Journal: Chemical Research in Toxicology

Article Title: Ginger Compound [6]-Shogaol and Its Cysteine-Conjugated Metabolite (M2) Activate Nrf2 in Colon Epithelial Cells in Vitro and in Vivo

doi: 10.1021/tx500211x

Figure Lengend Snippet: Effects of 6S on expression of Nrf2 and Nrf2 target genes in HCT-116 cells. (A) Effect of 6S on expression of AKR1B10, GGTLA4, FTL, HMOX1, GCLC, GCLM, and MT1 in HCT-116 cells. (B) Effect of 6S on the expression of Keap1, Nrf2, and phosphorylated Nrf2 (p-Nrf2). The protein levels of AKR1B10, GGTLA4, FTL, HMOX1, GCLC, GCLM, MT1, Keap1, Nrf2, and p-Nrf2 were determined by western blotting at the indicated time points after treatment of HCT-116 cells with 6S (20 μM). β-Actin was used as an internal standard. (C) Time-dependent effect of 6S on Nrf2 nuclear translocation. HCT-116 cells were treated with 20 μM 6S for 0, 2, 4, 6, 12, and 24 h. (D) Dose-dependent effect of 6S on Nrf2 nuclear translocation. HCT-116 cells were treated with 0, 5, 10, 20, and 40 μM 6S for 6 h. Lamin B and β-actin were used as internal controls for nuclear and cytoplasmic fractions, respectively. (E) IF staining of Nrf2. HCT-116 cells were treated with 20 μM 6S for 12 or 24 h and then fixed and labeled with anti-Nrf2 and appropriate FITC-conjugated secondary antibodies. Cells were counterstained with DAPI for visualization of the nuclei. Slides were viewed using fluorescent microscopy (DAPI, blue; Nrf2, red).

Article Snippet: Human recombinant Keap1 (no. NM_012289, OriGene, Rockville, MD) was incubated with 6S [molar ratios of 1:10 (Keap1/6S)] or DMSO in 120 μL of 25 mM Tris-HCl buffer (pH 8) for 2 h at room temperature.

Techniques: Expressing, Western Blot, Translocation Assay, Staining, Labeling, Microscopy

Journal: Chemical Research in Toxicology

Article Title: Ginger Compound [6]-Shogaol and Its Cysteine-Conjugated Metabolite (M2) Activate Nrf2 in Colon Epithelial Cells in Vitro and in Vivo

doi: 10.1021/tx500211x

Figure Lengend Snippet: Effects of M2 on expression of Nrf2 and Nrf2 target genes in HCT-116 cells. (A) Effect of M2 on the expression of AKR1B10, GGTLA4, FTL, HMOX1, GCLC, GCLM, and MT1. (B) Effect of M2 on the expression of Keap1, Nrf2, and p-Nrf2. The protein levels of AKR1B10, GGTLA4, FTL, HMOX1, GCLC, GCLM, MT1, Keap1, Nrf2, and p-Nrf2 were determined by western blotting at the indicated time points after the treatment of HCT-116 cells with M2 (20 μM). β-Actin was used as internal standard. (C) Time-dependent effect of M2 on Nrf2 nuclear translocation. HCT-116 cells were treated with 20 μM M2 for 0, 2, 4, 6, 12, and 24 h. (D) Dose-dependent effect of M2 on Nrf2 nuclear translocation. HCT-116 cells were treated with 0, 5, 10, 20, and 40 μM M2 for 6 h. Cytosolic and nucleic Nrf2 were determined using western blotting with the appropriate specific antibodies. Lamin B and β-actin were used as internal controls for nuclear and cytoplasmic fractions, respectively. (E, F) Effects of kinase inhibitors on M2-induced Nrf2 translocation and phosphorylation (E) and HMOX1 (F) expression. PD098059 (50 μM, a MEK1 inhibitor), LY294002 (50 μM, a PI3K inhibitor), or SB202190 (50 μM, a p38 inhibitor) was used to pretreat the cells for 30 min before they were exposed to 20 μM M2. After another 24 h of incubation, whole-cell lysates were prepared and assessed for Nrf2, p-Nrf2, and HMOX1 expression by western blotting. * p < 0.05.

