mesoplasma photuris  (ATCC)

Journal: Frontiers in Genetics

Article Title: Mesoplasma florum : a near-minimal model organism for systems and synthetic biology

doi: 10.3389/fgene.2024.1346707

Figure Lengend Snippet: Forty years of research on Mesoplasma florum . (A) Important milestones in M. florum research timeline. Representative picture of an M. florum L1 colony displaying the typical “fried-egg” morphology (adapted from , Vol. 44, No. 17, pp. 8501–8511, by permission of Oxford University Press; scale bar: 100 µm) as well as M. florum L1 cells observed by scanning electron microscopy are also depicted. (B) and (C) Maximum-likelihood phylogenetic trees of the Mollicutes (B) and the Mesoplasma/Entomoplasma genera (C) inferred using concatenated alignments of 109 and 229 conserved proteins, respectively. Trees were constructed using RAxML with 150 bootstrap replicates as determined using the autoFC bootstopping criterion. Bootstrap replicate values are of 100 unless specified otherwise. Bacillus subtilis and S. citri were used as outgroups. See for additional information on strains and genomes included in the Mesoplasma/Entomoplasma phylogenetic tree.

Article Snippet: Mesoplasma photuris , - , PUPA-2 (ATCC 49581) , ; , Photuris sp , Gut of firefly larva , GCF_000702725.1 , DOE Joint Genome Institute , 11/06/2014 , Contig , 778,966.

Techniques: Electron Microscopy, Construct

Journal: Frontiers in Genetics

Article Title: Mesoplasma florum : a near-minimal model organism for systems and synthetic biology

doi: 10.3389/fgene.2024.1346707

Figure Lengend Snippet: List of Mesoplasma and Entomoplasma strains with genome assemblies deposited on the RefSeq database.

Article Snippet: Mesoplasma photuris , - , PUPA-2 (ATCC 49581) , ; , Photuris sp , Gut of firefly larva , GCF_000702725.1 , DOE Joint Genome Institute , 11/06/2014 , Contig , 778,966.

Techniques: Isolation

Journal: Frontiers in Genetics

Article Title: Mesoplasma florum : a near-minimal model organism for systems and synthetic biology

doi: 10.3389/fgene.2024.1346707

Figure Lengend Snippet: Genome engineering tools and projects to transform Mesoplasma florum into an optimized cell chassis for systems and synthetic biology. (A) Whole genome cloning and transplantation procedure. Bacterial genomes containing a yeast vector are first transformed in yeast to allow genetic modifications using the available molecular tools. Modified genomes are then carefully isolated and transplanted into a compatible recipient bacterium. Recipient cells adopt the phenotype conferred by the transplanted genome (see ; ). (B) Serine integrase mediated genome engineering methodology. DNA fragments such as genes of interest (GOI) can be inserted or exchanged with the genome by the expression of a serine integrase. Serine integrases such as Bxb1 or PhiC31 catalyze the recombination between specific DNA sequences, namely, the attP and attB sites, resulting in attL and attR sites ( attL sites not shown, see for more details). (C) Integration of multiple data types by computer-aided design (CAD) tools to guide the design and optimize the synthesis, amplification, and assembly of large DNA fragments according to user-defined constraints. In silico models such as genome-scale models (GEMs) can be used to predict the fitness of designed genomes. (D) to (G) Example of synthetic genome projects. (D) Genome reduction, in which non-essential genes are removed from the genome, resulting in a reduced cell complexity and simplified metabolism. (E) Gene recoding at the genome scale. Within a given open reading frame, codons can be exchanged for synonymous ones, modifying the DNA sequence but not the corresponding amino acid sequence. (F) Streamlined genome using a swapped amino acid genetic code. By removing non-essential genes and recoding all remaining genes, specific codons can be removed from the genome, allowing anticodon swap between given tRNAs and creating an artificial genetic code. (G) Streamlined genome with gene of similar function regrouped in modules. Regrouping genes with related functions into modules can facilitate genome engineering efforts, and can be used to test specific hypotheses about gene regulation and genome organization.

