Journal: Journal of Clinical and Translational Hepatology

Article Title: ATOX1 Promotes Hepatocellular Carcinoma Carcinogenesis via Activation of the c-Myb/PI3K/AKT Signaling Pathway

doi: 10.14218/JCTH.2024.00422

Figure Lengend Snippet: (A) Analysis of ATOX1 transcripts in HCC tissues and adjacent normal tissues was conducted using the TCGA-LIHC database. (B) The mRNA expression of ATOX1 was analyzed in six pairs of HCC and adjacent normal tissues. (C) The protein expression levels of ATOX1 were assessed in three pairs of HCC and adjacent normal tissues (‘N’ represents adjacent normal tissue and ‘C’ represents HCC tissue). (D) ATOX1 expression in tumor tissues from 50 HCC patients was analyzed by IHC staining, with images from four representative patients presented (‘N’ represents adjacent normal tissue and ‘T’ represents tumor tissue); scale bar, 100 µm; magnification, ×20. ** p < 0.01, *** p < 0.001, and **** p < 0.0001. HCC, hepatocellular carcinoma; LIHC, liver hepatocellular carcinoma; TCGA, The Cancer Genome Atlas; IHC, immunohistochemistry; ATOX1, antioxidant-1.

Article Snippet: Sections were then blocked with goat serum for 30 m and incubated overnight with a rabbit anti-ATOX1 antibody (dilution 1:200, NOVUS).

Techniques: Expressing, Immunohistochemistry

Journal: Journal of Clinical and Translational Hepatology

Article Title: ATOX1 Promotes Hepatocellular Carcinoma Carcinogenesis via Activation of the c-Myb/PI3K/AKT Signaling Pathway

doi: 10.14218/JCTH.2024.00422

Figure Lengend Snippet: (A) ATOX1 overexpression was confirmed by Western blot analysis in Huh7 and HepG2 cells. (B) The viability of Huh7 and HepG2 cells overexpressing ATOX1 was evaluated using the CCK-8 assay. (C) The number of colonies in cells overexpressing ATOX1 was assessed by the colony formation assay. (D) Cell migration in Huh7 and HepG2 cells post-ATOX1 overexpression was examined using the Transwell assay. Bar graphs depict the number of cells that migrated to the lower chambers (scale bar, 100 µm; magnification, ×10). (E) The protein expression levels of E-cadherin and N-cadherin were analyzed in Huh7 and HepG2 cells transfected with control or OE-ATOX1 plasmids. The histogram shows a relative quantitative analysis. * p < 0.05, ** p < 0.01, *** p < 0.001. HCC, hepatocellular carcinoma; CCK-8, Cell Counting Kit-8; OE-ATOX1, ATOX1 overexpression; ATOX1, antioxidant-1.

Article Snippet: Sections were then blocked with goat serum for 30 m and incubated overnight with a rabbit anti-ATOX1 antibody (dilution 1:200, NOVUS).

Techniques: Over Expression, Western Blot, CCK-8 Assay, Colony Assay, Migration, Transwell Assay, Expressing, Transfection, Control, Cell Counting

Journal: Journal of Clinical and Translational Hepatology

Article Title: ATOX1 Promotes Hepatocellular Carcinoma Carcinogenesis via Activation of the c-Myb/PI3K/AKT Signaling Pathway

doi: 10.14218/JCTH.2024.00422

Figure Lengend Snippet: (A) ATOX1 knockdown was confirmed by Western blot analysis in Huh7 and HepG2 cells. (B and C) The proliferative capacity of Huh7 and HepG2 cells post-ATOX1 knockdown was evaluated by the CCK-8 (B) and colony formation (C) assays. (D) The Transwell assay was performed to assess the migratory ability of Huh7 and HepG2 cells with ATOX1 knockdown (scale bar, 100 µm; magnification, ×10). (E) The protein expression of E-cadherin and N-cadherin was analyzed in Huh7 and HepG2 cells transfected with siRNA-NC or siRNA-ATOX1. (F) After subcutaneous injection of Hep3B sh-NC or Hep3B sh-ATOX1 cells in BALB/c nude mice for four weeks, tumor volume and weight were measured. * p < 0.05, ** p < 0.01, *** p < 0.001. HCC, hepatocellular carcinoma; CCK-8, Cell Counting Kit-8; si-NC, small interfering RNA targeting negative control; si-ATOX1, small interfering RNA targeting ATOX1; ATOX1, antioxidant-1.

