Journal: Journal of Veterinary Science

Article Title: Epigallocatechin-3-gallate suppresses hemin-aggravated colon carcinogenesis through Nrf2-inhibited mitochondrial reactive oxygen species accumulation

doi: 10.4142/jvs.22097

Figure Lengend Snippet: WST, water-soluble tetrazolium salt; EGCG, epigallocatechin-3-gallate; Nrf2, nuclear factor erythroid-2-related factor 2. * p < 0.05.

Article Snippet: Nrf2 (NBP1-32822) and Keap1 (NBP2-03319) antibodies were purchased from Novus Biologicals (Centennial, CO, USA).

Techniques:

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting of a novel interplay between MET tyrosine kinase and NRF2 enhances sensitivity to Paclitaxel in triple negative breast cancer

doi: 10.1186/s13046-025-03625-y

Figure Lengend Snippet: High expression levels of RTKs and NRF2 correlate with worst prognosis in TNBC patients. A Volcano plot for Spearman correlation between NRF2 expression and indicated RTKs in BC samples from TCGA-BRCA dataset. Significant positive correlations are shown in red, and negative correlations in blue. Common RTKs are highlighted. B Expression levels of MET and EGFR across the four different BC subtypes from the TCGA dataset. Kaplan–Meier curves showing the probability of overall survival in TNBC patients with different expression levels of MET/NRF2 (left) and EGFR/NRF2 (right) ( C ), AXL , TGFBR1 , PDGFRA , and NRF2 ( D ). Statistical analysis: ( B ) One-way ANOVA followed by Tukey’s multiple comparison statistical test was performed. C-D Survival data derived from GSE31519 dataset for TNBC patients. P values were computed using the Logrank (Mantel Cox) test. ns: not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: For transient silencing of MET expression (MGT-13 and BT-549) and NRF2 expression (MGT-13), cells were seeded the day before and transfected for 24 h with LipofectamineTM RNAiMAX Transfection Reagent (Invitrogen) by using murine and human MET siRNA (Santa Cruz Biotechnology; sc-35924, sc-29397) or NRF2 siRNA (Santa Cruz Biotechnology; sc-37049) according to the manufacturer’s instructions.

Techniques: Expressing, Comparison, Derivative Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting of a novel interplay between MET tyrosine kinase and NRF2 enhances sensitivity to Paclitaxel in triple negative breast cancer

doi: 10.1186/s13046-025-03625-y

Figure Lengend Snippet: Inhibition of the MET receptor reduces NRF2 nuclear localization and activity in TNBC MMTV-R26 Met cell lines. A Immunoblotting (left) of pY 1234/35 MET, MET, NRF2, and relative densitometric analysis (right) of NRF2 protein levels in all MGT cell lines derived from the MMTV-R26 Met tumours. Actin was used as a loading control. B Immunoblotting (left) of pY 1234/35 MET, MET, NRF2, and relative densitometric (right) analysis of NRF2 protein levels in the MGT-13 cell line upon 16 h of PHA treatment. Actin was used as a loading control. C Immunofluorescence (left) and relative quantification (right) of NRF2 (red) nuclear intensity in MGT-13 cells upon 16 h of PHA treatment. DNA (Hoechst, blue). D Immunoblotting (top) and relative densitometric analysis (bottom) of NRF2 cytosolic and nuclear fractions in MGT-13 cells upon 16 h of PHA treatment. Vinculin and Lamin A/C were used as loading and quality controls. ( E ) RT-qPCR of NRF2 target genes in MGT-13 cells after 16 h of PHA treatment. Actin was used as housekeeping gene. F Workflow of RNA-seq experiments. p-value ≤ 0.5 and Log 2 foldchange (log₂FC) > 0.7 were used as threshold for analysis. G Activation z-score for transcription factors (CTRL vs. PHA). Values lower than − 2 (blue bars) suggest an inhibition of the pathway related to the corresponding transcription factor in the PHA treatment. Values over 2 (red bars) suggest an activation of the pathway in PHA samples. NFE2L2 (NRF2) is indicated in bold. Data from IPA. H Heatmap of z-scores for genes involved in the NRF2 pathway (according to IPA) for CTRL vs. PHA (3 samples for each condition, see x axis). Positive values of z-score (red) indicate upregulation within the sample, negative values (blue) downregulation. Results represent the mean of at least three independent experiments (± SEM or ± SD). Statistical analysis: ( A-B-D ) Unpaired t -test. C Mann-Whitney test according to the normal distribution. E Multiple t- test. PHA: PHA-665752 1 µM, MET inhibitor. ns: not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: For transient silencing of MET expression (MGT-13 and BT-549) and NRF2 expression (MGT-13), cells were seeded the day before and transfected for 24 h with LipofectamineTM RNAiMAX Transfection Reagent (Invitrogen) by using murine and human MET siRNA (Santa Cruz Biotechnology; sc-35924, sc-29397) or NRF2 siRNA (Santa Cruz Biotechnology; sc-37049) according to the manufacturer’s instructions.

