Figure 6

Endogenous ROS regulates antioestrogen effect of TAM-induced gene expression and ER phosphorylation. MCF-7 cells were treated for 30 min with either antioestrogen, tamoxifen (TAM), or E2 (367 pM). (A) Treatment with TAM (1 μ M) increased oxidation of DCF (green). Mitochondria were labelled with MitoTracker Red. Colocalisation of both probes (yellow) was used as a marker of mitochondrial. (B) Analysis of TAM-induced ROS by the DCF assay. Values in the graph show significant inhibition of TAM-induced oxidation of DCF by treatment with ROS scavengers, 20 μ M ebselen (Eb), or 500 μg ml−1 of PEG-catalase (PEG-CAT). (C) Comparison of the effect of Eb on the phosphorylation of ERα (p-ERα) in E2-treated MCF-7 cells. Representative images show Eb reduced the level of p-ERα (blue) in E2-treated MCF-7 cells. Specificity of p-ERα antibody to the ERα was determined by colocalisation of p-ERα antibody (blue) and anti-ER antibody (red) resulting in merged photo in pink colour. (D) Graph shows reduced number of E2-treated MCF-7 cells stained with anti-p-ERα antibody when pretreated with Eb. Values in the graph represent the number of ERα and p-ERα fluorescent cells counted and expressed as %. (E) Analysis of TAM-induced CDC25C mRNA expression. Graph indicates significant reduction in E2-induced gene expression by TAM and/or Eb treatment. The relative mRNA levels measured for each gene were normalised to 18S RNA. The quantitative values are mean±s.d. Data shown are representative of three independent experiments. *P<0.05, significantly different from control (CTRL); **P<0.05, significantly different from E2.