Journal: Nature genetics

Article Title: Characterizing the molecular regulation of inhibitory immune checkpoints with multi-modal single-cell screens.

doi: 10.1038/s41588-021-00778-2

Figure Lengend Snippet: a) Single-cell mRNA expression heatmap showing 20 differentially expressed genes for each mixscape -classified perturbation. For visualization purposes we downsampled our dataset to include 30 cells from each class in the heatmap. b) Violin plots of PD-L1 protein expression for all identified regulators. BRD4 (p-value=4.37e −28 ), CUL3 (p-value=2.81e −11 ) and MYC (p-value=4.51e −7 ) are negative regulators, while the remaining are positive (p-value < 1e −6 in all cases, two-sided Wilcox Rank sum). NT, non-targeted. c) Flow cytometry measurements of PD-L1 protein expression across experimental conditions. JQ1 inhibitor treatment (24 hours, 1μM) reduces stimulation-induced PD-L1 expression. Control=20,000, stim=20,000 and JQ1+stim=20,000 cells. d) Flow cytometry measurements of PD-L1 protein expression in BRD4 gRNA expressing cells, validating our ECCITE-seq findings. BRD4g2=9,100, BRD4g5=15,000 and NT=4,800 cells. e) Violin plots showing elevated expression of PD-L1 transcript in CUL3 KO cells, in comparison to non-targeting controls. f) Barplot summarizing gene set enrichment analysis results for 300 genes upregulated in CUL3 KO cells. Analysis was performed using the Human WikiPathways database from the EnrichR package and shows strong enrichment for the NRF2 pathway.

Article Snippet: NRF2 over-expression plasmid was purchased from Addgene (#21549).

Techniques: Expressing, Flow Cytometry, Control, Comparison

Journal: Nature genetics

Article Title: Characterizing the molecular regulation of inhibitory immune checkpoints with multi-modal single-cell screens.

doi: 10.1038/s41588-021-00778-2

Figure Lengend Snippet: a) Schematic representation describing two complementary modes of CUL3-mediated PD-L1 regulation. The CUL3-SPOP complex directly regulates PD-L1 protein stability through ubiquitination. The CUL3-KEAP1 complex regulates NRF2 protein stability, indirectly modulating NRF2-mediated PD-L1 transcription. b) Validation pooled CRISPR screen results (2 biological replicates) targeting KEAP1 , SPOP , CUL3 , BRD4 , IFNGR1 and NRF2 (including 4 non-targeting gRNAs). gRNAs targeting KEAP1 , SPOP , CUL3 and BRD4 (green) were enriched in cells expressing high levels of PD-L1 protein while NRF2 and IFNGR1 gRNAs were depleted (red) NT, non-targeted. c) Boxplots showing the PD-L1 protein geometric mean fluorescence intensity (gMFI) (n =2 for each boxplot), (d) PD-L1 transcript and (e) NRF2 transcript levels (log1p(TPM)) in control (n = 3) and NRF2 overexpression (OE) (n = 4) cells. f) Density plot showing the average log2 fold change of three CUL3 KO cell DE gene subsets in the NRF2 OE dataset. g) Boxplots showing the PD-L1 protein geometric mean fluorescence intensity (gMFI) (n=2 KEAP1 KO and n=3 NT), (h) PD-L1 transcript and (i) KEAP1 transcript levels (log1p(TPM)) in NT (n = 8) and KEAP1 KO (n = 8) cells. In c-e and g-i boxplots the middle line is the median, the lower and upper hinges correspond to the first and third quartiles, the upper whisker extends from the hinge to the largest value no further than 1.5 × IQR from the hinge (IQR = inter-quartile range) and the lower whisker extends from the hinge to the smallest value at most 1.5 × IQR of the hinge.

Article Snippet: NRF2 over-expression plasmid was purchased from Addgene (#21549).

