nrf2 over expression plasmid  (Addgene inc)

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: Free radical biology & medicine

Article Title: Nrf2/ARE pathway attenuates oxidative and apoptotic response in human osteoarthritis chondrocytes by activating ERK1/2/ELK1-P70S6K-P90RSK signaling axis

doi: 10.1016/j.freeradbiomed.2018.01.013

Figure Lengend Snippet: Human OA chondrocytes were transfected with Nrf2 expression plasmid (pNrf2, 1μg) or pcDNA3.1 as control plasmid (1μg) using P3 Primary Cell 4D-Nucleofector™ X Kit on 4D on Amaxa Nucleofection System. Forty-eight hours after the transfection, OA chondrocytes were serum starved and then stimulated with or without IL-1β (10 ng/ml) for 48 h. (A). At end of treatment, cell lysates were prepared using RIPA buffer and protein levels of cleaved caspase-3, cleaved caspase-8, pro-caspase-9, cleaved PARP and Bid were investigated by immunoblotting using antibodies against indicated protein. β-actin was used as a control for equal loading. Immunoblot results are representatives of three blots performed on samples obtained from three individuals. (B) Fold change relative to β-actin for the expression these proteins were quantified by measuring specific signal intensities using ImageJ software. *p≤0.05, as compared to pcDNA, # ≤0.05, as compared to pcDNA+IL-1β. (C) After transfection, OA chondrocyte were stimulated IL-1β (1 ng/ml) for 30 minutes at 37°C and then stained with JC-1 (2.5 μM) and mitochondrial membrane potential loss was measured by ratio of red to green fluorescence. Values were expressed relative to unstimulated control and graph shows mean ± SD of four replicates from a representative experiment and three such independent experiments were carried out. *p≤0.05, as compared to control, # ≤0.05, as compared to IL-1β. (D) Cytosolic lysates were prepared after 48 h of IL-1β (10 ng/ml) stimulation in transfected chondrocytes and levels of cytosolic cytochrome-c was estimated by immunoblotting using validated antibody. β-actin was used as a control for equal loading. Immunoblot results are representatives of three blots performed on samples obtained from three individuals.

Article Snippet: Over-expression and knockdown of Nrf2 in OA chondrocytes Nrf2 over-expression plasmid (pcDNA3-EGFP-C4-Nrf2) was a gift from Yue Xiong (Addgene plasmid # 21549) [ 29 ].

Techniques: Transfection, Expressing, Plasmid Preparation, Control, Western Blot, Software, Staining, Membrane, Fluorescence

Journal: Free radical biology & medicine

Article Title: Nrf2/ARE pathway attenuates oxidative and apoptotic response in human osteoarthritis chondrocytes by activating ERK1/2/ELK1-P70S6K-P90RSK signaling axis

doi: 10.1016/j.freeradbiomed.2018.01.013

Figure Lengend Snippet: Cartilage from OA patients and healthy donors was processed for lysate preparation or RNA isolation using method described in method section. (A) Protein expression of Nrf2 and its signaling target HO-1 and SOD2 was investigated by immunoblotting using antibodies against indicated protein. β-actin was used as a control for equal loading. Fold change relative to β-actin for the expression these proteins were quantified by measuring specific signal intensities using ImageJ software. Immunoblot results are representatives of three blots performed on samples obtained from three individuals. *p≤0.05, as compared to normal cartilage. (B) Expression of Nrf2, HO-1 and SOD2 was measured by quantitative PCR using SYBR® green assay (Life Technologies). β-actin was used as endogenous expression control. (C–E) RNA was isolated from damaged or smooth cartilage for same patients and mRNA levels of (C) Nrf2, (D) HO-1 and (E) SOD2 were determined by SYBR® green assay (Life Technologies). Each symbol represents a single patient and horizontal lines shows the mean value of expression levels in the indicated number of samples. Bar graph represents mean±SD from three subjects. *p≤0.05, as compared to normal cartilage or smooth cartilage.

Article Snippet: Over-expression and knockdown of Nrf2 in OA chondrocytes Nrf2 over-expression plasmid (pcDNA3-EGFP-C4-Nrf2) was a gift from Yue Xiong (Addgene plasmid # 21549) [ 29 ].