Article Snippet: Human recombinant Keap1 (no. NM_012289, OriGene, Rockville, MD) was incubated with 6S [molar ratios of 1:10 (Keap1/6S)] or DMSO in 120 μL of 25 mM Tris-HCl buffer (pH 8) for 2 h at room temperature.

Techniques: Expressing, Western Blot, Translocation Assay, Phospho-proteomics, Incubation

Journal: Chemical Research in Toxicology

Article Title: Ginger Compound [6]-Shogaol and Its Cysteine-Conjugated Metabolite (M2) Activate Nrf2 in Colon Epithelial Cells in Vitro and in Vivo

doi: 10.1021/tx500211x

Figure Lengend Snippet: Cysteine Residues of Keap1 Modified by 6S as Determined by UPLC–MS/MS Analysis a

Article Snippet: Human recombinant Keap1 (no. NM_012289, OriGene, Rockville, MD) was incubated with 6S [molar ratios of 1:10 (Keap1/6S)] or DMSO in 120 μL of 25 mM Tris-HCl buffer (pH 8) for 2 h at room temperature.

Techniques: Modification

Journal: Oncogene

Article Title: NRF2 activation by cysteine as a survival mechanism for triple-negative breast cancer cells.

doi: 10.1038/s41388-024-03025-0

Figure Lengend Snippet: Fig. 6 NRF2 target genes downstream of its Cys-dependent activation. A Western blot analysis of NRF2 expression in Hs 578T cells transduced with lentiviral vectors for the expression of NRF2-FL (NRF2) or GFP (as control). Whole protein extracts were prepared after 8 h of growth in presence (w Cyss) or absence (w/o Cyss) of Cyss. The same cells as in (A) were processed for RNA-seq analysis. B DESeq2 mean of normalized counts for NRF2 gene (NFE2L2). Two-way ANOVA was followed by Sidak’s multiple comparisons test. C Euler diagram showing effect of the expression of exogenous NRF2 on DEGs upregulated in GFP control cells by Cyss depletion. D Euler diagram showing the effect of exogenous NRF2 expression on downregulated DEGs in GFP control cells by Cyss depletion. Adjusted p value (p adj) <0.1 and |log2FC| > 0.32 were used as thresholds for analysis. E Hierarchical clustering of selected DEGs in control cells following Cyss (GFP w/o Cyss vs GFP w Cyss, |log2FC| > 0.32, p-adj > 0.1), considering their expression fold change relative to all the compared conditions. Color scale refers to the fold change values of DE transcripts. F, G DESeq2 mean normalized counts for one gene from each subgroup of NRF2-modulated genes as example. The corresponding pie chart segments are drawn.

Article Snippet: Lentiviral plasmid for KEAP1 expression (#RC202189L3) and NRF2 (#RC204140L1) were purchased from ORIGENE (Rockville, MD, USA).

Techniques: Activation Assay, Western Blot, Expressing, Transduction, Control, RNA Sequencing

Journal: Oncogenesis

Article Title: Nrf2-Keap1 pathway promotes cell proliferation and diminishes ferroptosis

