Oxidative Medicine and Cellular Longevity / 2016 / Article / Fig 6

Research Article

NRF2 Regulates HER2 and HER3 Signaling Pathway to Modulate Sensitivity to Targeted Immunotherapies

Figure 6

Knockdown of NRF2 by SiRNA represses both basal and induced antioxidant response pathway in PEO1 and SKOV3 cell lines. (a) Immunoblotting analysis showing repression of NRF2 following NRF2 knockdown by SiRNA in PEO1 and SKOV3 cell lines. Cells were either transfected with scrambled SiRNA (Sc) or transfected with 75 pmol of NRF2 SiRNA (Si). After 48 h, cells were either left untreated or treated with 200 μM tBHQ (T) for 4 h, before being processed for immunoblotting using relevant antibodies (Table 1). Ponceau stain of the same blot was used as loading control. Bar chart shows NRF2 levels by quantifying immunoblot signal intensities obtained in (a) and normalised to the value of untreated (UT) control and expressed as fold change. (b) Knockdown of NRF2 causes inhibition of its transcriptional antioxidant program in both constitutive and tBHQ induced states. PEO1 and SKOV3 cells were transfected with either empty PGL3 basic vector or 1 μg PGL3 basic vector with a cloned 8 x cis-antioxidant response elements (ARE) driving NRF2 dependent expression of luciferase gene. Cotransfection with 0.2 μg pRL-CMV plasmid was performed as an internal transfection control. Where required, cotransfection with either scrambled RNA (Sc) or NRF2 SiRNA was performed using 20 pmol SiRNA. At 24 h after transfection, treatment with 200 μM tBHQ was performed where indicated for 4 h following which, cells were processed for dual luciferase reporter assay (Promega) to record luciferase activity in multiplate reader (MODULUS, Promega). Data are the means with ±S.D. of triplicates normalised to the value of scrambled SiRNA (Sc) and expressed as fold change with statistical significance determined by ONE WAY ANOVA followed by Tukey’s post hoc test. indicates significance of scramble versus treatment groups while indicates significance of tBHQ versus tBHQ + NRF2 SiRNA groups according to the scale symbolised by or :  , or :  , and or :  .

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