Techniques: Isolation, Expressing, Western Blot, Control, Software, Real-time Polymerase Chain Reaction, SYBR Green Assay

Journal: Free radical biology & medicine

Article Title: Nrf2/ARE pathway attenuates oxidative and apoptotic response in human osteoarthritis chondrocytes by activating ERK1/2/ELK1-P70S6K-P90RSK signaling axis

doi: 10.1016/j.freeradbiomed.2018.01.013

Figure Lengend Snippet: Primary human chondrocytes were isolated from cartilage of OA patients. (A) Nrf2/ARE luciferase reporter vector was transfected in OA chondrocytes and 48 hours later, chondrocytes were stimulated with IL-1β (1 ng/ml) for indicated time and luciferase activity was measured by Dual-Glo® reporter assay. Renilla luciferase was cotransfected for normalization purposes. Values are the mean±SD of 2 experiments each performed in triplicate. (B–C) Human OA chondrocytes were stimulated with IL-1β (1 ng/ml) for indicated time. At the end of treatment, chondrocytes were harvested and cell lysates were prepared using RIPA buffer for immunoblot analysis. (B) Protein expression of Nrf2, HO-1, NQO1 and SOD2 was investigated by immunoblotting using antibodies against indicated protein. β-actin was used as a control for equal loading. Immunoblot results are representatives of three blots performed on samples obtained from three individuals. (C) Fold change relative to β-actin for the expression these proteins were quantified by ImageJ software. *p≤0.05, as compared to control. (D) Human OA chondrocytes were pre-treated with anti-oxidants-NAC(10 mM) or GSH (10 mM) or DPI (5μM) for 2 h followed by treatment with IL-1β (1 ng/ml) for 16 h and RNA were isolated from harvested chondrocytes and expression of Nrf2 and HO-1 was measured by quantitative PCR using the SYBR® green assay (Life Technologies). β-actin was used as endogenous expression control. Bar graph represents mean±SD from three subjects. *p≤0.05, as compared to control, # ≤0.05, as compared to IL-1β.

Article Snippet: Over-expression and knockdown of Nrf2 in OA chondrocytes Nrf2 over-expression plasmid (pcDNA3-EGFP-C4-Nrf2) was a gift from Yue Xiong (Addgene plasmid # 21549) [ 29 ].

Techniques: Isolation, Luciferase, Plasmid Preparation, Transfection, Activity Assay, Reporter Assay, Western Blot, Expressing, Control, Software, Real-time Polymerase Chain Reaction, SYBR Green Assay

Journal: Free radical biology & medicine

Article Title: Nrf2/ARE pathway attenuates oxidative and apoptotic response in human osteoarthritis chondrocytes by activating ERK1/2/ELK1-P70S6K-P90RSK signaling axis

doi: 10.1016/j.freeradbiomed.2018.01.013

Figure Lengend Snippet: Human OA chondrocytes were transfected with siRNA specific for Nrf2 (siNrf2, 100 nM) or negative controls siRNA (siCNT, 100 nM) using X-tremeGENE™ siRNA transfection reagent or transfected with Nrf2 expression plasmid (pNrf2, 1μg) or pcDNA3.1 as control plasmid (1μg) using P3 Primary Cell 4D-Nucleofector™ X Kit on 4D on Amaxa Nucleofection System. (A, B) Forty-eight hours after the transfection with (A) siRNA or (B) expression vector, OA chondrocytes were stained with DHR123 (5 μM) for 0.5 h at 37°C, and stimulated with IL-1β (1 ng/ml) for 30 minutes at 37°C and ROS generation was estimated by measuring fluorescence emission at 535 nm. Bar graph shows arbitrary fluorescence units indicating ROS levels. (C–D) Forty-eight hours after the transfection with (C) siRNA or (D) expression vector, OA chondrocytes were stained with stimulated with IL-1β (1 ng/ml) for 30 minutes at 37°C, supernatants were collected and generation of H2O2 in the supernatant was estimated using Amplex red assay as described in method section. Data points represent mean±SD of three replicates from a representative experiment and three such independent experiments were carried out.*p≤0.05, as compared to control, # ≤0.05, as compared to IL-1β.

Article Snippet: Over-expression and knockdown of Nrf2 in OA chondrocytes Nrf2 over-expression plasmid (pcDNA3-EGFP-C4-Nrf2) was a gift from Yue Xiong (Addgene plasmid # 21549) [ 29 ].

Techniques: Transfection, Expressing, Plasmid Preparation, Control, Staining, Fluorescence, Amplex Red Assay

Journal: Free radical biology & medicine

Article Title: Nrf2/ARE pathway attenuates oxidative and apoptotic response in human osteoarthritis chondrocytes by activating ERK1/2/ELK1-P70S6K-P90RSK signaling axis