Techniques: Inhibition, Activity Assay, Western Blot, Derivative Assay, Control, Immunofluorescence, Quantitative Proteomics, Quantitative RT-PCR, RNA Sequencing, Activation Assay, MANN-WHITNEY

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting of a novel interplay between MET tyrosine kinase and NRF2 enhances sensitivity to Paclitaxel in triple negative breast cancer

doi: 10.1186/s13046-025-03625-y

Figure Lengend Snippet: Targeting of the MET receptor reduces NRF2 activity in human TNBC cell lines. A Immunoblotting analysis of pY 1234/35 MET, MET, and NRF2 in human BC cell lines. Ponceau was used as a loading control. B Immunofluorescence (left) and relative quantification analysis (right) of NRF2 (red) nuclear intensity in human TNBC cells upon 16 h of PHA treatment. DNA (Hoechst, blue). C RT-qPCR of NRF2 target genes in human TNBC cells after 16 h of PHA treatment. Actin was used as housekeeping. D Immunoblotting (top) analysis of pY 1234/35 MET, MET, and NRF2 and relative densitometric analysis (bottom) of NRF2 in human BC cell lines after 4 h of serum-free media and 10 min of HGF stimulation (50ng/mL). Vinculin was used as a loading control. E Clonogenic assays (top) and relative quantification of the number of colonies (bottom) on MDA-MB-231, BT-549 and MCF10-A cells treated with PHA and ML-385 for 72 h. Results represent the mean of at least three independent experiments (± SEM or ± SD). Statistical analysis: ( B ) Mann-Whitney test according to the normal distribution. C Multiple t -test. D Unpaired t -test. E One-way ANOVA statistical test. PHA: PHA-665752 1 or 2 µM, MET inhibitor. ML-385: 5 µM, NRF2 inhibitor. ns: not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: For transient silencing of MET expression (MGT-13 and BT-549) and NRF2 expression (MGT-13), cells were seeded the day before and transfected for 24 h with LipofectamineTM RNAiMAX Transfection Reagent (Invitrogen) by using murine and human MET siRNA (Santa Cruz Biotechnology; sc-35924, sc-29397) or NRF2 siRNA (Santa Cruz Biotechnology; sc-37049) according to the manufacturer’s instructions.

Techniques: Activity Assay, Western Blot, Control, Immunofluorescence, Quantitative Proteomics, Quantitative RT-PCR, MANN-WHITNEY

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting of a novel interplay between MET tyrosine kinase and NRF2 enhances sensitivity to Paclitaxel in triple negative breast cancer

doi: 10.1186/s13046-025-03625-y

Figure Lengend Snippet: MET or NRF2 targeting enhances Paclitaxel sensitivity of TNBC cells. Cell viability of MGT-13 ( A ) and BT-549 ( B ) cells exposed to PTX in combination with PHA or ML-385. Percentage of cell viability in presence of drugs compared to controls (untreated cells) is indicated using a colour scale (from green to red). C-D Top panel: matrix synergy plot representing the synergy/antagonism score of each combination and its statistical significance calculated by Loewe model. Bottom panel: mapped to d-r surface showing the synergy distribution of drug combinations. The synergistic analysis was performed on MGT-13 ( C ) and BT-549 ( D ) cell lines. E-F Clonogenic assays on MGT-13 cells exposed to PTX in combination with PHA ( E ) or ML-385 ( F ) and treated as in Fig. 4A. G-H Histograms of flow cytometry experiments representing the percentage of dead cells upon ANNEXIN V/DAPI + staining of MGT-13 cells exposed for 72 h to PHA and PTX ( G ) or ML-385 and PTX ( H ) treatments alone or in combination. Results represent the mean of at least three independent experiments (± SEM). Statistical analysis: ( A-B-G-H ) One-way ANOVA statistical test was performed for each combination compared to the drug alone. (In A-B, * indicate the significance respect to PTX treatment). C-D Loewe models were used for synergy score calculation by Combenefit software. PHA: PHA-665752: MET inhibitor; ML-385: NRF2 inhibitor. PTX: Paclitaxel. ns: not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: For transient silencing of MET expression (MGT-13 and BT-549) and NRF2 expression (MGT-13), cells were seeded the day before and transfected for 24 h with LipofectamineTM RNAiMAX Transfection Reagent (Invitrogen) by using murine and human MET siRNA (Santa Cruz Biotechnology; sc-35924, sc-29397) or NRF2 siRNA (Santa Cruz Biotechnology; sc-37049) according to the manufacturer’s instructions.