Article Snippet: Sections were then blocked with goat serum for 30 m and incubated overnight with a rabbit anti-ATOX1 antibody (dilution 1:200, NOVUS).

Techniques: Knockdown, Western Blot, CCK-8 Assay, Transwell Assay, Expressing, Transfection, Injection, Cell Counting, Small Interfering RNA, Negative Control

Journal: Journal of Clinical and Translational Hepatology

Article Title: ATOX1 Promotes Hepatocellular Carcinoma Carcinogenesis via Activation of the c-Myb/PI3K/AKT Signaling Pathway

doi: 10.14218/JCTH.2024.00422

Figure Lengend Snippet: (A) Enriched pathways through KEGG analysis in the ATOX1 knockdown group. (B) Western blot analysis of phosphorylation levels of AKT and mTOR in HCC cells with ATOX1 knockdown or overexpression. (C and D) Phosphorylation levels of AKT and mTOR in Huh7 and HepG2 cells transfected with the OE-ATOX1 plasmid for 24 h before treatment with LY294002 (20 µM) for 24 h were determined by Western blot (C). The histogram shows a relative quantitative analysis (D). (E) The effect of LY294002 on ATOX1 overexpression-induced cell proliferation was assessed using the CCK-8 assay. (F and G) The effect of LY294002 on ATOX1 overexpression-induced cell migration was measured using the Transwell assay (scale bar, 100 µm; original magnification, ×10). Bar graphs depict the number of cells that migrated to the lower chambers. * p < 0.05, ** p < 0.01, *** p < 0.001. KEGG, Kyoto Encyclopedia of Genes and Genomes; HCC, hepatocellular carcinoma; CCK-8, Cell Counting Kit-8; OE-ATOX1, ATOX1 overexpression; si-NC, small interfering RNA targeting negative control; si-ATOX1, small interfering RNA targeting ATOX1; ATOX1, antioxidant-1.

Article Snippet: Sections were then blocked with goat serum for 30 m and incubated overnight with a rabbit anti-ATOX1 antibody (dilution 1:200, NOVUS).

Techniques: Knockdown, Western Blot, Phospho-proteomics, Over Expression, Transfection, Plasmid Preparation, CCK-8 Assay, Migration, Transwell Assay, Cell Counting, Small Interfering RNA, Negative Control

Journal: Journal of Clinical and Translational Hepatology

Article Title: ATOX1 Promotes Hepatocellular Carcinoma Carcinogenesis via Activation of the c-Myb/PI3K/AKT Signaling Pathway

doi: 10.14218/JCTH.2024.00422

Figure Lengend Snippet: (A and B) Differential expression of hub genes associated with the PI3K/AKT signaling pathway in groups with ATOX1 knockdown (A) and overexpression (B) revealed by heatmap analysis. (C) PPI network of ATOX1, c-Myb, and the PI3K/AKT pathway was constructed. (D and E) The mRNA (D) and protein (E) expression levels of c-Myb were analyzed in Huh7 and HepG2 cells following ATOX1 knockdown. (F) The mRNA expression of c-Myb was analyzed in HCC cells transfected with the OE-ATOX1 plasmid for 24 h before treatment with siRNA targeting c-Myb for 24 h. (G) The PI3K/AKT pathway was analyzed by Western blot in Huh7 and HepG2 cells transfected with the OE-ATOX1 plasmid for 24 h before treatment with siRNA targeting c-Myb for 24 h. ** p < 0.01, *** p < 0.001. PPI, Protein-Protein Interaction Network; HCC, hepatocellular carcinoma; OE-ATOX1, ATOX1 overexpression; si-NC, targeting negative control; si-ATOX1, small interfering RNA targeting ATOX1; si-c-Myb, small interfering RNA targeting c-Myb; ATOX1, antioxidant-1.