doi: 10.1016/j.freeradbiomed.2018.01.013

Figure Lengend Snippet: Human OA chondrocytes were transfected with Nrf2 expression plasmid (pNrf2, 1μg) or pcDNA3.1 as control plasmid (1μg) using P3 Primary Cell 4D-Nucleofector™ X Kit on 4D on Amaxa Nucleofection System. Forty-eight hours after the transfection, OA chondrocytes were serum starved and then stimulated with or without IL-1β (10 ng/ml) for 48 h. (A) Cell viability was measured by MTT assays using method described in method sections. Unstimulated chondrocytes were served as control and viability was expressed relative to control cells. Data point was expressed as a mean ± SD of two replicates from a representative experiment and three such independent experiments were carried out. *p≤0.05, as compared to pcDNA (control), # ≤0.05, as compared to pcDNA+IL-1β group. (B) Apoptosis was estimated by TUNEL assay as described in method section. Flow cytometric histograms showing percent TUNEL positive chondrocytes were shown. Twenty thousand cells in each group were acquired using a flowcytometer and percent apoptosis was calculated using flow Jo software and expressed as a mean±SD of three replicates from a representative experiment and three such independent experiments were carried out. (C) Apoptosis was visualized by DNA fragmentation assay as described in method section and DNA samples were electrophoresed in 1.8% agarose gel and DNA fragmentation pattern in control and simulated groups were visualized by EtBr staining. Results are representatives of three experiments performed on samples obtained from three individuals.

Article Snippet: Over-expression and knockdown of Nrf2 in OA chondrocytes Nrf2 over-expression plasmid (pcDNA3-EGFP-C4-Nrf2) was a gift from Yue Xiong (Addgene plasmid # 21549) [ 29 ].

Techniques: Transfection, Expressing, Plasmid Preparation, Control, TUNEL Assay, Software, DNA Fragmentation Assay, Agarose Gel Electrophoresis, Staining

Journal: Free radical biology & medicine

Article Title: Nrf2/ARE pathway attenuates oxidative and apoptotic response in human osteoarthritis chondrocytes by activating ERK1/2/ELK1-P70S6K-P90RSK signaling axis

doi: 10.1016/j.freeradbiomed.2018.01.013

Figure Lengend Snippet: Human OA chondrocytes were transfected with Nrf2 expression plasmid (pNrf2, 1μg) or pcDNA3.1 as control plasmid (1μg) using P3 Primary Cell 4D-Nucleofector™ X Kit on 4D on Amaxa Nucleofection System. Forty-eight hours after the transfection, OA chondrocytes were serum starved and then stimulated with or without IL-1β (10 ng/ml) for 15 and 30 minutes at 37°C (A–C). At end of treatment, cell lysates were prepared using RIPA buffer and immunoblotting was performed to detect the protein levels of (A) p-ERK1/2 (Thr180/Tyr182), total ERK1/2, p-MEK1/2 (Ser217/221), total MEK1/2, p-ELK-1 (Ser383), p-P70S6K (Ser411), p-P90RSK (Ser380). β-actin was used as a control for equal loading. Immunoblot results are representatives of three blots performed on samples obtained from three individuals. (B–C) At end of transfection, chondrocytes were treated with ERK inhibitor PD98059 (30 μM) for 2 h and then stimulated with IL-1β (10 ng/ml) for (B) 15 minutes and (C) 16 h at 37°C and processed for (B) immunoblotting with p-ERK1/2 and total ERK1/2 and (C) TUNEL assay as described in method section. Bar graph shows mean±SD of three replicates from a representative experiment and three such independent experiments were carried out. *p≤0.05, as compared to pcDNA, # ≤0.05, as compared to pcDNA+IL-1β, $≤0.05, as compared to pNrf2+IL-1β.

Article Snippet: Over-expression and knockdown of Nrf2 in OA chondrocytes Nrf2 over-expression plasmid (pcDNA3-EGFP-C4-Nrf2) was a gift from Yue Xiong (Addgene plasmid # 21549) [ 29 ].

Techniques: Transfection, Expressing, Plasmid Preparation, Control, Western Blot, TUNEL Assay

Journal: Free radical biology & medicine

Article Title: Nrf2/ARE pathway attenuates oxidative and apoptotic response in human osteoarthritis chondrocytes by activating ERK1/2/ELK1-P70S6K-P90RSK signaling axis

doi: 10.1016/j.freeradbiomed.2018.01.013

Figure Lengend Snippet: Nrf2 plays a pivotal role in protecting osteoarthritis chondrocytes against IL-1β-induced oxidative stress and apoptotic events through inhibition of extrinsic apoptosis (caspase-8) and intrinsic apoptotic pathways (caspase-9 and 3), mitochondria dysfunction, release of cytochrome-c in cytosol, expression of pro-apoptotic proteins-Bax and Bad and stimulation of anti-apoptotic proteins-Bcl-2, Bcl-xl and Mcl-1 via activation of ERK1/2/ELK1-P70S6K-P90RSK signaling axis in human OA chondrocytes.

Article Snippet: Over-expression and knockdown of Nrf2 in OA chondrocytes Nrf2 over-expression plasmid (pcDNA3-EGFP-C4-Nrf2) was a gift from Yue Xiong (Addgene plasmid # 21549) [ 29 ].

Techniques: Inhibition, Expressing, Activation Assay