Article Snippet: Mesoplasma photuris , - , PUPA-2 (ATCC 49581) , ; , Photuris sp , Gut of firefly larva , GCF_000702725.1 , DOE Joint Genome Institute , 11/06/2014 , Contig , 778,966.

Techniques: Clone Assay, Transplantation Assay, Plasmid Preparation, Transformation Assay, Modification, Isolation, Expressing, Amplification, In Silico, Sequencing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: APE1 redox function is required for activation of Yes-associated protein 1 under reflux conditions in Barrett’s-associated esophageal adenocarcinomas

doi: 10.1186/s13046-022-02472-5

Figure Lengend Snippet: Primary and secondary antibodies used in this study

Article Snippet: The APE1 redox-specific inhibitor E3330 was purchased from Novus Biologicals (NBP1-49,581, Centennial, CO, USA).

Techniques:

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: APE1 redox function is required for activation of Yes-associated protein 1 under reflux conditions in Barrett’s-associated esophageal adenocarcinomas

doi: 10.1186/s13046-022-02472-5

Figure Lengend Snippet: APE1 regulates YAP1 expression and transcriptional activity in BE and EAC cell lines. A FLO-1 (left) and OE33 (right) cells were transfected with APE1-wild-type overexpression plasmid and analyzed for APE1 and YAP1 expression by Western blot. β-actin was used as a loading control. B Western blot was used to analyze APE1 and YAP1 expression following transient APE1 knockdown (si-APE1) in FLO-1 (left), OE33 (middle) and CPB (right) cells. β-actin was used as a loading control. C Representative immunofluorescent staining images of APE1 (red) and YAP1 (green) in FLO-1 cells showing downregulation of nuclear YAP1 expression with or without APE1 knockdown. Quantification of stain intensity by ImageJ is also reported. DAPI (blue) was used for nuclear staining. D 8xCTIIC luciferase reporter assay was used to measure YAP1 transcriptional activity in FLO-1, OE33 and CPB cells with or without APE1 knockdown. The luciferase reporter activity values were normalized to Renilla expression levels. E qRT-PCR analyses of YAP1 downstream target genes, CTGF, CYR61 and ANKRD1 in FLO-1, OE33 and CPB cells with or without APE1 knockdown. Values are represented as mean ± SEM of three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001, ns: no significance

Article Snippet: The APE1 redox-specific inhibitor E3330 was purchased from Novus Biologicals (NBP1-49,581, Centennial, CO, USA).

Techniques: Expressing, Activity Assay, Transfection, Over Expression, Plasmid Preparation, Western Blot, Control, Knockdown, Staining, Luciferase, Reporter Assay, Quantitative RT-PCR

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: APE1 redox function is required for activation of Yes-associated protein 1 under reflux conditions in Barrett’s-associated esophageal adenocarcinomas

doi: 10.1186/s13046-022-02472-5

Figure Lengend Snippet: ABS induces YAP1 expression, promotes its nuclear accumulation and facilitates its transcriptional activity. A Western blot analysis of APE1 and YAP1 levels in FLO-1 and OE33 cells. Cells were exposed to 200 µM ABS for 20 min followed by recovery in complete media for the indicated time points. β-actin was used as a loading control. B Representative immunofluorescent staining images of APE1 (red) and YAP1 (green) in FLO-1 cells exposed to ABS (200 µM, pH 4, 20 min) followed by 3 h recovery versus control untreated cells. DAPI (blue) was used for nuclear staining. Quantification of stain intensity by ImageJ is also reported. C Representative immunofluorescent staining images of APE1 (red) and YAP1 (green) in FLO-1 cells repeatedly exposed to ABS (200 µM, pH 5.5, 20 min per day for 14 days) versus control untreated cells. DAPI (blue) was used for nuclear staining. Quantification of stain intensity by ImageJ is also reported. D-E YAP1 transcriptional activity was measured using 8xCTIIC luciferase reporter assay in FLO-1 cells exposed or not to ABS treatment (200 µM, pH 4, 20 min) followed by 3 h recovery ( D ) or FLO-1 cells repeatedly exposed to ABS (200 µM, pH 5.5, 20 min per day for 14 days) versus control untreated cells ( E ). The luciferase reporter activity values were normalized to Renilla expression levels. F qRT-PCR analyses of YAP1 downstream target genes, CTGF, CYR61 and ANKRD1 in FLO-1 cells exposed to ABS (200 µM, pH 4, 20 min) followed by 3 h recovery versus control untreated cells. Values are represented as mean ± SEM of three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001. G Representative immunohistochemistry staining images of APE1 and YAP1 in L2-IL-1β transgenic mouse tissues with high-grade dysplasia (HGD) and esophageal adenocarcinoma (EAC) lesions (Untreated: 8mice; HGD and EAC: 8mice each). Quantification of stain intensity by ImageJ is shown