Techniques: Ubiquitin Proteomics, Biomarker Discovery, CRISPR, Expressing, Fluorescence, Control, Over Expression, Whisker Assay

Journal: Oncogenesis

Article Title: Increased expression of NAF1 contributes to malignant phenotypes of glioma cells through promoting protein synthesis and associates with poor patient survival

doi: 10.1038/s41389-019-0134-2

Figure Lengend Snippet: Upon knocking down of c-Myc ( a , b ) and NRF2 ( c , d ) in glioma cell lines SF295 and U87 using siRNAs, the protein and mRNA expression levels of NAF1, c-Myc, and NRF2 were measured by western blot and qRT-PCR assays. Shown is representative of three independently preformed western blot experiments. GAPDH and β-actin were used as the normalized controls for western blot and qRT-PCR assays, respectively. Data were shown as mean ± SD, * P < 0.05; ** P < 0.01 ( n = 3). Upon ectopic expression of c-Myc ( e , f ) and NRF2 ( g , h ) in glioma cell lines SF295 and U87, the protein and mRNA expression levels of NAF1, c-Myc, and NRF2 were determined by western blot and qRT-PCR assays. Shown is representative of three independently preformed western blot experiments. GAPDH and β-actin were used as the normalized controls for western blot and qRT-PCR assays, respectively. Data were shown as mean ± SD, * P < 0.05; ** P < 0.01; *** P < 0.001 ( n = 3). The Dual-Luciferase Reporter assay system was used to evaluate the impact of ectopic expression of c-Myc ( i ) and NRF2 ( j ) on the promoter activity of NAF1 in SF295 and U87 cells with the empty vector or control lentivirus as the controls. All the ratios of the Luc/Renilla activity were expressed as means ± SD. *** P < 0.001 ( n = 3). k SF295 cells expressing c-Myc and NRF2 and control cells were subjected to ChIP-qPCR assays using corresponding primary antibodies. P1–P4 indicated four different regions of NAF1 promoter (P1: −100/−26; P2: −519/−410; P3: −981/−543; P4: −1648/−1551) (left panel). Fold enrichment was expressed as mean ± SD (middle and right panels). * P < 0.05; ** P < 0.01 ( n = 3)

Article Snippet: NC16 pCDNA3.1 FLAG NRF2 (Plasmid #36971), pCDNA-3xHA-hTERT (Plasmid #51637), and corresponding empty vector were obtained from Addgene (Cambridge, MA).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Luciferase, Reporter Assay, Activity Assay, Plasmid Preparation, Control, ChIP-qPCR

Journal: Oncogenesis

Article Title: Increased expression of NAF1 contributes to malignant phenotypes of glioma cells through promoting protein synthesis and associates with poor patient survival

doi: 10.1038/s41389-019-0134-2

Figure Lengend Snippet: a , b Protein expression levels of c-Myc, NRF2, and TERT upon NAF1 knockdown or ectopic expression in SF295 and U87 cells were determined by western blot analysis with GAPDH as a loading control. c , d The qRT-PCR assay was carried out to detect mRNA expression of c-Myc , NRF2 , and TERT upon depletion or overexpression of NAF1 in SF295 and U87 cells. β-Actin was used as an endogenous control. Data were shown as mean ± SD. ** P < 0.01; *** P < 0.001 ( n = 3). e , f Western blot analysis was used to evaluate protein expression of POLR1A and POLR2A upon knockdown or ectopic expression of NAF1 in SF295 and U87 cells. GAPDH was used as an endogenous control. The western blot is representative of three independently preformed experiments

Article Snippet: NC16 pCDNA3.1 FLAG NRF2 (Plasmid #36971), pCDNA-3xHA-hTERT (Plasmid #51637), and corresponding empty vector were obtained from Addgene (Cambridge, MA).

Techniques: Expressing, Knockdown, Western Blot, Control, Quantitative RT-PCR, Over Expression

Journal: Oncogenesis

Article Title: Increased expression of NAF1 contributes to malignant phenotypes of glioma cells through promoting protein synthesis and associates with poor patient survival

doi: 10.1038/s41389-019-0134-2

Figure Lengend Snippet: NAF1 plays a crucial role in maintaining the yield of mature H/ACA RNPs. During malignant transformation of glioma cells, increased expression of NAF1 promotes U17 snoRNA processing, 18S rRNA maturation, and the assembly of 40 S subunits, thereby enhancing protein synthesis, including some key molecules associated with malignant progression of gliomas, such as c-Myc, NRF2, TERT, POLR1A, and POLR2A. Meanwhile, c-Myc, NRF2, and TERT in turn transcriptionally upregulate NAF1 expression, while POLR1A and POLR2A also can active the transcription of 45 S rRNA , c-Myc , NRF2 , TERT , and H/ACA snoRNA. These observations indicate that there exist positive feedback loops between NAF1 and these key molecules. In addition, NAF1 can maintain telomere length by increasing the levels of TERT and TERC at transcriptional or post-transcriptional levels. Altogether, these molecular events will contribute to glioma tumorigenesis and progression

Article Snippet: NC16 pCDNA3.1 FLAG NRF2 (Plasmid #36971), pCDNA-3xHA-hTERT (Plasmid #51637), and corresponding empty vector were obtained from Addgene (Cambridge, MA).

