nrf2  (R&D Systems)

Journal: Nutrients

Article Title: Antioxidant and Anti-Inflammatory Properties of Rubber Seed Oil in Lipopolysaccharide-Induced RAW 267.4 Macrophages.

doi: 10.3390/nu14071349

Figure Lengend Snippet: Figure 3. Effect of RSO on Nrf2-Keap1 pathway of LPS-induced RAW 264.7 cells. Relative mRNA expression of Nrf2 (A), HO-1 (B), NQO1 (C), and Keap1 (D) genes normalized to β-actin and expressed as fold change of the control group, n = 3. (E) The protein levels of Nrf2 and Keap1 were analyzed by immunoblotting with β-actin as the internal reference protein for normalization, n = 3. Relative protein expression of Nrf2 (F) and Keap1 (G) were expressed as fold change of the LPS-induced group. ** p < 0.01 vs control group; # p < 0.05, ## p < 0.01 vs LPS-induced group.

Article Snippet: Human/Mouse/Rat Keap1 antibody (MAB3024) and Human/Mouse Nrf2 antibody (AF3925) were from R&D Systems (Minneapolis, MI, USA).

Techniques: Expressing, Control, Western Blot

Journal: Frontiers in Molecular Biosciences

Article Title: Nrf2 Modulates the Hybrid Epithelial/Mesenchymal Phenotype and Notch Signaling During Collective Cancer Migration

doi: 10.3389/fmolb.2022.807324

Figure Lengend Snippet: Nrf2 regulates the Notch signaling pathway in the migrating front. (A – C) Immunocytochemistry of RT4 bladder cancer cells measuring Jagged1 (left column), Notch1 (center column), and Dll4 (right column) protein levels in a cell migration assay for (A) CRISPR/Cas9 NFE2L2-KO Pool RT4 cells (KO), (B) RT4 cells (Control), and (C) sulforaphane treated (7.5 μM, 24 h) RT4 cells (SFN), respectively. Scale bars, 50 μm. (D – F) Quantification of immunocytochemistry data. (D) Average intensity of each cell in the whole monolayer. One-Way ANOVA with post-hoc Tukey’s test was used to compare across groups (Jagged1: KO n = 307 cells, Control n = 526 cells, SFN n = 423 cells. Notch1: KO n = 307 cells, Control n = 370 cells, SFN n = 423 cells. Dll4: KO n = 460 cells, Control n = 1135 cells, SFN n = 570 cells). (E) Tracing of relative fluorescence intensity per tissue depth measured as number of cell layers. Data represent mean ± S.E.M. (F) Heatmap of representative cells at the leading edge for Jagged1, Notch1, and Dll4, respectively. Each cell at the leading edge is indicated by “Cell #” where cell #1 refers to the first measured cell from top to bottom. Results are from four experiments performed across different days.

Article Snippet: Primary antibodies used were Nrf2 (1:100, cat. #AF3925; R&D Biosystems), E-cadherin (1:50, cat. #M3612; Agilent Dako), ZEB1 (1:100, cat. #ab124512; Abcam), Jagged1 (1:50, cat. #sc-390177; Santa Cruz Biotechnology), Notch1 (1:100, cat. #ab8925; Abcam), Dll4 (1:100, cat. #PA585931; Thermofisher Scientific).

Techniques: Immunocytochemistry, Cell Migration Assay, CRISPR, Control, Fluorescence

Journal: Frontiers in Molecular Biosciences

Article Title: Nrf2 Modulates the Hybrid Epithelial/Mesenchymal Phenotype and Notch Signaling During Collective Cancer Migration

