Journal: International Journal of Molecular Sciences

Article Title: Modulation of the Receptor Tyrosine Kinase TIE2/ Tek Pathway by NRF2 Activation in Neurovascular Endothelial Cells

doi: 10.3390/ijms27020770

Figure Lengend Snippet: Reduced TIE2/ Tek levels with SFN are dependent on NRF2 activity. ( A ) bEnd.3 cells were maintained under low-serum conditions (16 h, 1% FBS) and subjected to SFN (5 µM, 16 h). Expression levels of 84 endothelial genes were analyzed by quantitative real-time PCR (qRT-PCR) and normalized to the geometric mean of Actb , Gapdh , B2m , Gusb , and Hsp90ab1 levels. Volcano plot comparing gene expression of vehicle- versus SFN-treated cells. A fold of change greater than 1.5 is represented by yellow (increased expression when compared to vehicle-treated cells) and blue (decreased expression when compared to vehicle-treated cells) dots. Data are the mean of n = 4. Statistical analysis was performed with the GeneGlobe Data Analysis Center from Qiagen. A p -value < than 0.05 was considered significant and is represented by a line. A red square highlights the Tek gene. ( B ) Representative immunoblots of NRF2 (arrowhead), TIE2, CLDN5, HO-1, and VCL, and GAPDH as a loading control from bEnd.3 cells maintained under low-serum conditions (16 h, 1% FBS) and treated with SFN (5 µM, 16 h). ( C ) Densitometric quantification of NRF2, HO-1, TIE2, and CLDN5 protein levels from representative immunoblots of B expressed as a ratio of VCL and GAPDH, respectively. Data are mean ± S.D. (n = 3). * p < 0.05 and *** p < 0.001 vs. vehicle according to Student’s t -test. ( D ) bEnd.3 cells were maintained under low-serum conditions (16 h, 1% FBS) and subjected to SFN (5 µM, 16 h). Transcript levels of Hmox1 , Nqo1 , Slc7a11 , Tek , Cldn5 , Cdh5 , Ocln , and Tjp1 were determined by qRT-PCR and normalized to the geometric mean of the levels of Gapdh , Tbp , and Actb . Data are mean ± S.D. (n = 3). * p < 0.05 and *** p < 0.001 vs. vehicle according to Student’s t -test. ( E ) Confocal analysis of double immunofluorescence with anti-TIE2 (green) and anti-CLDN5 (red) antibodies in vehicle (Veh.) or SFN-treated bEnd.3 cells. ( F ) Quantification of TIE2 and CLDN5 intensity signals in E. Values are mean ± SD (n = 3, with >50 cells counted per field). * p < 0.05 vs. VEH-treated cells according to Student’s t -test. ( G ) bEnd.3 cells were transduced with lentiviral vectors carrying short hairpin RNA against NRF2 (shNRF2) or a scramble sequence (shCTRL). Five days post-transduction, cells were maintained under low-serum conditions (16 h, 1% FBS) and subjected to SFN (5 µM, 16 h). Representative immunoblots of NRF2 (arrowhead), TIE2, CLDN5, HO-1, and LMNB, and GAPDH as a loading control. ( H ) Densitometric quantification of NRF2, HO-1, TIE2, and CLDN5 protein levels from representative immunoblots of G expressed as a ratio of LMNB and GAPDH, respectively. Data are mean ± S.D. (n = 3). * p < 0.05 and ** p < 0.01 vs. vehicle, or # p < 0.05 vs. shCTRL according to Student’s t -test. ( I ) Transcript levels of Nfe2l2 , Hmox1 , Nqo1 , and Slc7a11 and ( J ) of Tek , Cldn5 , Cdh5 , Ocln , and Tjp1 from bEnd.3 cells transduced with shNRF2 or shCTRL maintained under low-serum conditions (16 h, 1% FBS) and subjected to SFN (5 µM, 16 h) were determined by qRT-PCR and normalized to the geometric mean of the levels of Gapdh , Tbp , and Actb . Data are mean ± S.D. (n = 3). * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. vehicle, or # p < 0.05 and ### p < 0.001 vs. shCTRL according to Student’s t -test.

Article Snippet: Lentiviral vector pLKO.1 shRNA control (shCtrl) (Addgene Plasmid #10879) was purchased from Addgene; pLKO-puro shNRF2 (NM_010902) was purchased from Sigma-Aldrich.

