Journal: Cell Communication and Signaling : CCS

Article Title: E3 ligase TRIM15 facilitates non-small cell lung cancer progression through mediating Keap1-Nrf2 signaling pathway

doi: 10.1186/s12964-022-00875-7

Figure Lengend Snippet: TRIM15 stabilizes Nrf2 through binding with Keap1. A HEK293 cells transfected with Flag-TRIM15, HA-Keap1, and Myc-Nrf2 were subjected to immunoprecipitation with HA antibody. Lysates were analyzed by western blotting. B TRIM15 reduced the interaction between Nrf2 and Keap1. Cell lysates were immunoprecipitated with an anti-Keap1 antibody and blotted with an anti-Nrf2 antibody. C – F Subcellular fractionation was used to isolate cytoplasmic and nuclear proteins, and immunoblotting was performed to examine the localization of Nrf2 following the downregulation or overexpression of TRIM15. Nuclear and cytoplasmic levels of Nrf2 are quantified. G Effect of TRIM15 knockdown (H1299 cells) or overexpression (H1650 cells) on the mRNA expression of the Nrf2-regulated genes. NAD(P)H quinone dehydrogenase1(NOQ1), thioredoxin (TXN), peroxiredoxin 1(PRDX1), hemoxygenase 1(HMOX1), glutamate-cysteine ligase catalytic subunit (GCLC), glutathione S-transferase μ1(GSTM1), glutathione S-transferase μ3(GSTM3), ferritin light chain (FTL). H Representative IHC staining images of Nrf2 in the same set of NSCLC tissue slices. Correlation analysis of TRIM15 and Nrf2 expression in NSCLC samples. Spearman correlation coefficients are shown. Scale bars, 100 μm. Statistical analyses were performed by two-tailed unpaired Student’s t -test. ** P < 0.01

Article Snippet: TRIM15 (Cat: HG23822-UT), Flag-TRIM15, HA-Keap1 (Cat: HG11981-NY), Nrf2 (Cat: HG17384-ACR), and Myc-Nrf2 (Cat: MG56971-NM) expression plasmid was purchased from Sino Biological Inc.

Techniques: Binding Assay, Transfection, Immunoprecipitation, Western Blot, Fractionation, Over Expression, Knockdown, Expressing, Immunohistochemistry, Two Tailed Test

Journal: Cell Communication and Signaling : CCS

Article Title: E3 ligase TRIM15 facilitates non-small cell lung cancer progression through mediating Keap1-Nrf2 signaling pathway

doi: 10.1186/s12964-022-00875-7

Figure Lengend Snippet: TRIM15-mediated Nrf2 signaling regulates growth and invasion in NSCLC cells in vitro. A Western blot analyses of TRIM15, Nrf2, Keap1, and Nrf2 target NQO1 in H1299 cells with TRIM15 knockdown with or without subsequent Nrf2 overexpression and H1650 cells overexpressing TRIM15 with or without subsequent knockdown of Nrf2. B ARE Luc reporter activity assessed in H1299 cells expressing shTRIM15, sh TRIM15 + Nrf2 or H1650 cells overexpressing TRIM15 with or without subsequent knockdown of Nrf2. Up-regulating of Nrf2 expression in H1299 cells or down-regulating of Nrf2 expression in H1650 cells was set as a control. C – F Cell proliferation ( C , D ), invasion ( E ) and ROS formation ( F ) in H1299 cells with or without shTRIM15 and Nrf2 rescue or in H1650 cells with or without TRIM15 overexpression and shNrf2 rescue. Statistical analyses were performed by two-tailed unpaired Student’s t -test. ** P < 0.01

Article Snippet: TRIM15 (Cat: HG23822-UT), Flag-TRIM15, HA-Keap1 (Cat: HG11981-NY), Nrf2 (Cat: HG17384-ACR), and Myc-Nrf2 (Cat: MG56971-NM) expression plasmid was purchased from Sino Biological Inc.

Techniques: In Vitro, Western Blot, Knockdown, Over Expression, Activity Assay, Expressing, Control, Two Tailed Test

Journal: Cell Communication and Signaling : CCS

Article Title: E3 ligase TRIM15 facilitates non-small cell lung cancer progression through mediating Keap1-Nrf2 signaling pathway

