fibroblasts. According to the intensity of the bands of remaining supercoiled DNA in serial dilutions in the assays of three fibroblast strains, these changes are significant. The P- values are 0.045 and 0.027 at the one-fourth dilution for comparisons between SUMO1 siRNA vs. non-target siRNA, or vs. without siRNA transfected fibroblasts, respectively (Student's t -test). This is representative of three SSc fibroblast strains examined in SUMO1 siRNA studies. * A , supercoiled DNA; B , relaxed DNA.

Article Snippet: Briefly, supercoiled DNA substrate (0.25 μg) (TopoGen, Inc.) was reacted with nuclear proteins containing topo I at serial dilutions.

Techniques: Cell Culture, Transfection, Serial Dilution, Transformation Assay, Inhibition

Journal: Arthritis Research & Therapy

Article Title: Decreased catalytic function with altered sumoylation of DNA topoisomerase I in the nuclei of scleroderma fibroblasts

doi: 10.1186/ar3435

Figure Lengend Snippet: Measurement of catalytic function of recombinant human topo I with and without sumoylation reaction . Recombinant human topo I proteins were sumoylated with either mutant sumo1 or wild type sumo1 or negative control (without sumoylation), and then were examined for their catalytic function in a serial dilution. Sumoylation of topo I with wild type sumo1 showed a reduction of efficiency in catalytic function (supercoiled DNA disappeared at the dilution of topo I concentration of 30) compared to the topo I protein sumoylated with mutant sumo1 or negative control (supercoiled DNA disappeared at topo I concentration of 22.5). This is representative of three assays. * A , standard supercoiled DNA band; B , standard relaxed DNA bands.

Article Snippet: Briefly, supercoiled DNA substrate (0.25 μg) (TopoGen, Inc.) was reacted with nuclear proteins containing topo I at serial dilutions.

Techniques: Recombinant, Mutagenesis, Negative Control, Serial Dilution, Concentration Assay