Fig. 2.
(A) Copper loading increases FPN1 mRNA levels in J774 cells in a dose-dependent manner. J774 cells were incubated with the indicated concentrations of CuSO4 for 20 h. Total RNA was isolated, electrophoresed, transferred to a Nytran N membrane, and hybridized with a 32P-labeled mouse FPN1 cDNA probe as described in Experimental Procedures. To control for sample loading, the membrane was stripped and reprobed for β-actin. Relative FPN1 mRNA levels after copper treatment were quantified by using quantity one software (Bio-Rad) and normalized to values for β-actin mRNA levels obtained in the same blot to determine fold induction compared to control (untreated cells). (Right) The relative FPN1 mRNA levels as a function of copper concentration (μM) after treatment with CuSO4 in two independent experiments (filled circles) and after treatment with CuCl2 in a separate experiment (open circles). (B) Copper loading increases in J774 cell FPN1 mRNA in a time-dependent manner. After incubation with 100 μM CuSO4 for the indicated times, total RNA was isolated and Northern analysis was performed. The fold induction of FPN1 mRNA was quantified as described for A, and Right shows mean values (±SE) determined as a function of time (h) in three separate experiments. (C) TRIEN blocks copper-mediated induction of FPN1 mRNA. J774 cells were treated with 20 μM CuSO4, 80 μM TRIEN, or both together for 20 h. After RNA isolation, Northern analysis was performed to detect FPN1 and β-actin mRNA. These results are representative of three independent experiments.