Techniques: Flow Cytometry, Staining, Software

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting of a novel interplay between MET tyrosine kinase and NRF2 enhances sensitivity to Paclitaxel in triple negative breast cancer

doi: 10.1186/s13046-025-03625-y

Figure Lengend Snippet: MET-NRF2 axis inhibition increases ROS levels and DNA damage accumulation. Immunoblotting ( A ) and relative densitometric analysis ( B ) of p62 and HO-1 in MGT-13 and BT-549 cells exposed for 24 h to PTX in combination with PHA. Actin was used as a loading control. C-F Cytofluorimetric analyses (top) and relative histograms (bottom) of reactive oxygen species (ROS) upon DHE staining on MGT-13 ( C-D ) and BT-549 ( E-F ) cells exposed for 24 h to PTX in combination with PHA or ML-385. Confocal microscopy analysis ( G ) and relative quantification ( H ) of 8-oxo-DG (red) foci/cell in MGT-13 cells treated alone or in combination with PTX and PHA. DNA (Hoechst, blue). Confocal microscopy analysis ( I ) and relative quantification ( J ) of γH2AX (green) positive cells in MGT-13 cells treated alone or in combination with PTX and PHA. K Immunoblotting analysis of γH2AX in MGT-13 and BT-549 treated as in ( I ). Vinculin and GAPDH were used as loading control. Results represent the mean of at least three independent experiments (± SEM or ± SD). Statistical analysis: ( B-D-F-H-J ) One-way ANOVA statistical test was performed for each combination compared to the drug alone. PHA: PHA-665752 1 or 3 µM, MET inhibitor. ML-385: 5 µM, NRF2 inhibitor. PTX: Paclitaxel 3nM or 30nM. ns: not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: For transient silencing of MET expression (MGT-13 and BT-549) and NRF2 expression (MGT-13), cells were seeded the day before and transfected for 24 h with LipofectamineTM RNAiMAX Transfection Reagent (Invitrogen) by using murine and human MET siRNA (Santa Cruz Biotechnology; sc-35924, sc-29397) or NRF2 siRNA (Santa Cruz Biotechnology; sc-37049) according to the manufacturer’s instructions.

Techniques: Inhibition, Western Blot, Control, Staining, Confocal Microscopy, Quantitative Proteomics

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting of a novel interplay between MET tyrosine kinase and NRF2 enhances sensitivity to Paclitaxel in triple negative breast cancer

doi: 10.1186/s13046-025-03625-y

Figure Lengend Snippet: MET modulates NRF2 signalling via SRC kinase-dependent p62/KEAP1 pathway. Immunoblotting ( A ) of pY 1234/35 MET, MET, pY 416 SRC, SRC and relative densitometric analyses ( B ) of pY 416 SRC normalized on total SRC in TNBC cell lines after 16 h of PHA treatment. GAPDH was used as loading control. C Immunoblotting pY 1234/35 MET, MET, pY 416 SRC, SRC, and NRF2 in MGT-13 cells transiently transfected with empty vector (pSGT) and active SRC (SRC Y527F ) after 16 h of PHA treatment. Actin was used as loading control. Immunoblotting ( D ) and relative densitometric analyses ( E ) of KEAP1, pS 349 p62 and p62 in human TNBC cell lines after 16 h of PHA treatment. Actin was used as loading control. F Confocal microscopy analyses and relative quantification of colocalizing dots of KEAP1 (red) and p62 (green) in human TNBC cell lines upon 16 h of PHA treatment. DNA (Hoechst, blue). 3X digital magnification showing merged signals. Results represent the mean of at least three independent experiments (± SEM or ± SD). Statistical analysis: ( B-E ) Unpaired t -test. F Mann-Whitney test according to the normal distribution. PHA: PHA-665752 1 µM, MET inhibitor. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: For transient silencing of MET expression (MGT-13 and BT-549) and NRF2 expression (MGT-13), cells were seeded the day before and transfected for 24 h with LipofectamineTM RNAiMAX Transfection Reagent (Invitrogen) by using murine and human MET siRNA (Santa Cruz Biotechnology; sc-35924, sc-29397) or NRF2 siRNA (Santa Cruz Biotechnology; sc-37049) according to the manufacturer’s instructions.