Article Snippet: Sections were then blocked with goat serum for 30 m and incubated overnight with a rabbit anti-ATOX1 antibody (dilution 1:200, NOVUS).

Techniques: Quantitative Proteomics, Knockdown, Over Expression, Construct, Expressing, Transfection, Plasmid Preparation, Western Blot, Negative Control, Small Interfering RNA

Journal: Journal of Clinical and Translational Hepatology

Article Title: ATOX1 Promotes Hepatocellular Carcinoma Carcinogenesis via Activation of the c-Myb/PI3K/AKT Signaling Pathway

doi: 10.14218/JCTH.2024.00422

Figure Lengend Snippet: (A) Huh7 cells overexpressing ATOX1 were treated with 100 µM CuSO 4 for 24 h and then cultured in a medium without CuSO 4 for 24 h. The total cellular copper content was quantified using ICP-MS analysis. (B and C) ROS levels in Huh7 and HepG2 cells with ATOX1 overexpression (B) or knockdown (C) were assessed. (D and E) The expression levels of apoptosis-related proteins, including cleaved Caspase-3, cleaved Caspase-9, BAX, and Bcl-2, were analyzed via Western blot analysis in HCC cells with ATOX1 overexpression or knockdown (D). The histogram shows a relative quantitative analysis (E). (F and G) Annexin V/PI double staining was performed to assess apoptosis in HCC cells. Bar graphs illustrate the quantitative analysis. (H) The protein expression of c-Myb in Huh7 cells transfected with siRNA targeting ATOX1 before treatment with acetylcysteine for 24 h. The histogram shows a relative quantitative analysis. * p < 0.05, ** p < 0.01, *** p < 0.001. HCC, hepatocellular carcinoma; ICP-MS, inductively coupled plasma mass spectrometry; ROS, reactive oxygen species; PI, propidium iodide; OE-ATOX1, ATOX1 overexpression; si-NC, small interfering RNA targeting negative control; si-ATOX1, small interfering RNA targeting ATOX1; ATOX1, antioxidant-1.

Article Snippet: Sections were then blocked with goat serum for 30 m and incubated overnight with a rabbit anti-ATOX1 antibody (dilution 1:200, NOVUS).

Techniques: Cell Culture, Over Expression, Knockdown, Expressing, Western Blot, Double Staining, Transfection, Clinical Proteomics, Mass Spectrometry, Small Interfering RNA, Negative Control

Journal: Journal of Clinical and Translational Hepatology

Article Title: ATOX1 Promotes Hepatocellular Carcinoma Carcinogenesis via Activation of the c-Myb/PI3K/AKT Signaling Pathway

doi: 10.14218/JCTH.2024.00422

Figure Lengend Snippet: (A) The viability of HCC cells treated with DCAC50 was analyzed by the CCK-8 assay. (B) The colony-forming ability of HCC cells treated with DCAC50 was examined via the colony formation assay. (C) ROS accumulation in HCC cells treated with DCAC50 was quantified. (D) The effect of DCAC50 on cell apoptosis was analyzed by flow cytometry. Bar graphs present quantitative analysis. * p < 0.05, ** p < 0.01, *** p < 0.001. HCC, hepatocellular carcinoma; CCK-8, Cell Counting Kit-8; ROS, reactive oxygen species; PI, propidium iodide; ATOX1, antioxidant-1.

