Fig. 1

Western blot and RT-PCR analysis. HepG2 cells were treated for 16 h in serum-free medium with 300 μM BCS and/or 40 ng/ml of IL-6. a Western blot analysis of pSTAT3, STAT3, and β-actin proteins on total cell extracts as described in methods. b Western blot, Coomassie Blue staining of soluble Cp isoform, relative to denaturing SDS-PAGE, and in gel nondenaturing SDS-PAGE enzymatic activity of concentrated and dialyzed culture medium. Equal amounts of total proteins were loaded per lane. c relative densitometric analysis. d representative image of soluble Cp isoform RT-PCR product: after 16 h of treatment, RNA was isolated, reverse transcribed and subjected to PCR. The amplicons relative to soluble Cp isoform and β-actin were analysed by agarose gel electrophoresis and intensity of bands was determined by ImageJ 1.47v software (http://imagej.nih.gov/ij). The values of intensity relative to soluble Cp were normalized by using the β-actin housekeeping gene, (e) densitometric analysis of Cp RT-PCR results. f graph relative to intracellular copper concentration in HepG2 cells. Cells were extensively washed, lysed as described in methods and used for atomic absorption analysis. The copper content was normalized by cellular total protein concentration. All values are expressed as means ± SEM (n = 6). All indicated differences were statistically significant (p < 0.05). *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. Expression levels of control condition were normalized to one, and all values are expressed as relative units