multiplate absorbance reader modulus  (Promega)

Journal: Oxidative Medicine and Cellular Longevity

Article Title: NRF2 Regulates HER1 Signaling Pathway to Modulate the Sensitivity of Ovarian Cancer Cells to Lapatinib and Erlotinib

doi: 10.1155/2017/1864578

Figure Lengend Snippet: Pharmacological (bexarotene) and genetic inhibition (siRNA) of NRF2 causes transcriptional and translational downregulation of HER1. Luciferase assay showing transcriptional downregulation of HER1 following NRF2 inhibition by (a) bexarotene or (b) siRNA in PEO1, OVCAR3, and SKOV3 cell lines. Exponentially growing PEO1, SKOV3, and OVCAR3 excluding MCF7-AREc32 cell lines were transfected with either empty PGL3 basic vector or 1 μ g PGL3 basic vector with cloned HER1 driving the expression of luciferase gene. Cotransfection with 0.2 μ g pRL-CMV plasmid was performed as an internal transfection control as described in the Materials and Methods. At 24 h posttransfection, cells were (a) either left untreated or treated with 2.5 μ M bexarotene. (b) Cells were either transfected with scrambled siRNA (Sc) or transfected with 20 pmol of NRF2 siRNA (Si) for 24 h. Following treatments, lysates were prepared and luciferase activity was measured using dual luciferase reporter assay (Promega) in multiplate reader (MODULUS, Promega). (c) Immunoblot analysis following treatment with bexarotene demonstrated protein downregulation of HER1 receptor and decrease of NRF2, HO-1, and HER1. Exponentially growing cells were either left untreated (UT) or treated with 2.5 μ M bexarotene for 24 h before being harvested and processed for immunoblotting using relevant antibodies. Bar chart showing total NRF2, HO-1, and total HER1 levels in PEO1, OVCAR3, and SKOV3 cell lines by quantifying immunoblot signal intensities obtained in (c). (d) Immunoblot analysis following knockdown of NRF2 demonstrated protein downregulation of both HER1 receptor and decrease of NRF2 and HO-1 in PEO1, OVCAR3, and SKOV3 cell lines. Cells were either transfected with scrambled siRNA (Sc) or transfected with 75 pmol of NRF2 siRNA (Si). After 24 h and 48 h, cells were harvested and processed for immunoblotting using relevant antibodies. β -Actin of the same blot was used as loading control. Bar chart shows the levels of relevant proteins by quantifying immunoblot signal intensities obtained and expressed as fold change. Data shown in (a) and (b) are the means ± S.D. of triplicates, normalised to UT or scramble expressed in fold change with statistical significance determined by Student's t -test ( ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001).

Article Snippet: The absorbance values at 595 nm were then recorded using a multiplate absorbance reader (MODULUS, Promega) and the data was used after normalising the fluorescence values.

Techniques: Inhibition, Luciferase, Transfection, Plasmid Preparation, Clone Assay, Expressing, Cotransfection, Control, Activity Assay, Reporter Assay, Western Blot, Knockdown

Journal: Oxidative Medicine and Cellular Longevity

Article Title: NRF2 Regulates HER1 Signaling Pathway to Modulate the Sensitivity of Ovarian Cancer Cells to Lapatinib and Erlotinib

doi: 10.1155/2017/1864578

Figure Lengend Snippet: Treatment with tBHQ reduces the knockdown effect of siRNA. (a) siRNA-mediated knockdown of NRF2 causes inhibition of its transcriptional antioxidant program and repression of HER1 level in both constitutive and tBHQ-induced states. 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 μ g PGL3 basic vector with promoters of HER1-cloned driving HER1 expression of luciferase gene. Cotransfection with 0.2 μ g 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 100 μ M 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). (b) Immunoblotting analysis showing repression of NRF2 following NRF2 knockdown by siRNA in PEO1, OVCAR3, 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 100 μ M tBHQ (T) for 4 h, before being processed for immunoblotting using relevant antibodies. β -Actin of the same blot was used as loading control. Bar chart shows NRF2 levels by quantifying immunoblot signal intensities obtained and expressed as fold change. Data in (a) are the means with ±S.D. of triplicates, normalised to scramble with statistical significance determined by one-way ANOVA followed by Tukey's post hoc test. ∗∗ p < 0.01 and ∗∗∗ p < 0.001.

Article Snippet: The absorbance values at 595 nm were then recorded using a multiplate absorbance reader (MODULUS, Promega) and the data was used after normalising the fluorescence values.

Techniques: Knockdown, Inhibition, Stable Transfection, Clone Assay, Expressing, Luciferase, Transfection, Plasmid Preparation, Cotransfection, Control, Reporter Assay, Activity Assay, Western Blot