Techniques: Western Blot, Control, Transfection, Plasmid Preparation, Confocal Microscopy, Quantitative Proteomics, MANN-WHITNEY

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting of a novel interplay between MET tyrosine kinase and NRF2 enhances sensitivity to Paclitaxel in triple negative breast cancer

doi: 10.1186/s13046-025-03625-y

Figure Lengend Snippet: SRC expression sustains NRF2 nuclear accumulation and activity in TNBC. A Immunoblotting (top) of NRF2, pY 416 SRC, SRC and relative densitometric analysis (bottom) of NRF2 protein levels in MDA-MB-231 cells upon 16 h of DAS treatment. GAPDH was used as a loading control. B Immunofluorescence (left) and relative quantification (right) of NRF2 (red) nuclear intensity in MDA-MB-231 cells upon 16 h of DAS treatment. DNA (Hoechst, blue). C Immunoblotting (top) and relative densitometric analysis (bottom) of NRF2 cytosolic and nuclear fractions in MDA-MB-231 cells upon 16 h of DAS treatment. Vinculin and Lamin A/C were used as loading and quality controls. D RT-qPCR of NRF2 target genes in MDA-MB-231 cells after 16 h of DAS treatment. Actin was used as housekeeping gene. E Expression levels of SRC in log 2 across the four different breast cancer subtypes from the TCGA dataset. F-G Kaplan–Meier curves show the overall survival probability of TNBC ( n = 579; F ) and non-TNBC ( n = 544; G ) patients with different expression levels of SRC and NRF2 . Results represent the mean of at least three independent experiments (± SEM or ± SD). Statistical analysis: ( A-C ) Unpaired t- test. B Mann-Whitney test according to the normal distribution. D Multiple t -test. E One-way ANOVA followed by Tukey’s multiple comparison statistical test was performed. F-G Survival data obtained from Gene Expression Omninbus (GEO) Id GSE31519 for TNBC patients and TCGA dataset for non-TNBC patients. P values were computed using the Logrank (Mantel Cox). DAS: Dasatinib 50 nM; SRC inhibitor. ns: not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: For transient silencing of MET expression (MGT-13 and BT-549) and NRF2 expression (MGT-13), cells were seeded the day before and transfected for 24 h with LipofectamineTM RNAiMAX Transfection Reagent (Invitrogen) by using murine and human MET siRNA (Santa Cruz Biotechnology; sc-35924, sc-29397) or NRF2 siRNA (Santa Cruz Biotechnology; sc-37049) according to the manufacturer’s instructions.

Techniques: Expressing, Activity Assay, Western Blot, Control, Immunofluorescence, Quantitative Proteomics, Quantitative RT-PCR, MANN-WHITNEY, Comparison, Gene Expression

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting of a novel interplay between MET tyrosine kinase and NRF2 enhances sensitivity to Paclitaxel in triple negative breast cancer

doi: 10.1186/s13046-025-03625-y

Figure Lengend Snippet: MET, SRC or NRF2 targeting improves Paclitaxel efficacy in TNBC patient-derived organoids. Organoid viability assays of PDO-21, PDO-43, and PDO-46 after PHA and PTX ( A ), ML-385 and PTX ( B ), DAS and PTX ( C ) combination treatments. D 4X digital magnification of bright-field images and relative quantification of organoids size of PDO-21 culture treated as described in ( A-B-C ). Scale bar: 400 μm. E Percentage of small, medium, and large PDO-21 treated as in ( A - B - C ). F Working model. Results represent the mean of at least three independent experiments (± SEM). Statistical analysis: ( A-B-C ) One-way ANOVA statistical test was performed for each combination compared to the drug alone. E Two-way ANOVA followed by Tukey’s multiple comparison test (* respect to CTR, $ respect to inhibitors and # respect to PTX). PHA: PHA-665752, MET inhibitor. DAS: Dasatinib. ML-385: NRF2 inhibitor. PTX: Paclitaxel. ns: not significant; *, $, # p < 0.05; **, $$, ## p < 0.01; ***, $$$, ### p < 0.001; ****, $$$$, #### p < 0.0001

