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. 2017 Dec 19;2017:1864578. doi: 10.1155/2017/1864578

Figure 1.

Figure 1

NRF2 regulates both basal and inducible expression of HER1. (a) Cells exhibit different basal expression. Exponentially growing PEO1, SKOV3, and OVCAR3 cells were transfected with either empty PGL3 basic vector or 1 μg PGL3 basic vector with cloned 1.5 kb fragments of either HER1 (prHER1) or HER2 (prHER3) promoter driving the expression of luciferase gene. Cotransfection with 0.2 μg pRL-CMV plasmid was performed as an internal transfection control. (b) tBHQ causes transcriptional induction of HER1 and induction of ARE in a concentration-dependent manner. MCF7-AREc32 which already contains stably cloned 8 × cis-antioxidant response elements (ARE) driving NRF2-dependent expression of luciferase gene was left without any transfection while PEO1, OVCAR3, and SKOV3 cells were transfected with either empty PGL3 basic vector or 1 μg PGL3 basic vector with promoters of HER1-cloned driving HER1 expression of luciferase gene. Cotransfection with 0.2 μg pRL-CMV plasmid was performed as an internal transfection control. Where required PEO1, SKOV3, and OVCAR3 cell lines and MCF7-AREc32 stable cell line were treated in quadruplicate with different concentrations of tBHQ as indicated for 24 h. (c) Immunoblot analysis following treatment with tBHQ demonstrated protein induction of HER1 receptor and also activation of total and with an increase of pAKT. Briefly, exponentially growing cells were either left untreated (UT) or treated with 100 μM tBHQ for 24  h before being harvested and processed for immunoblotting using relevant antibodies. Bar chart showing total NRF2, total HER1, and phospho-Akt levels in PEO1, OVCAR3, and SKOV3 cell lines by quantifying immunoblot signal intensities obtained in the blot image and normalised to the value of UT and expressed as fold change. Data shown in (a) and (b) are the means ± S.D. of triplicates normalised to the value of PGL3 or UT and expressed as fold change with statistical significance determined by one-way ANOVA followed by Tukey's post hoc test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001).