Journal: Oxidative Medicine and Cellular Longevity

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

doi: 10.1155/2016/4148791

Figure Lengend Snippet: Knockdown of NRF2 by SiRNA causes repression of phospho-NRF2 and HO-1 levels and elevation of reactive oxygen species (ROS). (a) Optimization of SiRNA-mediated NRF2 knockdown. Exponentially growing cells were transfected either with scrambled RNA (scrmbl) or with different amounts of SiRNA for either 24 h or 48 h before being processed for immunoblotting. (b) NRF2 knockdown results in repression of its substrates. The same lysates as in (a) were blotted for phospho-NRF2 and HO-1 levels. Bar charts in (a) and (b) show total NRF2, phospho-NRF2, and HO-1 levels in SKOV3 cell lines by quantifying immunoblot signal intensities obtained in respective blots and normalised to the value of UT and expressed as fold change. (c) NRF2 knockdown leads to ROS accumulation. SKOV3 cells were seeded in triplicate for 18 h and transfected with NRF2 SiRNA. Following 48 h incubation, cells were assayed for total ROS by loading them with DCFDA for 45 min and measuring fluorescence using fluorescence multiplate reader (MODULUS, Promega) with excitation and emission spectra of 485 nm/535 nm. The fluorescence reading was normalised to total cell abundance within the same wells as described in Materials and Methods. Data are the means with ±S.D. of triplicates, normalised to untreated (UT) control and expressed as fold change with statistical significance determined by Student's t -test according to the scale ∗ : p < 0.05, ∗∗ : p < 0.01, and ∗∗∗ : p < 0.001. (d) Immunofluorescent labelling of endogenous phospho-NRF2 and HO-1 exhibits repression following NRF2 knockdown. Cells were transfected as in (a) and processed for immunocytochemistry. Relevant primary antibodies followed by Alexa Fluor conjugated secondary antibodies were used for immunolabelling for phospho-NRF2 and HO-1 (red fluorescence). Nuclear reference was provided by costaining with 4′,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI). Images were captured with Leica DMiRe2 electronic microscope with 100x objective while merging, colocalisation, and further analysis were performed by using integrated features of Andor iQ Core software (ANDOR Technologies Ltd.). Scale bar indicates 10 μ m.

Article Snippet: On the day of assay, 20 μ L of the 5 mg/mL MTT stock was added to each well and plate was further incubated for 4 h. Following this, the old media with MTT were removed, cells were gently washed with prewarmed PBS, and 100 μ L of DMSO was added to solubilise the internalised MTT by shaking over an orbital shaker for 15 min. Absorbance of the released dye was measured and recorded using multiplate reader (MODULUS, Promega) at 540 nm.

Techniques: Knockdown, Transfection, Western Blot, Incubation, Fluorescence, Control, Immunocytochemistry, Microscopy, Software

Journal: Oxidative Medicine and Cellular Longevity

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

doi: 10.1155/2016/4148791

Figure Lengend Snippet: 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 μ M tBHQ (T) for 4 h, before being processed for immunoblotting using relevant antibodies . 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 μ g PGL3 basic vector with a cloned 8 x cis -antioxidant response elements (ARE) driving NRF2 dependent 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 200 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). 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 #: p < 0.05, ∗∗ or ##: p < 0.01, and ∗∗∗ or ###: p < 0.001.

Article Snippet: On the day of assay, 20 μ L of the 5 mg/mL MTT stock was added to each well and plate was further incubated for 4 h. Following this, the old media with MTT were removed, cells were gently washed with prewarmed PBS, and 100 μ L of DMSO was added to solubilise the internalised MTT by shaking over an orbital shaker for 15 min. Absorbance of the released dye was measured and recorded using multiplate reader (MODULUS, Promega) at 540 nm.

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

Journal: Oxidative Medicine and Cellular Longevity

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

doi: 10.1155/2016/4148791

Figure Lengend Snippet: NRF2 knockdown causes downregulation of HER2 and HER3 levels, repression of pAKT, and sensitisation to targeted immunotherapeutics. (a) Immunoblotting analysis showing inhibition of RTK signaling following depletion of NRF2 mRNA by SiRNA in SKOV3 cell line. Exponentially growing cells were either transfected with scrambled SiRNA (Scrmbl) or transfected with 75 pmol of NRF2 SiRNA for either 24 or 48 h or 100 pmol of NRF2 SiRNA for 48 h and processed for immunoblotting using relevant antibodies . β -actin was used as a loading control. Bar chart shows protein levels by quantifying immunoblot signal intensities obtained and normalised to the value of untreated (UT) control and expressed as fold change. (b) Immunofluorescent labelling of endogenous total HER2 or phospho-AKT exhibits repression following NRF2 knockdown. Cells were transfected as in (a) and processed for immunocytochemistry. Relevant primary antibodies were used to stain HER2 or phospho-AKT followed by Alexa Fluor conjugated secondary antibody (red fluorescence). Nuclear reference was provided by costaining with 4′,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI). Images were captured with Leica DMiRe2 electronic microscope at 100x objective while merging, colocalisation, and further analysis were performed by using integrated features of Andor iQ Core software (ANDOR Technologies Ltd.). Scale bar indicates 10 μ m. (c and d) HER2 and HER3 downregulation following NRF2 knockdown is caused by their transcriptional repression. Exponentially growing PEO1 cells (c) or SKOV3 cells (d) were transfected with either empty PGL3 basic vector or 1 μ g PGL3 basic vector with cloned 1.5 kb fragments of either HER2 (prHER2) or HER3 (prHER3) upstream promoter regions driving the expression of luciferase gene. Cotransfection with 0.2 μ g pRL-CMV plasmid was performed as an internal transfection control. At 24 h after transfection, cells were either left untreated (UT) or treated with 200 μ M tBHQ as indicated for 4 h following which, cells were processed for dual luciferase reporter assay (Promega) to record luciferase activity in multiplate reader (MODULUS, Promega). (d) The same was done for SKOV3 cell lines. (e) Knockdown of NRF2 through SiRNA sensitises cancer cell to RTK inhibitors while parallel knockdown of KEAP1 partially relieves this sensitisation. Cells were transfected with scrambled SiRNA or SiRNA targeting NRF2 either alone or with the inclusion of KEAP1 SiRNA. Following further 24 h incubation, cells were either left untreated or treated with 25 μ g/mL of HER2 inhibitors Pertuzumab and Trastuzumab. Cytotoxicity assay was performed as in (a). In (c–e), data are the means with ±S.D. of triplicates and expressed as fold change with statistical significance determined by ONE WAY ANOVA followed by Tukey's post hoc test (for c and d), or Student's t -test (for e) according to the scale ∗ : p < 0.05, ∗∗ : p < 0.01, and ∗∗∗ : p < 0.001.

Article Snippet: On the day of assay, 20 μ L of the 5 mg/mL MTT stock was added to each well and plate was further incubated for 4 h. Following this, the old media with MTT were removed, cells were gently washed with prewarmed PBS, and 100 μ L of DMSO was added to solubilise the internalised MTT by shaking over an orbital shaker for 15 min. Absorbance of the released dye was measured and recorded using multiplate reader (MODULUS, Promega) at 540 nm.

Techniques: Knockdown, Western Blot, Inhibition, Transfection, Control, Immunocytochemistry, Staining, Fluorescence, Microscopy, Software, Plasmid Preparation, Clone Assay, Expressing, Luciferase, Cotransfection, Reporter Assay, Activity Assay, Incubation, Cytotoxicity Assay

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