Techniques: Transformation Assay, Expressing

Journal: Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association

Article Title: Antiferroptotic properties of allicin and related organosulfur compounds-diallyl disulfide and diallyl trisulfide-from Garlic.

doi: 10.1016/j.fct.2024.115124

Figure Lengend Snippet: Fig. 4. The antiferroptotic effect of allicin is independent of Nrf2-regulated phase II enzyme expression. A. Western blot analysis showing that allicin upregulated expression of the phase II enzymes GCLM, GCLC, and HO-1 in WT HT22 cells but not Nrf2-KD HT22 cells. Cells were incubated with allicin (1 or 10 μM) for 16 h (Upper) Representative immunoblot. (Lower) Band intensities as quantified using ImageJ. Results are presented as the mean ± SD. *P < 0.05; ****P < 0.0001; ns, not significant. B. Allicin protected both WT and Nrf2-KD HT22 cells against erastin-induced death. Cells were treated with the indicated concentrations of allicin in the absence or presence of erastin for 24 h. Cell death was measured using an LDH assay. Results are presented as the mean ± SD. ###P < 0.001; ####P < 0.0001 compared to the corresponding erastin alone by ANOVA with post hoc Tukey’s test.

Article Snippet: Nrf2-knockdown (KD) HT22 cells were generated using the Nrf2 CRISPR/Cas9 KO plasmid (Santa Cruz Biotechnology, Dallas, TX, USA, cat# sc-421869) and the Nrf2 HDR plasmid (Santa Cruz Biotechnology, cat# sc-421869-HDR) as previously described (Hirata et al., 2020).

Techniques: Expressing, Western Blot, Incubation, Lactate Dehydrogenase Assay

Journal: PLoS ONE

Article Title: The toxic effects and possible mechanisms of Brusatol on mouse oocytes

doi: 10.1371/journal.pone.0177844

Figure Lengend Snippet: A) Oocytes at MII stage were immunostained with α -tubulin antibody to visualize spindle (green) and counterstained with Hoechst 33342 for chromosomes (blue). Representative confocal images showing the spindle morphology and chromosome alignment in control (a) and Brusatol treated (b-c) oocytes. Arrowheads indicate the abnormal spindle and arrows indicate the misaligned and decondensed chromosomes. B) Quantification of control, Brusatol treated and Brusatol+Nrf2-plasmid treated oocytes with spindle/chromosome defects. Data are expressed as mean percentage ± SD from three independent experiments in which at least 120 oocytes were analyzed. Scale bar, 20 μm. *p < 0.05.

Article Snippet: Nrf2 CRISPR Activation Plasmid (Cat#: sc-421869-ACT) was obtained from Santa Cruz Biotechnology (San Jose, CA, USA).

Techniques: Control, Plasmid Preparation

Journal: PLoS ONE

Article Title: The toxic effects and possible mechanisms of Brusatol on mouse oocytes

doi: 10.1371/journal.pone.0177844

Figure Lengend Snippet: A) Western blot showing decline of Nrf2 after Bruastol treatment with actin as a loading control. B) The relative mRNA level of Nrf2 was determined by qRT-PCR in control and Brusatol treated oocytes. mRNA level in control oocytes were set as 1. C) PBS (control group) or Nrf2 CRISPR Activation Plasmid (activation group) was microinjected into GV oocytes, which were arrested for 20 h with milrinone to allow synthesis of new Nrf2 protein. Then the oocytes were collected for Western blot. Results indicated that Nrf2 protein was efficiently overexpressed. D,E) Quantitative analysis of GVBD and Pb1 extrusion in different groups. Data are expressed as mean percentage ± SD from three independent experiments in which at least 80 oocytes were analyzed. *p < 0.05.

Article Snippet: Nrf2 CRISPR Activation Plasmid (Cat#: sc-421869-ACT) was obtained from Santa Cruz Biotechnology (San Jose, CA, USA).