Article Snippet: The APE1 redox-specific inhibitor E3330 was purchased from Novus Biologicals (NBP1-49,581, Centennial, CO, USA).

Techniques: Expressing, Activity Assay, Western Blot, Control, Staining, Luciferase, Reporter Assay, Quantitative RT-PCR, Immunohistochemistry, Transgenic Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: APE1 redox function is required for activation of Yes-associated protein 1 under reflux conditions in Barrett’s-associated esophageal adenocarcinomas

doi: 10.1186/s13046-022-02472-5

Figure Lengend Snippet: APE1 mediates ABS-induced YAP1 activation. A Representative immunofluorescence images of APE1 (red) and YAP1 (green) in FLO-1 cells exposed to ABS (200 µM, pH 4, 20 min) followed by 3 h recovery with or without APE1 knockdown. DAPI (blue) was used for nuclear staining. B Quantification of immunofluorescence stain intensity using ImageJ. C Cytosolic and nuclear fractions from FLO-1 (left) and OE33 (right) cells transfected with si-Ctrl or si-APE1 and treated with ABS (200 µM, pH 4, 20 min) followed by recovery in complete media for indicated timepoints were evaluated by western blot for the levels of APE1 and YAP1. β-tubulin and p84 were used as a loading control for cytosolic and nuclear fractions, respectively. D 8xCTIIC luciferase reporter assay was used to measure YAP1 transcriptional activity in FLO-1 and OE33 cells transfected with si-Ctrl or si-APE1 and exposed or not to ABS treatment (200 µM, pH 4, 20 min) followed by 3 h recovery. The luciferase reporter activity values were normalized to Renilla expression levels. Values are represented as mean ± SEM of three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: The APE1 redox-specific inhibitor E3330 was purchased from Novus Biologicals (NBP1-49,581, Centennial, CO, USA).

Techniques: Activation Assay, Immunofluorescence, Knockdown, Staining, Transfection, Western Blot, Control, Luciferase, Reporter Assay, Activity Assay, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: APE1 redox function is required for activation of Yes-associated protein 1 under reflux conditions in Barrett’s-associated esophageal adenocarcinomas