doi: 10.1038/oncsis.2017.65

Figure Lengend Snippet: Nrf2 and Keap1 regulates xCT expression. ( a ) The impact of Nrf2/Keap1 on xCT expression in mRNA level. Quantitive real-time PCR tested the fold change of Nrf2, xCT and Keap1 mRNA level in F98 glioma cells transfected with different vectors (Nrf2 KD, Nrf2 OE, Keap1 KD, Keap OE), control vector transfected F98 glioma cells served as control and normalized to 1. Values were given as mean±s.d. Statistical significance was tested by Two-way ANOVA, * P <0.05, ns: not significant, n =3. ( b ) The impact of Nrf2/Keap1 on xCT protein levels. Western blot of Nrf2, Keap1 and xCT protein in F98 gliomas with different Nrf2 or Keap1 levels. Control vector transfected F98 glioma cells served as controls. β-actin was used as a loading control. ( c ) Quantitative real-time PCR analysis of Nrf2, xCT and Keap1 mRNA level in U87 glioma cells transfected with different vectors (Nrf2 KD, Nrf2 OE, Keap1 KD, Keap OE). Control vector transfected U87 glioma cells served as controls and were normalized to 1. Values were given as mean±s.d. Statistical significance was tested by two-way ANOVA, * P <0.05, ns: not significant, with n =3. ANOVA, analysis of variance.

Article Snippet: Mouse monoclonal Keap1 antibody (MAB3024) was purchased from R&D System (Minneapolis, MN, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Transfection, Control, Plasmid Preparation, Western Blot

Journal: Oncogenesis

Article Title: Nrf2-Keap1 pathway promotes cell proliferation and diminishes ferroptosis

doi: 10.1038/oncsis.2017.65

Figure Lengend Snippet: Nrf2-Keap1 pathway promotes oncogenic progression. ( a ) Proliferation analysis of rodent F98 glioma cells with different Nrf2/Keap1 expresssion. Keap1 KD and Nrf2 OE cells showed a significant higher proliferation rate compared to control cells (Ctrl vector). In contrast, proliferation is lower in Nrf2 DN, Nrf2 KD and Keap1 OE cells. Values are given as mean±s.d. Statistical significance was tested with one-way ANOVA, * P <0.05, with n =16. ( b ) Analysis of cell numbers in Keap1 KD and Nrf2 OE cells. Quantification of cell numbers was performed 4 and 7 days following plating. Values are given as mean±s.d. Statistical significance was tested with one-way ANOVA, * P <0.05, n =3. ( c ). Scratch migration assay of F98 glioma cells with different Nrf2/Keap1 levels. Representative images show that Nrf2 OE and Keap1 KD cells tend to migrate slower than all other groups; scale bar, 200 μm. ( d ) Quantification of the distance of scratch in 0 and 24 hour time points. Values are given as mean±s.d. Statistical significance was tested by two-way ANOVA, * P <0.05, ns: not significant, n =6. ( e ) Colony formation assay of F98 glioma cells with different Nrf2/Keap1 levels. Representative images of colonies formed in each group; scale bar, 200 μm. ( f ) Quantification of total colonies area in comparison to controls. Values are given as mean±s.d. Statistical significance was tested by one-way ANOVA, * P <0.05, ns: not significant, n =13. ( g ) Extracellular glutamate concentrations measured by high-performance liquid chromatography: Nrf2 OE and Keap1 KD cell groups show higher levels of glutamate in comparison to control cells. Values were given as mean±s.d. Statistical significance was tested by one-way ANOVA, * P <0.05, with n =3. ANOVA, analysis of variance.

Article Snippet: Mouse monoclonal Keap1 antibody (MAB3024) was purchased from R&D System (Minneapolis, MN, USA).

Techniques: Control, Plasmid Preparation, Migration, Colony Assay, Comparison, High Performance Liquid Chromatography

Journal: Oncogenesis

Article Title: Nrf2-Keap1 pathway promotes cell proliferation and diminishes ferroptosis

