Fig. 1: Copper interferes with hepatic selenium homeostasis.
From: Excessive copper impairs intrahepatocyte trafficking and secretion of selenoprotein P
Intracellular Cu and Se concentrations were determined using TXRF with 1 mg/L Yttrium as standard for 1000 s and normalized to protein content of HepG2 cells (a, b, g, k). Extra- and intracellular SELENOP was determined using Western or dot blot and normalized to Ponceau (P) staining (d, e, h–j). a, b Intracellular Cu and Se content of cells treated with 0 or 100 µM CuSO4 without (-Se) or with 50 nM selenite or 200 nM SeMet (n = 4). c Extracellular SELENOP was determined in medium of HepG2 cells treated for 72 h with or without selenite and increasing Cu concentrations 24 h prior to harvest (n = 3). d Representative Western Blots of extra- and intracellular SELENOP and Ponceau staining of cells treated as described for a–b. e Quantification of extra- and intracellular SELENOP bands from Western Blots (shown in d) presented as Cu-induced fold changes (n = 4). Respective cells without Cu treatment were set as 1 (dotted line). f mRNA expression of SELENOP (n = 7) and LRP8 (n = 4) was determined by qPCR after 24 h incubation with 0 or 100 µM CuSO4 and normalized to RPL13A. g, h Intracellular Cu concentration and extracellular SELENOP of primary, murine hepatocytes treated for 24 h after isolation with 50 nM selenite in combination with 0 or 10 µM CuSO4 (n = 6). i–k A siRNA-mediated knockdown of SELENOP was generated and extra- (n = 10) and intracellular SELENOP (n = 12) and intracellular Se concentrations (n = 8) were analyzed after 72 h treatment with selenite and/or Cu. l MTT reduction capacity of SELENOP knockdown and control cells treated for 48 h with selenite including a 24 h incubation with increasing Cu concentrations. Cytotoxicity was related to cells without Cu treatment (n = 3). Data are depicted as mean ± SD. Biological replicates are indicated by individual dots. Statistical analyses were based on two-way ANOVA with Bonferroni’s post-test (a–c, i–l) or on two-tailed t test compared to cells without Cu treatment (e–h). *p < 0.05; **p < 0.01; ***p < 0.001 vs. -Cu, ###p < 0.001 vs. -Se; +++p < 0.001 vs. siControl. Source data are provided as a source Data file.