Article Snippet: For transient silencing of MET expression (MGT-13 and BT-549) and NRF2 expression (MGT-13), cells were seeded the day before and transfected for 24 h with LipofectamineTM RNAiMAX Transfection Reagent (Invitrogen) by using murine and human MET siRNA (Santa Cruz Biotechnology; sc-35924, sc-29397) or NRF2 siRNA (Santa Cruz Biotechnology; sc-37049) according to the manufacturer’s instructions.

Techniques: Derivative Assay, Quantitative Proteomics, Comparison

Journal: Free radical biology & medicine

Article Title: NAD(P)H:quinone oxidoreductase 1 activity reduces hypertrophy in 3T3-L1 adipocytes.

doi: 10.1016/j.freeradbiomed.2012.05.047

Figure Lengend Snippet: Fig. 2. Keap1 protein decreases in differentiated adipocytes, while steady-state mRNA expression for Nrf2 and Keap1 increases. 3T3-L1 cells were differentiated as described under Materials and methods and harvested on Days 0, 4, 8, 11, and 14. (A) Nrf2 and (C) Keap1 (70 kDa) protein expression was examined by immunoblotting. Black bars, full-length (FL) Nrf2 (56 kDa); gray bars, degraded (Deg) Nrf2 (40 kDa); white bars, total (Tot) Nrf2; the sums of full-length Nrf2 and degraded Nrf2 were determined to obtain relative total Nrf2 protein expression. (B) Relative steady-state Nrf2 and (D) Keap1 mRNA levels were measured by SYBRGreen qPCR and normalized to 18S rRNA. Significance was determined by one-way ANOVA Dunnett’s for protein and one-way ANOVA Bonferroni’s for mRNA; * Pr0.05 as compared to Day 0, bars indicate Pr0.05 between samples. Data are expressed as mean7SD; n¼3–4.

Article Snippet: We also tried the Santa Cruz N-terminal Nrf2 antibody (No. sc-13032) that gave 6–7 signals ranging from 30 to 100 kDa and another C-terminal Nrf2 antibody from R & D Systems (No. MAB3925) that gave 20þ signals ranging from 20 to 100þ kDa (data not shown).

Techniques: Expressing, Western Blot

Journal: Free radical biology & medicine

Article Title: NAD(P)H:quinone oxidoreductase 1 activity reduces hypertrophy in 3T3-L1 adipocytes.

doi: 10.1016/j.freeradbiomed.2012.05.047

Figure Lengend Snippet: Fig. 3. NQO1 protein increases during limited clonal expansion and postmitotic growth arrest of differentiation. 3T3-L1 cells were differentiated as described under Materials and methods and harvested on Days 0, 1, 2, 3, and 4. (A) NQO1 and (D) Keap1 protein expressions were examined by immunoblotting. (B) Relative NQO1, (C) Nrf2, and (E) Keap1 mRNA levels were measured by SYBRGreen qPCR and normalized to 18S rRNA. Significance was determined by one-way ANOVA Dunnett’s for protein and one-way ANOVA Bonferroni’s for mRNA; * Pr0.05 as compared to Day 0, bars indicate Pr0.05 between samples. Data are expressed as mean7SD; n¼3–4.

Article Snippet: We also tried the Santa Cruz N-terminal Nrf2 antibody (No. sc-13032) that gave 6–7 signals ranging from 30 to 100 kDa and another C-terminal Nrf2 antibody from R & D Systems (No. MAB3925) that gave 20þ signals ranging from 20 to 100þ kDa (data not shown).

Techniques: Western Blot

Journal: Free radical biology & medicine

Article Title: NAD(P)H:quinone oxidoreductase 1 activity reduces hypertrophy in 3T3-L1 adipocytes.

doi: 10.1016/j.freeradbiomed.2012.05.047

Figure Lengend Snippet: Fig. 4. NQO1 and Keap1 proteins, but not mRNA, decreases as adipocyte differentiation progresses. 3T3-L1 cells were differentiated as described under Materials and methods and harvested on Days 4, 5, 6, 7, and 8. (A) NQO1 and (D) Keap1 protein expressions were examined by immunoblotting. (B) Relative NQO1, (C) Nrf2, and (E) Keap1 mRNA levels were measured by SYBRGreen qPCR and normalized to 18S rRNA. Significance was determined by one-way ANOVA Dunnett’s for protein and one-way ANOVA Bonferroni’s for mRNA; * Pr0.05 as compared to Day 4. Data are expressed as mean7SD; n¼3.