Article Snippet: Sections were then blocked with goat serum for 30 m and incubated overnight with a rabbit anti-ATOX1 antibody (dilution 1:200, NOVUS).

Techniques: CCK-8 Assay, Colony Assay, Flow Cytometry, Cell Counting

Journal: Biomedicines

Article Title: Evaluation of ATOX1 as a Potential Predictive Biomarker for Tetrathiomolybdate Treatment of Breast Cancer Patients with High Risk of Recurrence

doi: 10.3390/biomedicines9121887

Figure Lengend Snippet: ATOX1 immunostaining of breast cancer tissue sections from patients in the phase II clinical trial for tetrathiomolybdate (TM) treatment. ( A ) ATOX1 immunohistochemical staining of a whole tumor tissue section (scale bar indicates 5 mm). ( B ) Examples of negative (score 0), weak (score 1), intermediate (score 2) and strong (score 3) ATOX1 staining intensities (scale bars indicate 100 µm).

Article Snippet: After blocking endogenous peroxidase, the slides were incubated with the mouse monoclonal anti-ATOX1 primary antibody (H00000475-M01, Novus Biologicals, Littleton, CO, USA) followed by incubation with secondary antibody horseradish peroxidase (HRP)-Probe and tertiary antibody HRP-Polymer.

Techniques: Immunostaining, Immunohistochemical staining, Staining

Journal: Biomedicines

Article Title: Evaluation of ATOX1 as a Potential Predictive Biomarker for Tetrathiomolybdate Treatment of Breast Cancer Patients with High Risk of Recurrence

doi: 10.3390/biomedicines9121887

Figure Lengend Snippet: Swimmer plot representing the clinical trial cohort (n patients = 47) with data on clinical response (POD = progression of disease), duration of therapy (in terms of TM treatment cycles), breast tumor subtype and stage of breast cancer for each patient, whereby each bar represents one patient in the study cohort. ATOX1 staining intensity levels in nucleus and cytoplasm are shown in the columns located to the left.

Article Snippet: After blocking endogenous peroxidase, the slides were incubated with the mouse monoclonal anti-ATOX1 primary antibody (H00000475-M01, Novus Biologicals, Littleton, CO, USA) followed by incubation with secondary antibody horseradish peroxidase (HRP)-Probe and tertiary antibody HRP-Polymer.

Techniques: Staining

Journal: Biomedicines

Article Title: Evaluation of ATOX1 as a Potential Predictive Biomarker for Tetrathiomolybdate Treatment of Breast Cancer Patients with High Risk of Recurrence

doi: 10.3390/biomedicines9121887

Figure Lengend Snippet: Graphical presentation of percentage of patients with low versus high ATOX1 intensity levels in the tumor cell cytoplasm or nucleus in the whole tumor tissue sections with differentiation between subtypes (( A , B ), respectively) and stages of disease (( C , D ), respectively). (‘Low ATOX1’ = negative + weak ATOX1 staining intensities and ‘High ATOX1’ = intermediate + strong ATOX1 staining intensities).

Article Snippet: After blocking endogenous peroxidase, the slides were incubated with the mouse monoclonal anti-ATOX1 primary antibody (H00000475-M01, Novus Biologicals, Littleton, CO, USA) followed by incubation with secondary antibody horseradish peroxidase (HRP)-Probe and tertiary antibody HRP-Polymer.

Techniques: Staining

Journal: Biomedicines

Article Title: Evaluation of ATOX1 as a Potential Predictive Biomarker for Tetrathiomolybdate Treatment of Breast Cancer Patients with High Risk of Recurrence

doi: 10.3390/biomedicines9121887

Figure Lengend Snippet: Kaplan–Meier curves for correlation between event-free survival (EFS) and ATOX1 intensity in ( A ) tumor cell cytoplasm and ( B ) in tumor cell nucleus of the breast tumor tissues from the phase II clinical trial for tetrathiomolybdate (TM) treatment. (‘Low ATOX1’ = negative and low ATOX1 staining intensities; ‘High ATOX1’ = intermediate and strong ATOX1 staining intensities).