doi: 10.1038/oncsis.2017.65

Figure Lengend Snippet: The effect of xCT in glioma cell proliferation, migration and colony formation. ( a ) Quantitive real-time PCR tested the fold change of xCT, Nrf2 and Keap1 mRNA level in F98 glioma cells transfected with xCT KD and xCT OE vectors. Control vector transfected cells served as controls and were normalized to 1. Values are given as mean±s.d. Statistical significance was tested by two-way ANOVA, * P <0.05, n =3. ( b , left) MTT proliferation assay of F98 cell with different xCT expresssion: xCT OE cells showed a slightly higher proliferation rate than control cells (Ctrl vector), while xCT KD cells proliferate as the same rate as control cells. (right) MTT-based proliferation assay of F98 cell with different Nrf2 levels and in addition with transfection of xCT OE or xCT KD vectors. Note that xCT overexpression in Nrf2 KD cells slightly increased the cell proliferation, while xCT KD in Nrf2 OE cells could not significantly depress cell proliferation. Values are given as mean±s.d. Statistical significance was tested by one-way ANOVA, * P <0.05, with n =8. ( c ). Scratch migration assay of F98 glioma cells with different xCT levels. (left) Representative images show that xCT KD cells migrate slower than other groups; scale bar, 200 μm. (right) Quantification of the distance of scratch in 0 and 24 hour time points also showed that the scratch of xCT KD cells remained larger in distance compared with control cells, however, xCT OE cells remained the same distance as control. Values are given as mean±s.d. Statistical significance was tested by Two-way ANOVA, * P <0.05, with n =10. ( d ) Colony formation assay of F98 glioma cells with different xCT levels. Left, Representative images of colonies forming in each group; scale bar, 200 μm. Right, Quantification of total colonies number in comparison to control cells. Values were given as mean±s.d. Statistical significance was tested by One-way ANOVA, * P <0.05, with n =24. ANOVA, analysis of variance.

Article Snippet: Mouse monoclonal Keap1 antibody (MAB3024) was purchased from R&D System (Minneapolis, MN, USA).

Techniques: Migration, Real-time Polymerase Chain Reaction, Transfection, Control, Plasmid Preparation, Proliferation Assay, Over Expression, Colony Assay, Comparison

Journal: Oncogenesis

Article Title: Nrf2-Keap1 pathway promotes cell proliferation and diminishes ferroptosis

doi: 10.1038/oncsis.2017.65

Figure Lengend Snippet: Nrf2-Keap1 pathway induces resistance to ferroptosis in F98 gliomas. ( a , left) Cell viability analysis following 10 μ m erastin treatment in F98 glioma cells expressing different Nrf2/Keap1 vectors. The group with 0.05% (v/v) DMSO treatment (vehicle) served as controls. The O.D. value of vehicle treated cells was set to 100%. Normalized data showed that Nrf2 KD, Nrf2 DN and Keap1 OE glioma cells were significantly more vulnerable to erastin treatment (55%, 70% and 54% decrease respectively vs control 33% decrease in cell viability), while Nrf2 OE and Keap1 KD glioma cells showed more resistant (25% and 13% decrease respectively vs control 33% decrease in cell viability). Values are given as mean±s.d. Statistical significance was tested by One-way ANOVA, * P <0.05, n =6. (right) Cell viability under another ferroptosis inducer, RSL3 (0.1 μ m ), was tested by MTT assay in F98 glioma cells expressing different Nrf2/Keap1 vectors, which showed consistent result as erastin treatment. Values are given as mean±s.d. Statistical significance was tested by One-way ANOVA, * P <0.05, with n =6. ( b ) Ferroptosis inhibitor, ferrostatin-1 (Ferro) (1 μ m ) treatment rescued erastin-induced ferroptosis in each group. Normalized quantification of O.D. value indicated an increase in cell viability of each group respectively: Ctrl vector (21%), Nrf2 KD (20.3%), Nrf2 OE (18.6%), Keap1 KD (34.3%) and Keap1 OE (39%). Values were given as mean±s.d. Statistical significance was tested by one-way ANOVA, * P <0.05, n =8. ( c ) 1 μ m ferrostatin-1 treatment rescued RSL3-induced ferroptosis in each group. Normalized quantification of O.D. value indicated an increase in cell viability of each group respectively: Ctrl vector (21%), Nrf2 KD (20.3%), Nrf2 OE (18.6%), Keap1 KD (34.3%) and Keap1 OE (39%). Values are given as mean±s.d. Statistical significance was tested by One-way ANOVA, * P <0.05, with n =6. ANOVA, analysis of variance.