doi: 10.3389/fmolb.2022.807324

Figure Lengend Snippet: Nrf2 modulations impair collective migration in a 2D model. (A) Bright-field images of CRISPR/Cas9 NFE2L2-KO Pool RT4 cells (KO), RT4 cells (Control) and sulforaphane treated (7.5 μM, 24 h) RT4 cells (SFN) for 0 and 48 h migration time points. Scale bars, 100 μm. (B) Violin plot of migration rate for KO, control, and SFN, respectively. One-way ANOVA with post-hoc Tukey test was used to compare across groups and the ROUT method was used to identify outliers (KO n = 44 leading edges, Control n = 91 leading edges, SFN n = 78 leading edges). (C) Representative images illustrating the formation of migration tips. Scale bars, 100 μm. (D) Violin plot of protrusions/mm for KO, control, and SFN, respectively. One-way ANOVA with post-hoc Tukey test was used to compare across groups and the ROUT method was used to identify outliers (KO n = 43 leading edges, Control n = 110 leading edges, SFN n = 86 leading edges). Results are from four experiments performed across different days.

Article Snippet: Primary antibodies used were Nrf2 (1:100, cat. #AF3925; R&D Biosystems), E-cadherin (1:50, cat. #M3612; Agilent Dako), ZEB1 (1:100, cat. #ab124512; Abcam), Jagged1 (1:50, cat. #sc-390177; Santa Cruz Biotechnology), Notch1 (1:100, cat. #ab8925; Abcam), Dll4 (1:100, cat. #PA585931; Thermofisher Scientific).

Techniques: Migration, CRISPR, Control

Journal: Frontiers in Molecular Biosciences

Article Title: Nrf2 Modulates the Hybrid Epithelial/Mesenchymal Phenotype and Notch Signaling During Collective Cancer Migration

doi: 10.3389/fmolb.2022.807324

Figure Lengend Snippet: Spatial patterning of cells in the multicell model of cell migration. (A) Left: In the multicell model, cells are arranged on a hexagonal lattice. Cells at the leftmost region (leading edge) are highly exposed to an external EMT-inducing signal (indicated by the blue shading), while cells in the interior are weakly exposed. Right: The signaling dynamics within each cell is described by the coupled biochemical network of Nrf2, EMT and Notch. Binding between Notch ligands and receptors of neighboring cells give rise to cell-cell communication. (B) Snapshot of the multicell pattern after 120 h of simulation starting from a randomized initial condition for a case of weak Nrf2 activation ( g N R F 2 = 0 molecules/hour). Green, yellow and red hexagons depict epithelial (E), hybrid E/M and mesenchymal (M) cells, respectively. (C) Fraction of E, E/M, and M cells as a function of distance from the leading edge for weak Nrf2 activation. Black dashed line indicates the M-E/M crossover point. (D , E) Same as (B , C) for intermediate Nrf2 activation ( g N R F 2 = 0.5 × 10 5 molecules/hour). (F , G) Same as (B , C) for strong Nrf2 activation ( g N R F 2 = 10 5 molecules/hour). (H) Crossover point where the fraction of hybrid E/M cells becomes larger than the fraction of M cells as a function of Nrf2 production rate. (I) Comparison of ZEB1 levels between simulation (red line) and control experiment (black line) in the first 13 cell layers. (J) Fold-change in ZEB1 levels between the first and second cell layers as a function of Nrf2 production rate. Result for panels C-E-G-H-I-J are averaged over five independent simulations.

Article Snippet: Primary antibodies used were Nrf2 (1:100, cat. #AF3925; R&D Biosystems), E-cadherin (1:50, cat. #M3612; Agilent Dako), ZEB1 (1:100, cat. #ab124512; Abcam), Jagged1 (1:50, cat. #sc-390177; Santa Cruz Biotechnology), Notch1 (1:100, cat. #ab8925; Abcam), Dll4 (1:100, cat. #PA585931; Thermofisher Scientific).