Article Snippet: We also tried the Santa Cruz N-terminal Nrf2 antibody (No. sc-13032) that gave 6–7 signals ranging from 30 to 100 kDa and another C-terminal Nrf2 antibody from R & D Systems (No. MAB3925) that gave 20þ signals ranging from 20 to 100þ kDa (data not shown).

Techniques: Western Blot

Journal: Free radical biology & medicine

Article Title: NAD(P)H:quinone oxidoreductase 1 activity reduces hypertrophy in 3T3-L1 adipocytes.

doi: 10.1016/j.freeradbiomed.2012.05.047

Figure Lengend Snippet: Fig. 6. LiCl treatment increases NQO1 protein, but not NQO1 mRNA. 3T3-L1 cells were differentiated as described under Materials and methods, and treated with varying concentrations of LiCl at Day 8 and harvested on Day 11. A 40 mM NaCl was used as a vehicle control (VC). (A) NQO1 was examined by immunoblotting. (B) Relative NQO1 and (C) Nrf2 mRNA levels were measured by SYBRGreen qPCR and normalized to 18S rRNA. Significance was determined by one-way ANOVA Dunnett’s; * Pr0.05 as compared to VC for protein and mRNA. Data are expressed as mean7SD; n¼3.

Article Snippet: We also tried the Santa Cruz N-terminal Nrf2 antibody (No. sc-13032) that gave 6–7 signals ranging from 30 to 100 kDa and another C-terminal Nrf2 antibody from R & D Systems (No. MAB3925) that gave 20þ signals ranging from 20 to 100þ kDa (data not shown).

Techniques: Control, Western Blot

Journal: Free radical biology & medicine

Article Title: NAD(P)H:quinone oxidoreductase 1 activity reduces hypertrophy in 3T3-L1 adipocytes.

doi: 10.1016/j.freeradbiomed.2012.05.047

Figure Lengend Snippet: Fig. 7. Sulforaphane increases NQO1 protein and mRNA levels. 3T3-L1 cells were differentiated as described under Materials and methods, and treated with varying concentrations of sulforaphane (SFN). A 0.3% DMSO was used as a VC. (A) NQO1 was examined by immunoblotting. (B) Relative NQO1 and (C) Nrf2 mRNA levels were measured by SYBRGreen qPCR and normalized to 18S rRNA. Significance was determined by one-way ANOVA Dunnett’s; * Pr0.05 as compared to VC for protein and mRNA. Data are expressed as mean7SD; n¼3.

Article Snippet: We also tried the Santa Cruz N-terminal Nrf2 antibody (No. sc-13032) that gave 6–7 signals ranging from 30 to 100 kDa and another C-terminal Nrf2 antibody from R & D Systems (No. MAB3925) that gave 20þ signals ranging from 20 to 100þ kDa (data not shown).

Techniques: Western Blot

Journal: The Journal of biological chemistry

Article Title: Nuclear factor Nrf2 promotes glycosidase OGG1 expression by activating the AKT pathway to enhance leukemia cell resistance to cytarabine.

doi: 10.1016/j.jbc.2022.102798

Figure Lengend Snippet: Figure 1. High expression of Nrf2 associates with AML relapse. A, analysis of gene expression profile of Nrf2 in AML samples (n = 173) and matched normal samples (n = 70) according to GEPIA website. B, the expression level of Nrf2 mRNA in normal (n = 17), complete remission (n = 33), and relapsed AML patients (n = 46) was identified by RT–PCR. C, the expression of Nrf2 was detected by RT–PCR in the same AML patients before and after relapse (n = 8). D, Western blot was adopted for detecting the expression level of Nrf2 in normal (n = 9), complete remission (n = 9), and relapsed (n = 9) AML samples.

Article Snippet: The Nrf2 antibody was acquired from the abcam, and the OGG1 antibody was purchased from the Novus.