Techniques: Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Gene Expression, Western Blot, Control, Immunofluorescence, Transduction, shRNA, Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Modulation of the Receptor Tyrosine Kinase TIE2/ Tek Pathway by NRF2 Activation in Neurovascular Endothelial Cells

doi: 10.3390/ijms27020770

Figure Lengend Snippet: Overexpression of NRF2 alters TIE2/ Tek levels. ( A ) bEnd.3 cells were transduced with lentiviral vectors carrying overexpressed NRF2 insensitive to KEAP1 degradation (NRF2 ∆ETGE ) or a lentivirus empty vector. Five days post-transduction, cells were maintained under low-serum conditions (16 h, 1% FBS). Representative immunoblots of NRF2 (arrowhead), TIE2, CLDN5, HO-1, and VCL, and GAPDH as a loading control. ( B ) Densitometric quantification of NRF2, HO-1, TIE2, and CLDN5 protein levels from representative immunoblots of A expressed as a ratio of VCL and GAPDH, respectively. Data are mean ± S.D. (n = 3). * p < 0.05 and *** p < 0.001 vs. empty vector according to Student’s t -test. ( C ) Transcript levels of Hmox1 , Nqo1 , and Slc7a11 and ( D ) of Tek , Cldn5 , Cdh5 , Ocln , and Tjp1 from bEnd.3 cells transduced with NRF2 ∆ETGE or empty vector maintained under low-serum conditions (16 h, 1% FBS) were determined by qRT-PCR and normalized to the geometric mean of the levels of Gapdh , Tbp , and Actb . Data are mean ± S.D. (n = 3). ** p < 0.01 and *** p < 0.001 vs. empty vector according to Student’s t -test. ( E ) bEnd.3 cells were transduced with lentiviral vectors NRF2 ∆ETGE or an empty vector. Five days post-transduction, cells were maintained under low-serum conditions (16 h, 1% FBS) and were subjected to ANGPT1 (400 ng/mL) purified from supernatant HEK293T-stable expression (CMP-ANGPT1) to the indicated time points. Representative immunoblots of NRF2 (arrowhead), TIE2, CLDN5, HO-1, and VCL, and GAPDH as a loading control. ( F ) Densitometric quantification of NRF2, TIE2, and CLDN5 protein levels from representative immunoblots of E expressed as a ratio of VCL and GAPDH, respectively. Data are mean ± S.D. (n = 3). * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. time 0 according to a one-way ANOVA followed by a Bonferroni post hoc test.

Article Snippet: Lentiviral vector pLKO.1 shRNA control (shCtrl) (Addgene Plasmid #10879) was purchased from Addgene; pLKO-puro shNRF2 (NM_010902) was purchased from Sigma-Aldrich.

Techniques: Over Expression, Transduction, Plasmid Preparation, Western Blot, Control, Quantitative RT-PCR, Purification, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Modulation of the Receptor Tyrosine Kinase TIE2/ Tek Pathway by NRF2 Activation in Neurovascular Endothelial Cells

doi: 10.3390/ijms27020770

Figure Lengend Snippet: Repression of TIE2/ Tek levels by NRF2 is not dependent on BACH1. ( A ) Subcellular fraction from bEnd.3 maintained under low-serum conditions (16 h, 1% FBS) and subjected to SFN (5 µM) and hemin (10 µM) treatments for 16 h. Representative immunoblots of NRF2 (arrowhead), BACH1, TIE2, CLDN5, HO-1, and LMNB, and GAPDH as a loading control. Densitometric quantification of BACH1 ( B ), NRF2 nuclear ( C ), TIE2 cytosol ( D ), and HO-1 cytosol ( E ) protein levels from representative immunoblots of A expressed as a ratio of LMNB for nuclear fraction and GAPDH for cytosol fraction, respectively. Data are mean ± S.D. (n = 3). * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. vehicle according to Student’s t -test. ( F , G ) bEnd.3 cells were maintained under low-serum conditions (16 h, 1% FBS) and subjected to SFN (5 µM) and hemin (10 µM) treatments for 16 h. Transcript levels of Hmox1 , Nqo1 , and Slc7a11 ( F ) and Tek , Bach1 , and Cldn5 ( G ) were determined by qRT-PCR and normalized to the geometric mean of the levels of Gapdh , Tbp , and Actb . Data are mean ± S.D. (n = 3). * p < 0.05, ** p < 0.05 and *** p < 0.001 vs. vehicle according to Student’s t -test. ( H ) bEnd.3 cells were transduced with lentiviral vectors carrying short hairpin RNA against BACH1 (shBACH1) or a scramble sequence (shCTRL). Five days post-transduction, cells were maintained under low-serum conditions (16 h, 1% FBS) and subjected to SFN (5 µM, 16 h). Representative immunoblots of NRF2 (arrowhead), BACH1, TIE2, CLDN5, HO-1, and VCL, and GAPDH as a loading control. ( I ) Densitometric quantification of NRF2, BACH1, HO-1, TIE2, and CLDN5 protein levels from representative immunoblots of H expressed as a ratio of VCL and GAPDH, respectively. Data are mean ± S.D. (n = 3). * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. vehicle, or ### p < 0.001 vs. shCTRL according to Student’s t -test. ( J ) Transcript levels of Hmox1 , Nqo1 , Bach1 , Tek , and Cldn5 from bEnd.3 cells transduced with shBACH1 or shCTRL maintained under low-serum conditions (16 h, 1% FBS) and subjected to SFN (5 µM, 16 h) were determined by qRT-PCR and normalized to the geometric mean of the levels of Gapdh , Tbp , and Actb . Data are mean ± S.D. (n = 3). * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. vehicle, or # p < 0.05 and ### p < 0.001 vs. shCTRL according to Student’s t -test. ns indicates not significant.