Techniques: Migration, Binding Assay, Activation Assay, Comparison, Control

Journal: Frontiers in Molecular Biosciences

Article Title: Nrf2 Modulates the Hybrid Epithelial/Mesenchymal Phenotype and Notch Signaling During Collective Cancer Migration

doi: 10.3389/fmolb.2022.807324

Figure Lengend Snippet: Nrf2 modulates EMT near the leading edge during collective migration. (A – C) Immunocytochemistry of RT4 bladder cancer cells measuring Nrf2 (left column), ZEB1 (center column), and E-cadherin (right column) protein levels in a cell migration assay for (A) CRISPR/Cas9 NFE2L2-KO Pool RT4 cells (KO), (B) RT4 cells (Control), and (C) sulforaphane treated (7.5 μM, 24 h) RT4 cells (SFN), respectively. Scale bars, 50 μm. (D – F) Quantification of immunocytochemistry data. (D) Average intensity of each cell in the whole monolayer. One-way ANOVA with post-hoc Tukey’s test was used to compare across groups (NRF2: KO n = 598 cells, Control n = 503 cells, and SFN n = 564 cells. ZEB1: KO n = 385 cells, Control n = 1267 cells, and SFN n = 1166 cells. E-cadherin: KO n = 385 cells, Control n = 1268 cells, and SFN n = 1166 cells). (E) Intensity distribution in the migrating monolayer measured as number of cell layers (position from the leading edge). Data represent mean ± S.E.M. (F) Heatmap of representative cells at the leading edge for Nrf2, E-cadherin, and ZEB1, respectively. Each cell at the leading edge is indicated by “Cell #” where cell #1 refers to the first measured cell from top to bottom. Results are from six experiments performed across different days.

Article Snippet: Primary antibodies used were Nrf2 (1:100, cat. #AF3925; R&D Biosystems), E-cadherin (1:50, cat. #M3612; Agilent Dako), ZEB1 (1:100, cat. #ab124512; Abcam), Jagged1 (1:50, cat. #sc-390177; Santa Cruz Biotechnology), Notch1 (1:100, cat. #ab8925; Abcam), Dll4 (1:100, cat. #PA585931; Thermofisher Scientific).

Techniques: Migration, Immunocytochemistry, Cell Migration Assay, CRISPR, Control

Journal: Frontiers in Molecular Biosciences

Article Title: Nrf2 Modulates the Hybrid Epithelial/Mesenchymal Phenotype and Notch Signaling During Collective Cancer Migration

doi: 10.3389/fmolb.2022.807324

Figure Lengend Snippet: Nrf2 upregulation modulates microRNA miR-200c-3p and Notch1 and Dll4 mRNA in the migrating front. (A – C) Live single cell gene expression measurements with the dsLNA probes in RT4 cells. From left to right: fluorescence images of control and SFN (7.5 μM, 24 h) cases, tracing of relative fluorescence intensity per tissue depth measured as number of cell layers, and heatmap of representative cells at the leading edge measuring (A) microRNA miR-200c-3p, (B) Notch1 mRNA, and (C) Dll4 mRNA levels in a cell migration assay. (D – F) FISH assay in fixed RT4 cells. From left to right: fluorescence images of control and SFN cases, tracing of relative fluorescence intensity per tissue depth measured as number of cell layers, and heatmap of representative cells at the leading edge measuring (D) microRNA miR-200c-3p, (E) Notch1 mRNA, and (F) Dll4 mRNA levels in a scratch cell migration assay. Scale bars, 50 μm. Each cell at the leading edge is indicated by “Cell #” where cell #1 refers to the first measured cell from top to bottom. Data represent mean ± S.E.M. The two-way ANOVA with the post-hoc Tukey test was performed to compare across groups. At least 500 cells were analyzed for each condition (miRNA-200c-3p, Notch1 mRNA, and Dll4 mRNA). Results are from three experiments performed across different days.

Article Snippet: Primary antibodies used were Nrf2 (1:100, cat. #AF3925; R&D Biosystems), E-cadherin (1:50, cat. #M3612; Agilent Dako), ZEB1 (1:100, cat. #ab124512; Abcam), Jagged1 (1:50, cat. #sc-390177; Santa Cruz Biotechnology), Notch1 (1:100, cat. #ab8925; Abcam), Dll4 (1:100, cat. #PA585931; Thermofisher Scientific).