Techniques: Western Blot, Control, Quantitative RT-PCR, CRISPR, Activation Assay, Plasmid Preparation

Journal: PLoS ONE

Article Title: The toxic effects and possible mechanisms of Brusatol on mouse oocytes

doi: 10.1371/journal.pone.0177844

Figure Lengend Snippet: A) Brusatol reduced the mRNA level of Nrf2 target genes. oocytes were treated with 200 nM of brusatol for 4h, 8h, 14h and subjected to qRT-PCR. B) Representative images of CM-H2DCFDA fluorescence in control, and Brusatol treated oocytes to show the ROS levels. C) Quantitative analysis of fluorescence intensity shown in B. D) Brusatol reduced the mRNA level of mitochondrial related genes. E) To mark the mitochondrion, representative images of mito-tracker fluorescence in control, and Brusatol treated oocytes were taken. F) Quantitative analysis of fluorescence intensity shown in E. Scale bar, 20 μm. *p < 0.05.

Article Snippet: Nrf2 CRISPR Activation Plasmid (Cat#: sc-421869-ACT) was obtained from Santa Cruz Biotechnology (San Jose, CA, USA).

Techniques: Quantitative RT-PCR, Fluorescence, Control

Journal: PLoS ONE

Article Title: The toxic effects and possible mechanisms of Brusatol on mouse oocytes

doi: 10.1371/journal.pone.0177844

Figure Lengend Snippet: A) The relative mRNA level of cyclin B1 was determined by qRT-PCR in control and Brusatol treated oocytes. mRNA level in control oocytes were set as 1. B) Oocytes at MI stage were immunostained with cyclin B1 antibody (green) and counterstained with Hoechst 33342 for chromosomes (blue). C) Western blot showing decline of cyclin B1 after Bruastol treatment with actin as a loading control. C) Western blot showing increase of cyclin B1 after Nrf2 overexpression with actin as a loading control. Error bars indicate ± sd Scale bar, 20 μm. *p < 0.05.

Article Snippet: Nrf2 CRISPR Activation Plasmid (Cat#: sc-421869-ACT) was obtained from Santa Cruz Biotechnology (San Jose, CA, USA).

Techniques: Quantitative RT-PCR, Control, Western Blot, Over Expression

Journal: iScience

Article Title: Suppression of the KRAS - NRF2 axis shifts arginine into the phosphocreatine energy system in pancreatic cancer cells

doi: 10.1016/j.isci.2023.108566

Figure Lengend Snippet: Characterization of Panc-1 NRF2 −/ − cells (A) Luciferase driven by the full (pL) or truncated (pS) NRF2 promoter after ectopic expression of wild-type KRAS or KRASG12D. DMNQ, 1,4-naphthoquinone, is a molecule that produces ROS. (B) Volcano plot of DEGs in NRF2 −/ − compared to WT-Panc-1 cells. (C and D) Gene Set Enrichment Analysis (GSEA) plots showing the enrichment of NRF2 signaling and antioxidant response genes in WT versus NRF2 −/ − Panc-1 cells. (E) Heatmap showing fold expression of NRF2 signaling in NRF2 −/− Panc-1 cells compared to WT cells; genes directly regulated by NRF2 through direct binding to the proximal promoter are marked in green (lane 4). (F) Log 2 (fold change) of major DEGs (p < 0.05). (G) Level of basal ROS in NRF2 −/ − and WT Panc-1 cells. (H) GSH/total glutathione and total thiol groups in NRF2 −/ − and WT Panc-1 cells; Data represent the mean ± s.d. of at least 3 independent experiments: ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001 by Student’s t test.

Article Snippet: Plasmid pWZL-HYGRO NRF2 was obtained by subcloning through a restriction-ligation based approach the ORF of NRF2 (BamHI/SalI) from pBABE HYGRO mRFP1 NRF2 (Addgene plasmid #136579).

Techniques: Luciferase, Expressing, Binding Assay

Journal: iScience

Article Title: Suppression of the KRAS - NRF2 axis shifts arginine into the phosphocreatine energy system in pancreatic cancer cells

doi: 10.1016/j.isci.2023.108566

Figure Lengend Snippet: NRF2 depletion affects Panc-1 transcriptome and promotes a deep metabolic reprogramming (A) Functional enrichment analysis of WT cells compared to NRF2 −/− cells. NES = normal enrichment score, FDR = false discovery rate, p= p-value. (B–D) Heatmaps of DEGs relative to glycolysis, PPP and glutathione pathways. (E) DEGs of enzymes involved in glycolysis, PPP and glutathione cycle pathways. (F–H) Expression level of some key enzymes involved in the pathways determined by qRT-PCR. To demonstrate that the enzymes are controlled by the KRAS G12D - NRF2 axis, we re-expressed NRF2 in NRF2 −/− cells and obtained an expression profile similar to that of WT cells. (I) Western blot showing the level of tkt in WT, NRF2 −/− and NRF2 −/− cells in which NRF2 was re-expressed. Data represent the mean ± s.d. of at least 3 independent experiments: ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001 by Student’s t test.