Article Snippet: Mouse monoclonal Keap1 antibody (MAB3024) was purchased from R&D System (Minneapolis, MN, USA).

Techniques: Expressing, Control, MTT Assay, Plasmid Preparation

Journal: Oncogenesis

Article Title: Nrf2-Keap1 pathway promotes cell proliferation and diminishes ferroptosis

doi: 10.1038/oncsis.2017.65

Figure Lengend Snippet: Nrf2-Keap1 pathway induces resistance to ferroptosis in U87 glioma cells. ( a ) Analysis of cell viability under erastin treatment on human glioma cell line U87. DMSO treatment served as vehicle. The O.D. value of vehicle treated cells was set to 100%. Nrf2 KD, Nrf2 DN and Keap1 OE U87 cells showed significantly more vulnerability to 10 μ m erastin treatment (80%, 85% and 68% decrease, respectively, vs control’s 60% decrease in cell viability), while Nrf2 OE and Keap1 KD U87 cells were more resistant to erastin (46% and 38% decrease, respectively, vs control’s 60% decrease in cell viability). Values are given as mean±s.d. Statistical significance was tested by One-way ANOVA, * P <0.05, with n =6. ( b ) Ferrostatin-1 (1 μ m ) treatment rescued erastin-induced ferroptosis in each group of U87 cells. Normalized quantification of O.D. value indicated an increase in cell viability of each group respectively: Ctrl vector (56%), Nrf2 KD (56%), Nrf2 OE (43%), Keap1 KD (15%) and Keap1 OE (48%). Values were given as mean±s.d. Statistical significance was tested by one-way ANOVA, * P <0.05, with n =6. ANOVA, analysis of variance.

Article Snippet: Mouse monoclonal Keap1 antibody (MAB3024) was purchased from R&D System (Minneapolis, MN, USA).

Techniques: Plasmid Preparation

Journal: Oncogenesis

Article Title: Nrf2-Keap1 pathway promotes cell proliferation and diminishes ferroptosis

doi: 10.1038/oncsis.2017.65

Figure Lengend Snippet: Targeting xCT overcomes Nrf2/Keap1 mediated resistance to ferroptosis. ( a ) xCT overexpression in Nrf2 KD/ Keap1 OE cells rescued their ferroptotic resistance to erastin. Cell viability after 10 μ m erastin treatment was tested by MTT assay in F98 glioma cells expressing Nrf2 KD/ Keap1 OE as well as Nrf2 KD plus xCT OE or Keap1 OE plus xCT OE. Normalized data showed that xCT overexpression rescued cell viability by 27% under erastin treatment. Values are given as mean±s.d. Statistical significance was tested by One-way ANOVA, * P <0.05, with n =6. ( b ) Pharmacological inhibition of xCT by 400 μ m sulfasalazine (SAS) increases the sensitivity of Nrf2 OE/ Keap1 KD cells to erastin treatment. MTT assay showed a 47 and 52% decrease respectively in cell viability when erastin plus SAS treatment was applied. Values are given as mean±s.d. Statistical significance was tested by one-way ANOVA, * P <0.05, with n =6. ANOVA, analysis of variance.

Article Snippet: Mouse monoclonal Keap1 antibody (MAB3024) was purchased from R&D System (Minneapolis, MN, USA).

Techniques: Over Expression, MTT Assay, Expressing, Inhibition

Journal: Oncogenesis

Article Title: Nrf2-Keap1 pathway promotes cell proliferation and diminishes ferroptosis