doi: 10.1186/s13046-022-02472-5

Figure Lengend Snippet: ABS-induced YAP1 activation depends on APE1 redox activity. A FLO-1 and OE33 cells were pre-treated or not with NAC (200 µM, 2 h) then exposed to ABS (200 µM, pH 4, 20 min) and allowed to recover for 3 h in complete media. Western blot analysis was used to evaluate APE1 and YAP1 protein levels. β-actin was used as a loading control. B FLO-1 and OE33 cells with transient knockdown of APE1 using si-APE1 were reconstituted with wild type APE1 (flag APE1) or APE1 redox mutant (C65A). Western blot was used to analyze the protein levels of APE1 and YAP1. β-actin was used as a loading control. C FLO-1 and OE33 cells were pre-treated or not with APE1 redox inhibitor E3330 (100 µM, overnight) then exposed to ABS (200 µM, pH 4, 20 min) and allowed to recover for 3 h in complete media with or without E3330. Western blot analysis was used to evaluate APE1 and YAP1 protein levels. β-actin was used as a loading control. D FLO-1 and OE33 cells were transfected with 8xCTIIC luciferase reporter and Renilla. Cells were then pre-treated or not with APE1 redox inhibitor E3330 (100 µM, overnight) then exposed to ABS (200 µM, pH 4, 20 min) and allowed to recover for 3 h in complete media with or without E3330. The relative 8xCTIIC luciferase reporter activity was measured and reported as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001. E–F 8xCTIIC luciferase reporter assay was used to measure YAP1 transcriptional activity in FLO-1 ( E ) and OE33 ( F ) cells transfected or not with wild type APE1 (flag APE1) or APE1 redox mutant (C65A) with or without exposure to ABS (200 µM, pH 4, 20 min followed by 3 h recovery). The luciferase reporter activity values were normalized to Renilla expression levels. Values are represented as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: The APE1 redox-specific inhibitor E3330 was purchased from Novus Biologicals (NBP1-49,581, Centennial, CO, USA).

Techniques: Activation Assay, Activity Assay, Western Blot, Control, Knockdown, Mutagenesis, Transfection, Luciferase, Reporter Assay, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: APE1 redox function is required for activation of Yes-associated protein 1 under reflux conditions in Barrett’s-associated esophageal adenocarcinomas

doi: 10.1186/s13046-022-02472-5

Figure Lengend Snippet: E3330 reduces PDX tumor growth and YAP1 activation. A Average tumor volume of PDX with or without 20 mg/kg E3330 treatment for 28 days (Control: 5mice; E3330: 5mice). B Kaplan–Meier survival curve for PDX following treatment endpoint. C Average weight of control and E3330 treated PDXs over 28 days of treatment. D Representative immunohistochemistry staining images of APE1 and YAP1 in PDX mouse tissues with or without E3330 treatment. Quantification of immunofluorescence intensity by ImageJ is also shown. E Representative immunofluorescence images of CTGF (green) in PDX mouse tissues with or without E3330 treatment. DAPI (blue) was used for nuclear staining

Article Snippet: The APE1 redox-specific inhibitor E3330 was purchased from Novus Biologicals (NBP1-49,581, Centennial, CO, USA).

Techniques: Activation Assay, Control, Immunohistochemistry, Staining, Immunofluorescence

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: APE1 redox function is required for activation of Yes-associated protein 1 under reflux conditions in Barrett’s-associated esophageal adenocarcinomas

doi: 10.1186/s13046-022-02472-5

Figure Lengend Snippet: APE1 regulates YAP1 stability through β-TrCP ubiquitinase. A-B FLO-1 ( A ) and OE33 ( B ) cells with or without APE1 transient knockdown were treated with cycloheximide (CHX, 100 µg/mL) for the indicated timepoints. The levels of APE1 and YAP1 were determined using western blot. β-actin was used as a loading control. C FLO-1 and OE33 cells with or without APE1 transient knockdown were treated or not with MG132 (10 µM) for 16 h. The levels of APE1 and YAP1 were evaluated using western blot. β-actin was used as a loading control. D Western blot was used to analyze APE1 and β-TrCP expression following transient APE1 knockdown (si-APE1) in FLO-1 and OE33 cells. β-actin was used as a loading control. E FLO-1 and OE33 cells were transfected with wild type APE1 (flag APE1), immunoprecipitated with flag antibody and immunoblotted with β-TrCP and APE1 antibodies. F-G Representative images of proximity ligation assay for APE1 and β-TrCP in FLO-1 and OE33 cells ( F ) and YAP1 and β-TrCP in OE33 cells with and without APE1 knockdown ( G ). DAPI (blue) was used for nuclear staining. H In vitro ubiquitination assay in FLO-1 and OE33 cells transfected with HA-ubiquitin plasmid then treated or not with APE1 redox inhibitor E3330 (100 µM, overnight) followed by MG132 treatment (10 µM for 6 h). YAP1 antibody was used for immunoprecipitation and samples were then blotted with anti-ubiquitin and anti-YAP1

Article Snippet: The APE1 redox-specific inhibitor E3330 was purchased from Novus Biologicals (NBP1-49,581, Centennial, CO, USA).

