Sigmaplot 10

Kinetic parameters of BioF–CueO were determined by monitoring the oxidation of 2,6‐dimethoxyphenol (DMP) at 30°C, as described before (Grass and Rensing, 2001). Experiments (in duplicate) were carried out at 30°C in 20 mM Tris buffer, pH 6.5, plus 10 μM Cu²⁺ and using a protein concentration of 1 μg ml⁻¹. Addition of higher Cu²⁺ concentrations induced an intense nonspecific oxidation of the substrate that interfered with the monitoring of the enzymatic activity. Activity was determined spectrophotometrically at 468 nm in an Evolution 201 spectrophotometer (Thermo Scientific) following the appearance of the DMP oxidation product, 3,3′,5,5′‐tetramethoxydiphenoquinone (ε₄₆₈ = 14 800 M⁻¹ cm⁻¹) (Slomczynski et al., 1995). The kinetic parameters (K_m and k_cat) of the enzyme were calculated with the Michaelis Menten equation (Michaelis and Menten, 1913) using the SigmaPlot 10.0 utilities (Systat Software Inc.).
All graphs presented in this work were created with SigmaPlot 10.0. Data represent the mean of duplicate or triplicate experiments, depending on each case, and error bars represent the standard deviation.

Huh7.5 cells harboring a Con1 genotype 1b subgenomic replicon were seeded at a density of 5000 cells per well in 96-well plates. The cells were treated with increasing concentrations of the tested compounds in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum and 0.2% dimethylsulfoxid (DMSO) without G418 and cultured for 3 days. Total RNA was extracted using the RNeasy 96 kit (Qiagen, Valencia, CA, USA). HCV RNA levels were measured by means of a quantitative real-time polymerase chain reaction assay using the Taqman technology with HCV-specific primers (sense 5′- CGCCCAAACCAGAATACGA-3′ and antisense 5′- AGATAGTACACCCTTTTGCCAGATG-3′) and probe (5′-6-FAMCAATGTGTCAGTCGCG-TAMRA-3′) on an ABI 7003 device (Applied Biosystems, Foster City, CA, USA). Each data point represents the average of at least three replicates in cell culture. HCV RNA level reductions after treatment were assessed by comparing the level of HCV RNA in compound-treated cells to that of control cells treated with 1% DMSO. The effective concentration 50% (EC50), i.e. the compound concentration at which 50% of the maximal effect is achieved, was calculated using a four-parameter curve fitting method in the Sigma Plot 10 software (Systat Software, San Jose, CA, USA).

Results are presented as means ± SEM. The GraphPad Prism 5.0 software (GraphPad Software Inc., San Diego, CA, USA) was employed to carry out calculations. To calculate IC₅₀ and CC₅₀ values, the percentages of inhibition were plotted against the drug concentration and fitted with a straight line determined by a linear regression (Sigma Plot 10 software, (Systat Software, San Jose, CA, USA). Results presented are representative of three to four independent experiments.
The statistical significance was determined by the Kruskal-Wallis test performed with the GraphPad Prism 5.0 software (GraphPad Software Inc., San Diego, CA, USA). Each treatment was compared to controls. p values < 0.05 were considered significant.

Meibum was collected and lipid phase transitions were measured as described previously [21 (link)]. Curves were fit using Sigma plot 10 software (Systat Software, Inc., Chicago, IL, USA) and the confidence levels were obtained from a critical value table of the Pearson product–moment correlation coefficient. Averages were compared using the Student’s t test. A value of p < 0.05 was considered statistically significant. Data are reported as the mean plus or minus the standard error.

Curves were fitted using Sigma plot 10 software (Systat Software, Inc., Chicago IL, USA) and the confidence levels, p, were obtained from a critical value table of the Pearson product-moment correlation coefficient. A value of p < 0.05 was considered statistically significant.