doi: 10.1186/s12964-022-00875-7

Figure Lengend Snippet: TRIM15 mediated increase in Nrf2 regulates growth and invasion in vivo. A Nude mice were randomized into three groups and subcutaneously injected with H1650 cells that had been transfected with control (empty vector), TRIM15, or TRIM15 + shNrf2 plasmids. Tumors formed in nude mice were collected 30 days after grafting, and the tumor weight were measured. B Measurement of tumor volume in experimental groups over time. C Western blotting analysis was performed to evaluate the levels of TRIM15, Nrf2, Keap1, and NQO1 in harvested tumors. D , E Up-regulation of TRIM15 significantly promoted lung metastasis in H1650 xenograft nude mice models, whereas the suppression of Nrf2 prevented the tumor metastasis of TRIM15 overexpressing cells. Representative pictures of the lung metastases in nude mice by H&E staining. Quantification of lung metastases in all groups. Scale bar: 200 μm. F , G A representative image of tumor growth in nude mice subcutaneously inoculated with H1299 cells tranfected with shCtrl, shTRIM15 or shTRIM15 + Nrf2 plasmids. Tumor volumes were measured on the indicated days. H Western blotting analysis was performed to evaluate the levels of TRIM15, Nrf2, Keap1, and NQO1 in xenograft tumors. I Representative pictures of the lung metastases in nude mice by H&E staining. Quantification of lung metastases in all groups. Scale bar: 200 μm. J TRIM15 was significantly upregulated in NSCLC and that increased TRIM15 was associated with poor survival. TRIM15 promoted tumor proliferation and metastasis by activating Nrf2 signaling. Furthermore, TRIM15 regulated Nrf2 activity by modulating Keap1 and inducing its ubiquitination and degradation in NSCLC cells. Activation of Nrf2 facilitated tumor cell proliferation and invasion. N.S. represents no significant. Statistical analyses were performed by two-tailed unpaired Student’s t -test. ** P < 0.01

Article Snippet: TRIM15 (Cat: HG23822-UT), Flag-TRIM15, HA-Keap1 (Cat: HG11981-NY), Nrf2 (Cat: HG17384-ACR), and Myc-Nrf2 (Cat: MG56971-NM) expression plasmid was purchased from Sino Biological Inc.

Techniques: In Vivo, Injection, Transfection, Control, Plasmid Preparation, Western Blot, Staining, Activity Assay, Activation Assay, Two Tailed Test

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: bioRxiv

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

doi: 10.1101/2020.06.28.175596

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, CUL3 and MYC are negative regulators, while the remaining are positive (p-value < 1e -6 in all cases). (C) Flow cytometry measurements of PD-L1 protein expression across experimental conditions. JQ1 inhibitor treatment (24 hours, 1μM) reduces stimulation-induced PD-L1 expression. (D) Flow cytometry measurements of PD-L1 protein expression based on individual gRNA perturbations, validating our ECCITE-seq findings. (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 reveals a strong enrichment for the NRF2 pathway.

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

Techniques: Expressing, Flow Cytometry, Comparison

Journal: bioRxiv

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

doi: 10.1101/2020.06.28.175596

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). (C) Flow cytometry measurements of PD-L1 protein expression in NRF2 -overexpressing (green) or control (grey) THP-1 cells. Overexpression of NRF2 results in upregulation of PD-L1 protein when compared to control cells. Three independent replicates are shown.

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

Techniques: Ubiquitin Proteomics, Biomarker Discovery, CRISPR, Expressing, Flow Cytometry, Control, Over Expression

Journal: Biomolecules

Article Title: The Impact of NRF2 Inhibition on Drug-Induced Colon Cancer Cell Death and p53 Activity: A Pilot Study

doi: 10.3390/biom12030461

Figure Lengend Snippet: Sulforaphane (SFN) induces NRF2 protein levels and reduces drug-induced cell death. ( a ) HCT116 and RKO cells were exposed to increasing doses of SFN for 24 h, and cell viability was assessed by XTT assay. The histograms represent the mean plus S.D. from three independent experiments. ( b ) Cells treated as in ( a ) were analyzed by Western blot for NRF2 expression levels. Actin was used as protein loading control. The ratio of NRF2 levels vs. β-actin, following densitometric analysis using ImageJ software, is shown. ( c ) In the upper panel, HCT116 and RKO cell viability was measured by XTT assay at 492 nM and cell death was measured by Trypan blue staining after treatment with cisplatin (CDDP) (5 µg/mL) alone or in combination with SFN (2 µM) for 24 h. The results are expressed as cell death percentage ± S.D. In the lower panels, the expression levels of PARP cleavage (cl.) were assessed by Western blot. Actin was used as protein loading control and the ratio of cl.PARP vs. β-actin, is reported. ( d ) Cell viability, as measured by Trypan blue staining, of HCT116-p53 −/− cells treated as in ( c ) The results are expressed as cell death percentage ± S.D. * p ≤ 0.01.