Techniques: Knockdown, Western Blot, Control, Expressing, Transfection, Immunoprecipitation, Proximity Ligation Assay, Staining, In Vitro, Ubiquitin Assay, Plasmid Preparation

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: APE1 redox function is required for activation of Yes-associated protein 1 under reflux conditions in Barrett’s-associated esophageal adenocarcinomas

doi: 10.1186/s13046-022-02472-5

Figure Lengend Snippet: ABS enhances sphere formation and induces YAP1 through APE1. A Representative bright field images of OE33 spheres derived from control cells (UT) or cells repeatedly exposed to ABS (200uM, pH5.5) for 20 min per day for 14 days (rABS). Quantification of the number and diameter of spheres is also presented. B Western blot analysis of APE1 and YAP1 in OE33-derived spheres with or without repeated ABS. C Representative bright field images of spheres derived from repeated-ABS-treated OE33 cells with or without E3330 (50 µM) treatment. Quantification of the number and diameter of spheres is also presented. D Western blot analysis of APE1 and YAP1 in spheres derived from repeated-ABS-treated OE33 cells with or without E3330 (50 µM) treatment. E Representative bright field images of spheres derived from repeated-ABS-treated OE33 cells with or without transient APE1 knockdown. Quantification of the number and diameter of spheres is also presented. F–H Representative immunofluorescence images of spheres derived from repeated-ABS-treated OE33 cells with or without E3330 (50 µM) treatment showing APE1 (red) and YAP1 (green) staining in ( F ), CTGF (green) in ( G ) and CD44 (green) in ( H ). DAPI (blue) was used for nuclear staining. Quantification of immunofluorescence intensity by ImageJ is also shown. Values are represented as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: The APE1 redox-specific inhibitor E3330 was purchased from Novus Biologicals (NBP1-49,581, Centennial, CO, USA).

Techniques: Derivative Assay, Control, Western Blot, Knockdown, Immunofluorescence, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: APE1 redox function is required for activation of Yes-associated protein 1 under reflux conditions in Barrett’s-associated esophageal adenocarcinomas

doi: 10.1186/s13046-022-02472-5

Figure Lengend Snippet: A schematic diagram illustrating the role of APE1 in activating and stabilizing YAP1. Exposure of cells to acidic bile salts (ABS) induce APE1-dependent increase in YAP1, leading to its activation and translocation into the nucleus. Mechanistically, APE1 stabilizes YAP1 by binding to β-TrCP thereby disrupting YAP1-β-TrCP interaction and preventing YAP1 polyubiquitination and degradation

Article Snippet: The APE1 redox-specific inhibitor E3330 was purchased from Novus Biologicals (NBP1-49,581, Centennial, CO, USA).

Techniques: Activation Assay, Translocation Assay, Binding Assay

Journal: Redox Biology

Article Title: Activation of NRF2 by APE1/REF1 is redox-dependent in Barrett's related esophageal adenocarcinoma cells