Article Snippet: After blocking endogenous peroxidase, the slides were incubated with the mouse monoclonal anti-ATOX1 primary antibody (H00000475-M01, Novus Biologicals, Littleton, CO, USA) followed by incubation with secondary antibody horseradish peroxidase (HRP)-Probe and tertiary antibody HRP-Polymer.

Techniques: Staining

Journal: Biomedicines

Article Title: Evaluation of ATOX1 as a Potential Predictive Biomarker for Tetrathiomolybdate Treatment of Breast Cancer Patients with High Risk of Recurrence

doi: 10.3390/biomedicines9121887

Figure Lengend Snippet: Kaplan–Meier curves for correlation between event-free survival (EFS) and ATOX1 intensity in tumor cell cytoplasm of whole breast tumor tissues with differentiation between ( A ) different subtypes and ( B ) stages of breast cancer disease.

Article Snippet: After blocking endogenous peroxidase, the slides were incubated with the mouse monoclonal anti-ATOX1 primary antibody (H00000475-M01, Novus Biologicals, Littleton, CO, USA) followed by incubation with secondary antibody horseradish peroxidase (HRP)-Probe and tertiary antibody HRP-Polymer.

Techniques:

Journal: Cancers

Article Title: Dual-Targeting of ATOX1 and ROCK1: A Potent Strategy to Potentiate the Inhibition of Lung Adenocarcinoma Proliferation

doi: 10.3390/cancers17172887

Figure Lengend Snippet: Association of ATOX1 and ROCK1 expression in lung adenocarcinoma (LUAD) with patient survival and MCM protein family expression. ( A , B ) High ATOX1 expression was associated with worse disease-free survival (DFS, p = 0.01) and overall survival (OS, p = 0.10) in patients. ( C , D ) ROCK1 high expression was associated with worse DFS ( p = 0.01) and OS ( p = 0.02). ( E , F ) Patients with dual-low expression of ATOX1 and ROCK1 had the best DFS ( p = 0.01) and OS ( p = 8.2 × 10 −3 ). ( G ) Low expression of ROCK1 is associated with lower expression of MCMs, and is more pronounced at high ATOX1 expression.

Article Snippet: Antibodies mainly included Anti-ATOX1 antibody [ EPR10352 ] (ab154179, Abcam, Cambridge, MA, USA), Mouse Monoclonal ATOX1 Antibody (3E1) (H00000475-M05, Novus Biologicals, Littleton, CO, USA), Anti-ROCK1 antibody [EPR638Y]—BSA and Azide free (ab230799, Abcam, Cambridge, MA, USA), Anti-MCM2 antibody [SP85] (ab95361, Abcam, Cambridge, MA, USA), and Anti-MCM7/PRL antibody [EP1974Y] (ab52489, Abcam, Cambridge, MA, USA).

Techniques: Expressing

Journal: Cancers

Article Title: Dual-Targeting of ATOX1 and ROCK1: A Potent Strategy to Potentiate the Inhibition of Lung Adenocarcinoma Proliferation

doi: 10.3390/cancers17172887

Figure Lengend Snippet: Immunohistochemical staining of ATOX1 and ROCK1 in lung adenocarcinoma (LUAD) and normal paraneoplastic lung tissue. This figure shows the immunohistochemical results of ATOX1 and ROCK1 in 35 LUAD patient samples and normal paraneoplastic lung tissues (NATs). There are four typical expression images, namely, negative, weakly positive, moderately positive, and strongly positive, in LUAD.