Article Snippet: NRF2 CRISPR/Cas9 KO and NRF2 HDR plasmids were co-transfected into HCT116 colon cancer cells, using UltraCruz Transfection Reagent (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: XTT Assay, Western Blot, Expressing, Control, Software, Staining

Journal: Biomolecules

Article Title: The Impact of NRF2 Inhibition on Drug-Induced Colon Cancer Cell Death and p53 Activity: A Pilot Study

doi: 10.3390/biom12030461

Figure Lengend Snippet: NRF2 is involved in SFN-induced inhibition of CDDP cytotoxicity. ( a ) NRF2-proficient (NRF2-ctr) and NRF2-KO (NRF2-Cas9) cells were treated with SFN (2 µM) for 8 h and NRF2 and HO-1 protein levels analyzed by Western blot. Actin was used as protein loading control. Densitometric analysis of NRF2/β-actin and HO-1/β-actin is reported in the right panels. * p ≤ 0.01. ( b ) NRF2-ctr and NRF2-Cas9 cell proliferation (left panel) was measured by XTT assay and cell viability (right panel) was measured by Trypan blue staining after treatment with cisplatin (CDDP) (5 µg/mL) alone or in combination with SFN (2 µM) for 24 h. The results are expressed as cell death percentage ± S.D. * p ≤ 0.01.

Article Snippet: NRF2 CRISPR/Cas9 KO and NRF2 HDR plasmids were co-transfected into HCT116 colon cancer cells, using UltraCruz Transfection Reagent (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Inhibition, Western Blot, Control, XTT Assay, Staining

Journal: Biomolecules

Article Title: The Impact of NRF2 Inhibition on Drug-Induced Colon Cancer Cell Death and p53 Activity: A Pilot Study

doi: 10.3390/biom12030461

Figure Lengend Snippet: NRF2 inhibition restores CDDP-induced p53 activity impaired by SFN. ( a ) NRF2-ctr and NRF2-Cas9 cells were treated with cisplatin (CDDP) (5 µg/mL) alone or in combination with SFN (2 µM) for 24 h before assessing the phospho (p) Ser46, p53 and HO-1 levels by Western blot. Actin was used as protein-loading control. Densitometric analysis of pSer46/p53, p53/β-actin and HO-1/β-actin is reported in the right panels. * p ≤ 0.01. ( b ) Total mRNA was extracted from NRF2-ctr and NRF2-Cas9 cells untreated or treated as in ( a ). The indicated gene expression was assayed by semiquantitative RT-PCR. The histograms represent the mean plus S.D. of three independent experiments. Densitometric analysis using ImageJ software was applied to calculate the gene/28S ratio. * p ≤ 0.01.

Article Snippet: NRF2 CRISPR/Cas9 KO and NRF2 HDR plasmids were co-transfected into HCT116 colon cancer cells, using UltraCruz Transfection Reagent (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Inhibition, Activity Assay, Western Blot, Control, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Software

Journal: Biomolecules

Article Title: The Impact of NRF2 Inhibition on Drug-Induced Colon Cancer Cell Death and p53 Activity: A Pilot Study

doi: 10.3390/biom12030461

Figure Lengend Snippet: NRF2 activation impairs the CDDP-induced DNA damage. NRF2-ctr and NRF2-Cas9 cells were treated with CDDP (5 µg/mL) alone or in combination with SFN (2 µM) for 24 h. The indicated proteins’ expression was analyzed by Western blot and the densitometric analyses reported in the right panels with S.D. Actin was used as protein loading control. * p ≤ 0.01.

Article Snippet: NRF2 CRISPR/Cas9 KO and NRF2 HDR plasmids were co-transfected into HCT116 colon cancer cells, using UltraCruz Transfection Reagent (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Activation Assay, Expressing, Western Blot, Control

Journal: Biomolecules

Article Title: The Impact of NRF2 Inhibition on Drug-Induced Colon Cancer Cell Death and p53 Activity: A Pilot Study

doi: 10.3390/biom12030461

Figure Lengend Snippet: Proposed model for NRF2 role in cancer cell chemosensitivity and p53 activity. Hyperactivation of NRF2 (by SFN) counteracts the CDDP-induced DNA damage, impairing the p53 activity and reducing cell death; ZnCl 2 supplementation counteracts the effect of SFN/NRF2 rescuing the DNA damage, p53 activity, and cell death, induced by CDDP.

Article Snippet: NRF2 CRISPR/Cas9 KO and NRF2 HDR plasmids were co-transfected into HCT116 colon cancer cells, using UltraCruz Transfection Reagent (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Activity 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: STAR Protocols

Article Title: Protocol for scarless genome editing of human pluripotent stem cell based on orthogonal selective reporters

doi: 10.1016/j.xpro.2024.103084

Figure Lengend Snippet:

Article Snippet: The resulted plasmid is B2M-SDMutation-EPG (Addgene #215545). g. Digested the resulted plasmid with restriction enzyme HindIII and XbaI for changing the selection cassette from Puro-EGFP to Hygro-tdTomato. h. PCR amplify the Hygro-tdTomato sequence with primer pair Hygro-tdT-F/Hygro-tdT-R taking the P2A-mCherry-EhT backbone (Addgene #215549) as template. i. Purify the PCR products and digested plasmid with a commercial kit. j.

Techniques: Recombinant, DNA Extraction, Transformation Assay, Cloning, Software, Transferring, Spectrophotometry