doi: 10.1016/j.redox.2021.101970

Figure Lengend Snippet: Loss of APE1 resulted in increased oxidative stress, DNA damage, and promoted cell death in response to reflux conditions. (A–B) Flow cytometry analyses of OE33 cells to detect the intracellular ROS levels using CM-H2DCFDA dye in APE1 knockdown (sh-APE1) and control (sh-Ctrl) cells treated with or without ABS (pH 4.0). Representative flow cytometry profiles are shown in (A). The quantitative results from 3 independent experiments are shown in (B). (C–F) OE33 cells were transfected with si-Ctrl and si-APE1 for 48 h, followed by acidic bile salts (ABS) treatment for 20 min and recovery in full medium for 3 h. Cells treated with 100 μM H 2 O 2 were used as a positive control. 8-Oxoguanine (8-OxoG), an oxidative DNA damage marker, was used for immunofluorescence. Representative immunofluorescence images are shown in (C) and the quantitative data, using ImageJ, is shown in panel (D). p-H 2 AX (S139, γH2AX, green), a DNA double-strand breaks marker, and APE1 (red) were used for immunofluorescence. The representative images are shown in panels, (E) and the quantitative data is shown in panel (F). DAPI (blue) was used as a nuclear counterstain. (G) Flow cytometry analysis of annexin V staining in si-Ctrl and si-APE1 cells with or without ABS treatment. The quantitative analysis data from three independent experiments are shown. (H) Western blotting analysis of cleaved PARP in si-Ctrl and si-APE1 cells with ABS or control PBS treatment. *P < 0.05; **P < 0.01; ***P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The E3330, an APE1 redox inhibitor, was obtained from Novus Biologicals (NBP1-49581, Centennial, CO, USA) and GSK-3β inhibitors; lithium chloride (Calbiochem) and CHIR-98014 (Selleck chemicals).

Techniques: Reflux, Flow Cytometry, Transfection, Positive Control, Marker, Immunofluorescence, Staining, Western Blot

Journal: Redox Biology

Article Title: Activation of NRF2 by APE1/REF1 is redox-dependent in Barrett's related esophageal adenocarcinoma cells

doi: 10.1016/j.redox.2021.101970

Figure Lengend Snippet: APE1 and NRF2 were induced by acidic bile salts. (A) Western blot analysis of APE1, NRF2 and KEAP1 protein levels in esophageal cell lines from Barrett's (BAR10-T, CPA), dysplastic Barrett's (CPB) and EAC (FLO1, OE33, OE19 and SKGT4). (B) Immunohistochemistry staining of APE1 and NRF2 in a representative normal esophagus and an esophageal adenocarcinoma tissue samples (x10) with insets of higher magnification (×40). (C–D) Western blot analysis of APE1, NRF2 and KEAP-1 levels in OE33, and FLO1 cells. Cells were exposed to 100 μM or 200 μM (FLO1) ABS for 20 min with recovery in the complete media for the indicated time points. Whole-cell lysates were used for western blotting. (E, F) The ARE luciferase reporter assay for the NRF2 transcriptional activity in OE33 and FLO1 cells, following treatment with a mixture of bile salts (200 μM) or PBS (Ctrl). The luciferase reporter activity values were normalized to β-gal expression levels and are represented as percentage luciferase activity relative to control (set as 100%). (G, H) qRT-PCR analyses of NRF2 downstream target genes, HO-1 and TRXND1 in FLO1 and OE33 cells treated with ABS. Values are mean ± SD of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: The E3330, an APE1 redox inhibitor, was obtained from Novus Biologicals (NBP1-49581, Centennial, CO, USA) and GSK-3β inhibitors; lithium chloride (Calbiochem) and CHIR-98014 (Selleck chemicals).

Techniques: Western Blot, Immunohistochemistry, Staining, Luciferase, Reporter Assay, Activity Assay, Expressing, Quantitative RT-PCR

Journal: Redox Biology

Article Title: Activation of NRF2 by APE1/REF1 is redox-dependent in Barrett's related esophageal adenocarcinoma cells

doi: 10.1016/j.redox.2021.101970

Figure Lengend Snippet: Induction and regulation of NRF2 are APE1-dependent. (A–C) Knockdown of APE1 downregulated NRF2 protein levels in CPB, OE33, and FLO1 cells. Cells were transfected with si-APE1 or sh-APE1 and controls (si-Ctrl or sh-Ctrl). Whole-cell lysates were collected for western blotting analysis of APE1, NRF2, and KEAP-1. (D–F) Relative ARE luciferase activity was determined in CPB, OE33, and FLO1 cells with or without APE1 knockdown followed by transfection with PGL3- NRF2-ARE-luc and renilla for 48 h. (G–H) FLO1 and OE33 cells were knockdown of APE1 followed by transfection with PGL3- NRF2-ARE-luc and renilla for 24 h and treatment with 200 μM bile salts cocktails for another 24 h, ARE luciferase activity was determined as previously described. (I–J) mRNA expression of NRF2 downstream genes HO-1 and TRXND1 in OE33 cells treated with and without ABS. Values are mean ± SD of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: The E3330, an APE1 redox inhibitor, was obtained from Novus Biologicals (NBP1-49581, Centennial, CO, USA) and GSK-3β inhibitors; lithium chloride (Calbiochem) and CHIR-98014 (Selleck chemicals).