doi: 10.1038/oncsis.2017.65

Figure Lengend Snippet: Nrf2-Keap1 regulates erastin-induced ROS generation in glioma cells. ( a ) Fluorescence activated cell sorting-based analysis for total cellular ROS following erastin application in control, Nrf2 KD, Nrf2 OE, Keap1 KD and Keap1 OE glioma cells. Total cellular ROS was monitored via DCF fluorescence intensity. Ferrostatin-1 (1 μ m ) and DFO (50 μ μ ) treatment blocked erastin (10 μ m ) induced ROS generation. Dashed lines indicate the DCF median fluorescence intensity of vehicle treated group. ( b ) Quantification of the percentage of DCF fluorescence positive cell population in various experimental groups. Nrf2 KD and Keap1 OE cells showed 28–30% DCF fluorescence positive cell population after erastin treatment, while Nrf2 OE cells showed only 2% DCF positive population, Keap1 KD and control cells showed a medium 12–13% DCF-positive population. Values are given as mean±s.d. Statistical significance was tested by One-way ANOVA, * P <0.05, with n =3. ( c ) Fluorescence activated cell sorting-based analysis for cellular lipid ROS following 10 μ m erastin application in control, Nrf2 KD, Nrf2 OE, Keap1 KD and Keap1 OE glioma cells. Lipid ROS was monitored via C11-BODIPY staining. Nrf2 KD and Keap1 OE cells showed higher C11-BODIPY positive population after erastin treatment, while Nrf2 OE and Keap1 KD cells showed a lower C11-BODIPY-positive population compared to controls. Ferrostatin-1 (1 μ m ) treatment blocked erastin-induced lipid ROS generation. Oval circles indicate C11-BODIPY positive cell population. ANOVA, analysis of variance.

Article Snippet: Mouse monoclonal Keap1 antibody (MAB3024) was purchased from R&D System (Minneapolis, MN, USA).

Techniques: Fluorescence, FACS, Control, Staining

Journal: Redox Biology

Article Title: N-acetyl- l -cysteine ethyl ester (NACET) induces the transcription factor NRF2 and prevents retinal aging and diabetic retinopathy

doi: 10.1016/j.redox.2025.103914

Figure Lengend Snippet: NACET increases intracellular levels of Cys, which directly conjugate to Cys residues 226 and 613 of KEAP1 and induce NRF2 expression. a , Western blot analysis of KEAP1 and NRF2 expression in ARPE-19 cells following lentiviral-mediated expression of shRNAs targeting 3′UTR of KEAP1 mRNA (shKEAP1) or GFP (Ctrl). Where indicated. Cells were treated with 1 mM NACET for 2 h b , Schematic representation of the GSH synthesis pathway. The enzymes involved are boxed; abbreviations: ACY1, aminoacylase 1; GCL, glutamate-cysteine ligase; GSS, glutathione synthetase. c , Heatmap showing the status of sensor cysteine residues identified by mass spectrometry analysis of purified 3xFLAG-KEAP1 transfected into HEK 293 cells treated and not treated with NACET: unconjugated (purple squares), NACET-, NAC-, Cys- or GSH-conjugated in NACET-treated and untreated samples (cyan squares) or only in NACET-treated samples (yellow squares). d , AlphaFold model showing the domains of KEAP1 and the relative position of the 12 Cys residues found conjugated in the MS analysis. e , Scheme of the transfection protocol for the analysis of NRF2 transactivation activity. pRL-SV40 was used for the constitutive expression of Renilla Luciferase for normalization. f , NRF2 activity measured as transactivation of the ARE-luc reporter gene in cells silenced for endogenous KEAP1 expression. KEAP1 silenced cells were transfected with reporter plasmids and one plasmid for the expression of shRNA-resistant KEAP1 (wt or mutant, as indicated). 8 h post transfection, the cells were grown for 24 h in growth medium ±0.2 mM NACET. KEAP1 Cys residues were substituted with serine, glutamic acid (C288E) or tryptophan (C273W) as described by Suzuki et al. . Values represent the fold change in normalized luciferase activity in cells transfected for the expression of the corresponding KEAP1 variant and are expressed as means ± SD (N = 3–5). Differences were tested using one-way ANOVA followed by Dunnett's multiple comparisons test. g, Confluent ARPE-19 cells were pre-treated for 30 min with 100 μM BSO where indicated and then incubated with 0.2 mM NACET for the indicated time points, lysed by TCA-EDTA solution and the intracellular thiols were measured by HPLC. Thiol concentrations are normalized for the total protein content and expressed in nmol/mg protein. Measurements are the means ± SD of 4 independent experiments. Differences were tested using one-way ANOVA followed by Tukey's multiple comparisons test. ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. For clarity, not all the significant differences are indicated. h , Confluent ARPE-19 cells were incubated with 1 mM NAC for 1 h, and thiols were extracted and measured as in ( g ). i , Whole lysates of cells treated as in ( g ) were analyzed by Western blot with anti-NRF2 antibody. Anti-GAPDH antibody was used as a loading control. Densitometric analysis of bands is shown. j , Cartoon showing the proposed NACET's tripartite mechanism of action.