Article Snippet: Plasmid pWZL-HYGRO NRF2 was obtained by subcloning through a restriction-ligation based approach the ORF of NRF2 (BamHI/SalI) from pBABE HYGRO mRFP1 NRF2 (Addgene plasmid #136579).

Techniques: Functional Assay, Expressing, Quantitative RT-PCR, Western Blot

Journal: iScience

Article Title: Suppression of the KRAS - NRF2 axis shifts arginine into the phosphocreatine energy system in pancreatic cancer cells

doi: 10.1016/j.isci.2023.108566

Figure Lengend Snippet: Panc-1 NRF2 −/ − cells show higher mitochondrial function and a change in oncogenic program (A) Real-time oxygen consumption rate (OCR) was determined during successive treatments with oligomycin (ATP synthase inhibitor), FCCP (uncoupler of oxidative phosphorylation), rotenone (complex I inhibitor) and antimycin-A (complex III inhibitor) in WT, NRF2 −/ − and NRF2 −/ − +NRF2 cells. (B and C) The rates of basal respiration, ATP-coupled respiration, maximal respiration and OCR/ECAR ratio in NRF2 −/− and NRF2 −/ − +NRF2 cells were normalised to total protein content and quantified. (D) NRF2 −/ − cells show a 10-fold higher ROS level than WT cells. When NRF2 is re-expressed in the cells, the ROS level decreases to the level observed in WT cells. (E and F) GSEA plots showing the enrichment of KRAS in WT and MYC in NRF2 −/ − Panc-1 cells. (G) Expression of MYC in WT and NRF2 −/ − cells; XMD8-92 (5 μM) was used to inhibit ERK5 for 24 h. Densitometric analysis of 3 experiments is provided. (H) phosphorylation levels of AKT1 and ERK1/2 in WT, NRF2 −/ − and NRF2 −/ − + NRF2 cells. Densitometric analysis of 4 experiments is provided; data represent the mean ± s.d. of at least 3 independent experiments: ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001 by Student’s t test.

Article Snippet: Plasmid pWZL-HYGRO NRF2 was obtained by subcloning through a restriction-ligation based approach the ORF of NRF2 (BamHI/SalI) from pBABE HYGRO mRFP1 NRF2 (Addgene plasmid #136579).

Techniques: Phospho-proteomics, Expressing

Journal: iScience

Article Title: Suppression of the KRAS - NRF2 axis shifts arginine into the phosphocreatine energy system in pancreatic cancer cells

doi: 10.1016/j.isci.2023.108566

Figure Lengend Snippet: NRF2 depletion leads to the activation of alternative energy supply pathways (A and B) Heatmaps showing DEG clusters involved in arginine/proline and glutamine metabolism. (C) Metabolic network showing the fate of arginine in Panc-1 cells lacking the KRAS G12D -NRF2 axis. The cells respond to NRF2 deletion by channeling arginine toward creatine, polyamines and nitric oxide metabolism. Enzymes involved in the metabolic pathways are shown, ↓ = downregulated, ↑ = upregulated. (D) Log 2 (fold change) of some key DEGs (p < 0.05) involved in arginine metabolism. (E and F) Expression level of some key enzymes determined by qRT-PCR. As a control, we re-expressed NRF2 in NRF2 -/- cells. The enzymes CKM1A and CKM1B are ckb isozymes localized in the mitochondria. (G) Expression of GLUL was measured by western blot. Data represent the mean ± s.d. of at least 3 independent experiments: ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001 by Student’s t test.

Article Snippet: Plasmid pWZL-HYGRO NRF2 was obtained by subcloning through a restriction-ligation based approach the ORF of NRF2 (BamHI/SalI) from pBABE HYGRO mRFP1 NRF2 (Addgene plasmid #136579).

Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Control, Western Blot

Journal: iScience

Article Title: Suppression of the KRAS - NRF2 axis shifts arginine into the phosphocreatine energy system in pancreatic cancer cells

doi: 10.1016/j.isci.2023.108566

Figure Lengend Snippet: Arginine catabolism meets the energy needs of NRF2 −/ − cells (A) Creatine biosynthesis intermediates in WT, NRF2 −/ − and NRF2 −/ − added with NRF2 cells. (B) Creatine kinase enzyme activity in WT, NRF2 −/ − and NRF2 −/ − added with NRF2 cells. (C) Western blot showing suppression of CKB with esiRNAs against CKB. (D) ATP levels in WT, NRF2 −/ − and NRF2 −/ − cells re-expressing NRF2. Suppression of CKB leads to a strong decrease in ATP levels. (E) Urea cycle intermediates in WT, NRF2 −/ − and NRF2 −/ − re-expressing NRF2. Data represent the mean ± s.d. of 5 independent experiments: ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001 by Student’s t test (paired comparison) or Dunn’s multiple comparison test.

Article Snippet: Plasmid pWZL-HYGRO NRF2 was obtained by subcloning through a restriction-ligation based approach the ORF of NRF2 (BamHI/SalI) from pBABE HYGRO mRFP1 NRF2 (Addgene plasmid #136579).

Techniques: Activity Assay, Western Blot, Expressing, Comparison

Journal: iScience

Article Title: Suppression of the KRAS - NRF2 axis shifts arginine into the phosphocreatine energy system in pancreatic cancer cells

doi: 10.1016/j.isci.2023.108566

Figure Lengend Snippet: Depletion of NRF2 makes Panc-1 cells addicted to amino acids, especially arginine, but less sensitive to glucose restriction (A) % Cell viability relative to DMEM of WT, NRF2 −/− and NRF2 −/− +NRF2 Panc-1 cells in medium without glutamine (-Gln), low glucose (LG), LG without glutamine and 2-deoxyglucose (2DG). (B) Amount of ATP (pmol ATP/cell) in WT and NRF2 −/− cells, clone KO-134 and KO-16 cells in DMEM (high glucose) and LG. (C) % colonies in WT, NRF2 −/− and NRF2 −/− re-expressing NRF2 cells cultured in DMEM, LG without arginine and LG with added arginine. (D) Confocal microscopy images of WT and NRF2 −/− spheroids embedded in Matrigel and cultured in DMEM (high glucose) or LG for 4 days. Length scale bar = 10μm. (E and F) Growth of WT, NRF2 −/− and NRF2 −/− +NRF2 spheroids in DMEM or LG. The percentage of WT and NRF2 −/− spheroids showing invasive/branching structures when grown in DMEM and LG for 4 days; Data represent the mean ± s.d. of at least 3 independent experiments: ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001 by Student’s t test (paired comparison) or Dunn multiple comparisons test.

Article Snippet: Plasmid pWZL-HYGRO NRF2 was obtained by subcloning through a restriction-ligation based approach the ORF of NRF2 (BamHI/SalI) from pBABE HYGRO mRFP1 NRF2 (Addgene plasmid #136579).

Techniques: Expressing, Cell Culture, Confocal Microscopy, Comparison

Journal: iScience

Article Title: Suppression of the KRAS - NRF2 axis shifts arginine into the phosphocreatine energy system in pancreatic cancer cells

doi: 10.1016/j.isci.2023.108566

Figure Lengend Snippet: Effect of cyclocreatine on PDAC growth (A) Epifluorescence microscopy images of Hoechst + WT and NRF2 −/ − cells invading a 3D matrix after exposure to 20% FBS as chemoattractant for 16 h in the presence or absence of cyclocreatine. (B) Matrigel invasion rate in the absence and presence of CCr of 3 × 10 5 WT and NRF2 −/− cells grown in 0.1% FBS/DMEM or 0.1% FBS/DMEM + 5mM CCr resuspended and seeded in 24mm Transwell with 8.0 μm pore polycarbonate membrane coated with Matrigel solution. Matrix invasion into the lower chamber containing 20% FBS/DMEM was measured 16 h after seeding by staining the invading cells with DAPI. For each experiment, the invasion rate of 5 randomly selected high-power fields was evaluated. (C) ATP production (pmol ATP/cell) in WT and NRF2 −/ − cells treated with 1 and 5 mM cyclocreatine or 5 μM homoarginine for 48 h. (D) Effect on colony formation of anthrathiophenedione 1.6 μM 2a in the absence or presence of 5 μM cyclocreatine (CCr) by WT and NRF2 −/ − cells. (E) Immunoblot evaluation of NRF2 levels in WT (clone 202), NRF2 +/ − (clone 201) and NRF2 −/ − (clone 239) MIA PaCa-2 cells. (F) mRNA expression level of the indicated genes in MIA PaCa-2 clones compared to WT cells. (G) MIA PaCa-2 clones were treated with the indicated mM concentrations of sodium oxamate for 60 h and subjected to a resazurin/resorufin assay. IC50 was calculated using an unadjusted third-degree polynomial regression curve. (H) histogram showing the ATP levels of the indicated cells treated or not with 5 mM CCr for 72 h. Data refer to untreated WT cells. (I) Percentage of colony formation of WT and NRF2 −/ − MIA-PaCa-2 cells in the presence and absence of arginine. Data represent the mean ± s.d. of at least 3 independent experiments: ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001 by Student’s t test (paired comparison) or Dunn multiple comparison test.