Techniques: Transfection, Western Blot, Luciferase, Activity Assay, Expressing

Journal: Redox Biology

Article Title: Activation of NRF2 by APE1/REF1 is redox-dependent in Barrett's related esophageal adenocarcinoma cells

doi: 10.1016/j.redox.2021.101970

Figure Lengend Snippet: Acidic bile salts induced NRF2 nuclear accumulation where APE1 is required for NRF2 nuclear retention. CPB (A), OE33 (C) and FLO1 (E) cells were treated with ABS for 20 min, followed by recovery in complete media. The samples were collected at 0, 1, 3, 6, and 24 h time points post treatment. Cytosolic and nuclear fractions were isolated and evaluated by western blotting for the levels of NRF2, APE1 and KEAP1. CPB (B), OE33 (D) and FLO1 (F) cells were transfected with si-Ctrl and si-APE1 for 48 h. Cytosolic and nuclear fractions were isolated and evaluated by western blotting for the levels of NRF2, APE1 and KEAP1. β-tubulin and p84 were used as a loading control for cytosolic and nuclear fractions, respectively.

Article Snippet: The E3330, an APE1 redox inhibitor, was obtained from Novus Biologicals (NBP1-49581, Centennial, CO, USA) and GSK-3β inhibitors; lithium chloride (Calbiochem) and CHIR-98014 (Selleck chemicals).

Techniques: Isolation, Western Blot, Transfection

Journal: Redox Biology

Article Title: Activation of NRF2 by APE1/REF1 is redox-dependent in Barrett's related esophageal adenocarcinoma cells

doi: 10.1016/j.redox.2021.101970

Figure Lengend Snippet: APE1 was required for NRF2 stability. (A, C) OE33 and FLO1 cells were treated with or without ABS (100 μM) for 20 min, followed by recovery in complete media with cycloheximide (CHX, 100 μg/mL) at the indicated time points. (E, G) OE33 (E) and FLO1(G) cells with stable knockdown of APE1 using sh-APE1 or sh-Ctrl were treated with cycloheximide (CHX, 100 μg/mL) at the indicated time points. The levels of APE1 and NRF2 were determined by western blotting. The band intensity of NRF2 was measured and normalized with the actin using the Quantity One software (BioRad Laboratories, USA) and normalized to β-actin of the same samples. (B, D, F and H) Half-life time (t1/2) of NRF2 was calculated and plotted using GraphPad Prism software, corresponding to A, C, E and G, respectively.

Article Snippet: The E3330, an APE1 redox inhibitor, was obtained from Novus Biologicals (NBP1-49581, Centennial, CO, USA) and GSK-3β inhibitors; lithium chloride (Calbiochem) and CHIR-98014 (Selleck chemicals).

Techniques: Western Blot, Software

Journal: Redox Biology

Article Title: Activation of NRF2 by APE1/REF1 is redox-dependent in Barrett's related esophageal adenocarcinoma cells

doi: 10.1016/j.redox.2021.101970

Figure Lengend Snippet: APE1-dependent inhibition of GSK-3β promoted the increase in NRF2. (A and B) Knockdown of APE1 blocked ABS-induced NRF2 upregulation. CPB (A) and OE33 (B) cells were transfected with si-Ctrl and si-APE1 followed by exposure to 100 μM ABS for 20 min, then recovery for 1, 3, 6 h in complete media. Immunoblot results of APE1, NRF2, p -GSK-3β (S9), total GSK-3β and β-actin levels were shown. (C and D) CPB and OE33 cells were pretreated with or without 10 mM LiCl (GSK-3β inhibitor) overnight and on the next day cells were treated with ABS (100μM/20min) followed by recovery time points. Immunoblots of APE1, NRF2, and β-actin levels were shown. (E) OE33 cells were transfected with 1 μg of pcDNA flag-APE1, whereas control cells received an empty vector. After 24 h transfection, the cells were split and pretreated with 10 mM LiCl overnight. On the next day, the cells were treated with ABS, followed by different recovery time points in the presence and absence of LiCl. Immunoblots of APE1, NRF2 and β-actin levels were shown.