Techniques: Gene Expression, Fluorescence, Control, Cell Migration Assay

Journal: Frontiers in Molecular Biosciences

Article Title: Nrf2 Modulates the Hybrid Epithelial/Mesenchymal Phenotype and Notch Signaling During Collective Cancer Migration

doi: 10.3389/fmolb.2022.807324

Figure Lengend Snippet: Analysis of the leading edge by the multicell model. (A) To conduct leading edge analysis, the expression of miR-200, Notch, Dll4 and Jagged1 is analyzed in the leftmost layer of cells (i.e., the cell layer more exposed to EMT-inducing signal and thus comparable to the experimental leading edge). (B) Heatmap of expression levels for miR-200, Notch1, Dll4 and Jagged1 in the leading edge as a function of Nrf2 activation. Each column represents the lattice leading edge for a different level of Nrf2 production rate ( g N r f 2 ). (C) Log-normalized probability to observe cells with varying levels of Dll4 and Jagged1 in the model’s leading edge under low Nrf2 induction. (D) Fraction of Epithelial, hybrid E/M, and Mesenchymal cells in the leading edge as a function of Nrf2 production rate ( g N r f 2 ). For panels (C , D) , results are averaged over five simulations starting from randomized initial conditions.

Article Snippet: Primary antibodies used were Nrf2 (1:100, cat. #AF3925; R&D Biosystems), E-cadherin (1:50, cat. #M3612; Agilent Dako), ZEB1 (1:100, cat. #ab124512; Abcam), Jagged1 (1:50, cat. #sc-390177; Santa Cruz Biotechnology), Notch1 (1:100, cat. #ab8925; Abcam), Dll4 (1:100, cat. #PA585931; Thermofisher Scientific).

Techniques: Expressing, Activation Assay

Journal: Frontiers in Molecular Biosciences

Article Title: Nrf2 Modulates the Hybrid Epithelial/Mesenchymal Phenotype and Notch Signaling During Collective Cancer Migration

doi: 10.3389/fmolb.2022.807324

Figure Lengend Snippet: Leader cell formation at the leading edge. (A) Representative bright-field images of leader cells after a 24 h cell migration assay for CRISPR/Cas9 NFE2L2-KO Pool RT4 cells (KO), RT4 cells (Control) and sulforaphane treated (7.5 μM, 24 h) RT4 cells (SFN), respectively. (B) Plot showing leader cells per millimeter for CRISPR/Cas9 NFE2L2-KO Pool RT4 cells (KO), RT4 cells (Control) and sulforaphane treated RT4 cells (SFN), respectively. One-way ANOVA with post-hoc Tukey’s test was used to compare across groups and the ROUT method was used to identify outliers (KO n = 46, Control n = 100, SNF n = 86 leading edges). (C – H) Representative immunocytochemistry images of leader cells in the control case (RT4 cells) characterizing gene expression for (C) E-cadherin, (D) Jagged1, (E) ZEB1, (F) Notch1, (G) Nrf2, (H) Dll4. (I , J) Representative overlay images using the dsLNA biosensors to measure mRNA levels of (I) Dll4 and (J) Notch1, respectively. (K , L) Mean intensity of leader vs. follower cells for levels of (K) Dll4 mRNA ( n = 6 follower cells) and (L) Notch1 mRNA ( n = 6 follower cells). (K , L) represent the quantification of one leader cell and its follower cells. Immunocytochemistry data are from four experiments, and dsLNA biosensor data are from three experiments. Scale bars, 20 μm.

Article Snippet: Primary antibodies used were Nrf2 (1:100, cat. #AF3925; R&D Biosystems), E-cadherin (1:50, cat. #M3612; Agilent Dako), ZEB1 (1:100, cat. #ab124512; Abcam), Jagged1 (1:50, cat. #sc-390177; Santa Cruz Biotechnology), Notch1 (1:100, cat. #ab8925; Abcam), Dll4 (1:100, cat. #PA585931; Thermofisher Scientific).

Techniques: Cell Migration Assay, CRISPR, Control, Immunocytochemistry, Gene Expression