Article Snippet: Plasmid pWZL-HYGRO NRF2 was obtained by subcloning through a restriction-ligation based approach the ORF of NRF2 (BamHI/SalI) from pBABE HYGRO mRFP1 NRF2 (Addgene plasmid #136579).

Techniques: Epifluorescence Microscopy, Membrane, Staining, Western Blot, Expressing, Clone Assay, Comparison

Journal: iScience

Article Title: Suppression of the KRAS - NRF2 axis shifts arginine into the phosphocreatine energy system in pancreatic cancer cells

doi: 10.1016/j.isci.2023.108566

Figure Lengend Snippet: The KRAS/NRF2 axis is switched off and arginine metabolism is switched on in PDAC developing resistance to FOLFIRINOX (A) GSEA plot using as geneset genes downregulated in ID211 compared to ID188 ( Table S2 ) and our RNA-seq dataset. Functional enrichment was performed on core-enriched genes and expressed as FDR (False Discovery Rate). (B) GSEA plot obtained by using as geneset the top 500 genes downregulated in NRF2 −/ − Panc-1 cells and GSE193389 as the dataset. Functional enrichment was performed on the core-enriched genes and expressed as FDR. (C) GSEA plot obtained by using as geneset genes downregulated in PDOs obtained from FOLFIRINOX-treated patients and our RNA-seq dataset. Functional enrichment was performed on the core-enriched genes and expressed as FDR. (D) Heatmap of NRF2-regulated arginine-auxotrophic genes. (E) GSEA plot obtained by using as geneset the “NRF2-regulated arginine-auxotrophic signature” and GSE193389 as the dataset. (F) Heatmap of 177 TCGA-PDAC patients ranked accordingly to the expression of the seven indicated genes representing the “NRF2-regulated arginine-auxotrophic signature”. For each sample, the Winter hypoxia index (W.H.I.) and the fraction of the genome altered (F.G.A.) are indicated above the heatmap. (G) Dot plots showing the expression levels, expressed as z-scores, of NRF2 signaling ( G6PD, GSTM4, NQO1, GCLM, HMOX1, PRDX1 ), LDHA and Winter Hypoxia score in patients with increased expression of the “NRF2-regulated arginine auxotrophic signature” (z > 3, indicated in red) compared to other patients (indicated in gray). (H) Left, Kaplan-Meier survival analysis in 168 TCGA-PDAC patients characterized by high (above the median, in red) or low (below the median, in blue) expression of the “NRF2-regulated arginine auxotrophic signature”; right: histogram representing the frequency of genetic alterations of the indicated oncogenes and tumor suppressors in the two identified patient groups.

Article Snippet: Plasmid pWZL-HYGRO NRF2 was obtained by subcloning through a restriction-ligation based approach the ORF of NRF2 (BamHI/SalI) from pBABE HYGRO mRFP1 NRF2 (Addgene plasmid #136579).

Techniques: RNA Sequencing, Functional Assay, Expressing

Journal: iScience

Article Title: Suppression of the KRAS - NRF2 axis shifts arginine into the phosphocreatine energy system in pancreatic cancer cells

doi: 10.1016/j.isci.2023.108566

Figure Lengend Snippet:

Article Snippet: Plasmid pWZL-HYGRO NRF2 was obtained by subcloning through a restriction-ligation based approach the ORF of NRF2 (BamHI/SalI) from pBABE HYGRO mRFP1 NRF2 (Addgene plasmid #136579).

Techniques: Recombinant, GSH Assay, esiRNA