Techniques: Expressing, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: The Journal of biological chemistry

Article Title: Nuclear factor Nrf2 promotes glycosidase OGG1 expression by activating the AKT pathway to enhance leukemia cell resistance to cytarabine.

doi: 10.1016/j.jbc.2022.102798

Figure Lengend Snippet: Figure 2. High expression of OGG1 associates with high expression of Nrf2 in AML. A–D, RT–PCR analysis of the expressions of OGG1, XRCC1, AP site endonuclease 1 (APE1), and DNA polymerase β (POL-β) in Nrf2-low (n = 29) and Nrf2-high (n = 26) groups. E, the gene expression profiles of OGG1 in AML samples (n = 173) and matched normal samples (n = 70) were analyzed according to GEPIA website. F, RT–PCR analysis of OGG1 expression in normal (n = 28), complete remission (n = 29), and relapsed (n = 26) AML patients. G, the expression of OGG1 in the same AML patients before and after relapse was

Article Snippet: The Nrf2 antibody was acquired from the abcam, and the OGG1 antibody was purchased from the Novus.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Gene Expression

Journal: The Journal of biological chemistry

Article Title: Nuclear factor Nrf2 promotes glycosidase OGG1 expression by activating the AKT pathway to enhance leukemia cell resistance to cytarabine.

doi: 10.1016/j.jbc.2022.102798

Figure Lengend Snippet: Figure 3. High expression of Nrf2 in Ara-C-resistant AML cell lines. A, U937 and U937R cells were treated with different concentrations of Ara-C (4, 16, 64, 192, 386, 578, 768, and 1536 μM) for 24 h, and the cell viability was detected by CCK-8 colorimetry. B, THP-1 and THP-1R cells were treated with different concentrations of Ara-C (0.5, 4, 64, 192, 386, 578, 768, and 1536 μM) for 24 h, and the cell viability was detected by CCK-8 colorimetry. C, after U937, U937R, THP-1, and THP-1R were cocultured with Ara-C (2 μM) for 24 h, the apoptosis rate of AML cell lines was detected by FCM. D, analysis of apoptosis rate.

Article Snippet: The Nrf2 antibody was acquired from the abcam, and the OGG1 antibody was purchased from the Novus.

Techniques: Expressing, CCK-8 Assay, Colorimetric Assay

Journal: The Journal of biological chemistry

Article Title: Nuclear factor Nrf2 promotes glycosidase OGG1 expression by activating the AKT pathway to enhance leukemia cell resistance to cytarabine.

doi: 10.1016/j.jbc.2022.102798

Figure Lengend Snippet: Figure 4. High expression of Nrf2 decreases the sensitivity of leukemic cells to Ara-C. A, Western blot was used to verify the upregulation of Nrf2 in AML- sensitive celllines (U937andTHP-1). B, the relative grayvalues of Nrf2expression. C, Western blotwas appliedtodetect thedownregulation ofNrf2in AML-resistant cell lines (U937R andTHP-1R). D, the relativegray values of Nrf2 expression. E, RT–PCR verifiedthe expression ofmRNA after Nrf2 regulation.F, FCM was employed todetect the apoptosis rate of AML-sensitive cell lines cocultured with Ara-C (2 μM) for 24 h after Nrf2 overexpression. G, after downregulation of Nrf2, the apoptosis rate of AML drug–resistant cell lines cocultured with Ara-C (100 μM) for 24 h was detected by FCM. Each experiment was repeated three times independently and expressed as mean ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001. AML, acute myeloid leukemia; Ara-C, cytarabine; FCM, flow cytometry; Nrf2, nuclear factor E2–related factor 2.

Article Snippet: The Nrf2 antibody was acquired from the abcam, and the OGG1 antibody was purchased from the Novus.

Techniques: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Over Expression, Cytometry

Journal: The Journal of biological chemistry

Article Title: Nuclear factor Nrf2 promotes glycosidase OGG1 expression by activating the AKT pathway to enhance leukemia cell resistance to cytarabine.

doi: 10.1016/j.jbc.2022.102798

Figure Lengend Snippet: Figure 5. Overexpression of Nrf2 promotes the expression of OGG1 to mediate drug resistance in AML cells. A, detection of OGG1 expression in normal (bone marrow mononuclear cells from normal healthy donors) and AML-sensitive cell lines (U937 and THP-1) and drug-resistant cell lines (U937R and THP-1R) by IF staining. Scale bars represent 20 μm. B, quantification of OGG1 relative fluorescence intensity in normal and leukemia cells by ImageJ. C, Western blot was employed to detect OGG1 protein expression after upregulation of Nrf2. D, the relative gray values of Nrf2 and OGG1 expressions.