Article Snippet: The E3330, an APE1 redox inhibitor, was obtained from Novus Biologicals (NBP1-49581, Centennial, CO, USA) and GSK-3β inhibitors; lithium chloride (Calbiochem) and CHIR-98014 (Selleck chemicals).

Techniques: Inhibition, Transfection, Western Blot, Plasmid Preparation

Journal: Redox Biology

Article Title: Activation of NRF2 by APE1/REF1 is redox-dependent in Barrett's related esophageal adenocarcinoma cells

doi: 10.1016/j.redox.2021.101970

Figure Lengend Snippet: APE1 redox function was required for GSK-3β-mediated APE1 regulation of NRF2. (A–C) CPB, OE33 and FLO1 cells were pre-treated with or without E3330, an APE1 redox inhibitor for 24 h. Cells were then transfected with PGL3-NRF2-ARE-Luc and β-gal with or without E3330 for another 24 h. The relative ARE luciferase reporter activity was measured and is shown as a percentage relative to controls (set as 100%). Values are mean ± SD of three independent experiments. *P < 0.05; ***P < 0.001. (D and E) CPB and OE33 cells were pre-treated with or without E3330 (100 μM) for overnight followed by treatment with ABS (100 μM) for 20 min and recovery in full media for 1, 3 and 6 h post-treatment with or without E3330. Western blot analyses were used to determine the levels of APE1, NRF2, GSK-3β (Total) and p -GSK-3β (S9). β-actin was used as a loading control. (F) OE33 cells were transfected with wild type APE1 with the flag (flag-APE1) or mutant APE1 C65A (APE1-C65A, an APE1 redox mutant). Western blot was applied for the levels of NRF2, APE1, GSK-3β (Total) and pGSK-3β (S9). (G) OE33 cells with stable knockdown of APE1 using sh-APE1 were reconstituted with wild type APE1 (APE1) or mutant APE1 C65A (C65A). Western blot was used to analyze the protein levels of NRF2, APE1, GSK-3β (Total) and pGSK-3β (S9).

Article Snippet: The E3330, an APE1 redox inhibitor, was obtained from Novus Biologicals (NBP1-49581, Centennial, CO, USA) and GSK-3β inhibitors; lithium chloride (Calbiochem) and CHIR-98014 (Selleck chemicals).

Techniques: Transfection, Luciferase, Activity Assay, Western Blot, Mutagenesis

Journal: Redox Biology

Article Title: Activation of NRF2 by APE1/REF1 is redox-dependent in Barrett's related esophageal adenocarcinoma cells

doi: 10.1016/j.redox.2021.101970

Figure Lengend Snippet: A schematic diagram illustrating the role of APE1 in activating NRF2 under reflux conditions. Exposure of cells to reflux conditions (acidic bile salts, ABS) generates high levels of ROS. High ROS and oxidative stress levels induce APE1-dependent increase in NRF2, leading to its release and translocation into the nucleus. In the nucleus, NRF2 is stabilized and regulated by APE1 redox function that inactivates GSK-3β dependent degradation of NRF2. Loss of redox function of APE1 mediates inactivation of GSK-3-β to promote accumulation and activation of NRF2.

Article Snippet: The E3330, an APE1 redox inhibitor, was obtained from Novus Biologicals (NBP1-49581, Centennial, CO, USA) and GSK-3β inhibitors; lithium chloride (Calbiochem) and CHIR-98014 (Selleck chemicals).

Techniques: Reflux, Translocation Assay, Activation Assay