Figure 5

Induction of ERBB2 (NeuT) by the Tet-on system in MCF-7/NeuT cells influences TXNRD1 and TXNIP. (a) Oncogenic ERBB2 (NeuT) overexpression in MCF-7 cells, induced by exposure to doxycycline, triggers phosphorylation of ERK1/2 and AKT (PKB) as shown by immunoblotting. (b) ERBB2-mediated changes in the transcription of thioredoxin reductase 1 (TXNDR1) and thioredoxin interacting protein (TXNIP) are demonstrated both by Affymetrix Gene Array data and quantitative real-time PCR (qRT-PCR). The gene array graphs display the mean values (± standard deviation) of the Affymetrix probesets. The qRT-PCR graphs show a representative experiment of three with the mean value (± standard deviation) of a triplicate amplification. (c) TXNRD1 and TXNIP protein levels were analyzed by inmmunoblotting. Bands of ~55 kDa for TXNRD1 and ~50 kDa for TXNIP were observed. (d) An increase in accumulation of reactive oxygen species 14 days after onset of ERBB2 (NeuT) expression was shown by detection of malondialdehyde by a thiobarbituric acid reactive substance (TBARS) assay and an N-methyl-2-phenylindole (NMPI) assay. The results represent the mean (± standard deviation) of three experiments, each measured eight times. DETBA, 1,3-diethyl-2-thiobarbituric acid.