Article Snippet: The Nrf2 antibody was acquired from the abcam, and the OGG1 antibody was purchased from the Novus.

Techniques: Over Expression, Expressing, Staining, Western Blot

Journal: The Journal of biological chemistry

Article Title: Nuclear factor Nrf2 promotes glycosidase OGG1 expression by activating the AKT pathway to enhance leukemia cell resistance to cytarabine.

doi: 10.1016/j.jbc.2022.102798

Figure Lengend Snippet: Figure 7. Nrf2 overexpression promotes OGG1 expression by activating AKT signaling pathway. A, GeneMania protein–protein interaction network of OGG1, Nrf2, and AKT. B and C, Western blot was applied to identify the expressions of Nrf2, OGG1, AKT, p-AKT, Cleaved-caspase 9, and Total-caspase 9 proteins in AML-sensitive cell lines after overexpression of Nrf2. D and E, Western blot was adopted for detecting the expression of Nrf2, OGG1, AKT, p-AKT, Cleaved-caspase 9, and Total-caspase 9 proteins in AML drug–resistant cell lines after downregulation of Nrf2. Each experiment was repeated three times independently and denoted as mean ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001. Nrf2, nuclear factor E2–related factor 2; OGG1, 8-hydroxyguanine DNA glycosidase.

Article Snippet: The Nrf2 antibody was acquired from the abcam, and the OGG1 antibody was purchased from the Novus.

Techniques: Over Expression, Expressing, Western Blot

Journal: The Journal of biological chemistry

Article Title: Nuclear factor Nrf2 promotes glycosidase OGG1 expression by activating the AKT pathway to enhance leukemia cell resistance to cytarabine.

doi: 10.1016/j.jbc.2022.102798

Figure Lengend Snippet: Figure 8. Inhibition of AKT signaling pathway to reduce OGG1 expression. A, after U937 cells were treated with 2 μM MK-2206 for 24 h, the protein expression levels of Nrf2, OGG1, AKT, p-AKT, Cleaved-caspase 9, and Total-caspase 9 in Nrf2 overexpression and EV1 groups were detected by Western blot. B, the relative gray value of related proteins. C, after THP-1 cells were treated with 2 μM MK-2206 for 24 h, the protein expression levels of Nrf2, OGG1, AKT, p-AKT, Cleaved-caspase 9, and Total-caspase 9 in Nrf2 overexpression and EV1 groups were detected by Western blot. D, the relative gray values of related

Article Snippet: The Nrf2 antibody was acquired from the abcam, and the OGG1 antibody was purchased from the Novus.

Techniques: Inhibition, Expressing, Over Expression, Western Blot

Journal: The Journal of biological chemistry

Article Title: Nuclear factor Nrf2 promotes glycosidase OGG1 expression by activating the AKT pathway to enhance leukemia cell resistance to cytarabine.

doi: 10.1016/j.jbc.2022.102798

Figure Lengend Snippet: Figure 9. Downregulation of Nrf2 reduces the risk of drug resistance in vivo. A and B, tumor volume of mice after subcutaneous injection of U937R (n = 4), U937R-EV (n = 4), U937R-si-Nrf2 (n = 3), and U937R-EV + Ara-C (n = 4) and U937R-si-Nrf2 + Ara-C (n = 3) cells after 35 days growth. C, changes of tumor weight after subcutaneousinjectionofU937R(n=4),U937R-EV(n=4),U937R-si-Nrf2(n=3),andU937R-EV+Ara-C(n=4)andU937R-si-Nrf2+Ara-C(n=3)cells.DandE,volumegrowth curveofxenografttumor.F,analysisanddrawingofsurvivalcurveofxenograftedtumormicebyKaplan–Meiermethod.G–J,IHCwasappliedtoidentifytheexpressionsof Nrf2andOGG1insubcutaneoustransplantedtumortissuesections.Thescalebarsfromlefttorightrepresent100and50μm,respectively.*p< 0.05,**p< 0.01,and***p< 0.001. Ara-C, cytarabine; EV, empty vector; IHC, immunohistochemistry; Nrf2, nuclear factor E2–related factor 2; OGG1, 8-hydroxyguanine DNA glycosidase.

Article Snippet: The Nrf2 antibody was acquired from the abcam, and the OGG1 antibody was purchased from the Novus.

Techniques: In Vivo, Injection, Plasmid Preparation, Immunohistochemistry