Article Snippet: Plasmid for KEAP1 ( NM_012289 ) expression in KEAP1-silenced cells was purchased from ORIGENE (#SC111946; Rockville, MD, USA).

Techniques: Expressing, Western Blot, Mass Spectrometry, Purification, Transfection, Activity Assay, Luciferase, Plasmid Preparation, shRNA, Mutagenesis, Variant Assay, Incubation, Control

Journal: Theranostics

Article Title: Nrf2 and HIF1α converge to arsenic-induced metabolic reprogramming and the formation of the cancer stem-like cells

doi: 10.7150/thno.42903

Figure Lengend Snippet: Activation of Nrf2 and Nrf2-dependent HIF1α expression in the cells treated by iAs. A. Time-dependent activation of Nrf2 and HIF1α. B. Involvement of JNK in iAs-induced Nrf2 as well as HIF1α. C. Luciferase reporter gene activities of Nrf2 and HIF1α in the cells treated with 1 μM iAs for the indicated times. D. Lentiviral transfection of Keap1 inhibited iAs-induced Nrf2 activation and HIF1α expression. E. Knockout of Nrf2 by CRISPR-Cas9 gene editing prevented HIF1α induction by iAs. Red asterisk denotes the non-specific (N.S.) band.

Article Snippet: Human Keap1 overexpression clone lentiviral particle (RC202189L4V) and Lentiviral control particle (PS100093V) were purchased from Origene Technologies Inc. BEAS-2B cells were seeded in 24-wells plate (5 × 10 4 cells per well) and incubated for 20 hours, and then infected with Keap1 lentiviral overexpression particle and Lentiviral control particle for 20 hours, respectively.

Techniques: Activation Assay, Expressing, Luciferase, Transfection, Knock-Out, CRISPR

Journal: Theranostics

Article Title: Nrf2 and HIF1α converge to arsenic-induced metabolic reprogramming and the formation of the cancer stem-like cells

doi: 10.7150/thno.42903

Figure Lengend Snippet: iAs induces expression of a number of stemness genes through Nrf2 and/or HIF1α activation. A. Enrichment status of Nrf2 and HIF1α (pointed by red arrows) induced by iAs on these indicated stemness genes as determined by ChIP-seq. B. De novo and/or known Nrf2 motif(s) induced by iAs on the promoter or gene body of MYC, CD44, KLF4, and EGFR that had been linked to the stemness of CSCs. C. Nrf2 inhibition by lentiviral Keap1 transfection prevented induction of MYC and SOX2 by iAs. This Nrf2 inhibition also reduced KLF4 expression. D. Nrf2 knockout blocked iAs-induced expression of MYC, SOX2, BACH1, TBC1D7, and decreased expression of KLF4.

Article Snippet: Human Keap1 overexpression clone lentiviral particle (RC202189L4V) and Lentiviral control particle (PS100093V) were purchased from Origene Technologies Inc. BEAS-2B cells were seeded in 24-wells plate (5 × 10 4 cells per well) and incubated for 20 hours, and then infected with Keap1 lentiviral overexpression particle and Lentiviral control particle for 20 hours, respectively.

Techniques: Expressing, Activation Assay, ChIP-sequencing, Inhibition, Transfection, Knock-Out