Article Snippet: Lentiviral vector pLKO.1 shRNA control (shCtrl) (Addgene Plasmid #10879) was purchased from Addgene; pLKO-puro shNRF2 (NM_010902) was purchased from Sigma-Aldrich.

Techniques: Western Blot, Control, Quantitative RT-PCR, Transduction, shRNA, Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Modulation of the Receptor Tyrosine Kinase TIE2/ Tek Pathway by NRF2 Activation in Neurovascular Endothelial Cells

doi: 10.3390/ijms27020770

Figure Lengend Snippet: NRF2 does not modify Tek mRNA stability and does not bind its promoter. ( A ) bEnd.3 were maintained under low-serum conditions (16 h, 1% FBS), pre-treated with SFN (5 µM, 6 h), and subsequently subjected to actinomycin D (5 µg/mL) or sustained SFN (5 µM) at the indicated time points. The graph depicts the natural logarithm of the relative levels of the Tek mRNA as a function of actinomycin D or actinomycin D/SFN incubation time, normalized to the geometric mean of the levels of Gapdh , Tbp , and Actb . The mRNA half-life was determined using the linear part of the degradation curve. Data are mean ± S.D. (n = 3). No statistically significant differences ( p ≤ 0.05) were detected between vehicle- and SFN-treated groups at any of the analyzed time points according to a two-way ANOVA followed by a Bonferroni post hoc test. ( B ) Representative immunoblots of NRF2 (arrowhead) and TIE2, as well as VCL as a loading control, from bEnd.3 were maintained under low-serum conditions (16 h, 1% FBS), pre-treated with SFN (5 µM, 6 h), and subsequently subjected to CHX (100 µM) or sustained SFN (5 µM) at the indicated time points. ( C ) The graph depicts the natural logarithm of the relative levels of the TIE2 protein as a function of CHX or CHX/SFN incubation time from representative immunoblots of A normalized to VCL. The protein half-life was determined using the linear part of the degradation curve. Data are mean ± S.D. (n = 3). No statistically significant differences ( p ≤ 0.05) were detected between vehicle- and SFN-treated groups at any of the analyzed time points according to a two-way ANOVA followed by a Bonferroni post hoc test. ( D ) bEnd.3 cells were transduced with lentiviral vectors carrying overexpressed NRF2 insensitive to KEAP1 degradation (NRF2 ∆ETGE ) or a lentivirus empty vector. Five days post-transduction, cells were maintained under low-serum conditions (16 h, 1% FBS). Chromatin immunoprecipitation (ChIP) analysis was performed with anti-IgG or anti-PolII antibodies, and the potential AREs in Tek with the highest were analyzed by qRT-PCR. The figure shows representative data normalized as the fold of enrichment with the anti-PolII antibody vs. the IgG antibody. The presence of already known AREs in Hmox1 (ARE1), Hmox1 (ARE2), and Nqo1 was analyzed as a positive control, and Actb was amplified as a negative control.

Article Snippet: Lentiviral vector pLKO.1 shRNA control (shCtrl) (Addgene Plasmid #10879) was purchased from Addgene; pLKO-puro shNRF2 (NM_010902) was purchased from Sigma-Aldrich.

Techniques: Incubation, Western Blot, Control, Transduction, Plasmid Preparation, Chromatin Immunoprecipitation, Quantitative RT-PCR, Positive Control, Amplification, Negative Control