Article Snippet: Antibodies mainly included Anti-ATOX1 antibody [ EPR10352 ] (ab154179, Abcam, Cambridge, MA, USA), Mouse Monoclonal ATOX1 Antibody (3E1) (H00000475-M05, Novus Biologicals, Littleton, CO, USA), Anti-ROCK1 antibody [EPR638Y]—BSA and Azide free (ab230799, Abcam, Cambridge, MA, USA), Anti-MCM2 antibody [SP85] (ab95361, Abcam, Cambridge, MA, USA), and Anti-MCM7/PRL antibody [EP1974Y] (ab52489, Abcam, Cambridge, MA, USA).

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Cancers

Article Title: Dual-Targeting of ATOX1 and ROCK1: A Potent Strategy to Potentiate the Inhibition of Lung Adenocarcinoma Proliferation

doi: 10.3390/cancers17172887

Figure Lengend Snippet: Effects of ATOX1 and ROCK1 expression on patient survival and LLCs. ( A ) Comparison of overall survival of patients with different ATOX1/ROCK1 expression patterns; the best survival was observed in the group with low ATOX1/ROCK1 expression (log-rank test, p = 2.4 × 10 −3 ), and the expression of both was correlated with prognosis. ( B ) Immunofluorescence showed that ATOX1 and ROCK1 were highly expressed in LLCs and were localized in the cytoplasm. ( C , D ) Inhibition of ATOX1 alone (siATOX1: p = 1.34 × 10 −2 ; tetrathiomolybdate: p = 1.38 × 10 −2 vs. NC) or ROCK1 alone (siROCK1: p = 8.02 × 10 −1 ; Fasudil: p = 4.89 × 10 −1 vs. NC) did not significantly affect LLC proliferation by CCK-8, while co-inhibition profoundly suppressed proliferative capacity ((siRNA co-inhibition: p = 1.09 × 10 −4 ; drug co-inhibition: p = 4.27 × 10 −5 , vs. NC, mixed-effects model). ( E ) In LLCs, ATOX1 and ROCK1 negatively feedback up-regulate each other and interfere with expression affecting MCM2/7 levels. Statistical symbols: n.s., not significant ( p > 0.05); *, significant difference ( p < 0.05); **, highly significant difference ( p < 0.01); ***, extremely significant difference ( p < 0.001).

Article Snippet: Antibodies mainly included Anti-ATOX1 antibody [ EPR10352 ] (ab154179, Abcam, Cambridge, MA, USA), Mouse Monoclonal ATOX1 Antibody (3E1) (H00000475-M05, Novus Biologicals, Littleton, CO, USA), Anti-ROCK1 antibody [EPR638Y]—BSA and Azide free (ab230799, Abcam, Cambridge, MA, USA), Anti-MCM2 antibody [SP85] (ab95361, Abcam, Cambridge, MA, USA), and Anti-MCM7/PRL antibody [EP1974Y] (ab52489, Abcam, Cambridge, MA, USA).

Techniques: Expressing, Comparison, Immunofluorescence, Inhibition, CCK-8 Assay

Journal: Cancers

Article Title: Dual-Targeting of ATOX1 and ROCK1: A Potent Strategy to Potentiate the Inhibition of Lung Adenocarcinoma Proliferation

doi: 10.3390/cancers17172887

Figure Lengend Snippet: Inhibitory effects of ATOX1 and ROCK1 co-inhibition on LLC migration. ( A ). Transwell assay results for LLCs. Single treatments with si-ATOX1, si-ROCK1, tetrathiomolybdate, or Fasudil decreased cell migration. ( B ). Co-Co-inhibition of ATOX1 and ROCK1, either by combined si-RNAs or drugs, significantly enhanced the inhibition of LLC migration, showing the superiority of synergistic inhibition in regulating cell migration. Statistical symbols: **, highly significant difference ( p < 0.01); ***, extremely significant difference ( p < 0.001).

Article Snippet: Antibodies mainly included Anti-ATOX1 antibody [ EPR10352 ] (ab154179, Abcam, Cambridge, MA, USA), Mouse Monoclonal ATOX1 Antibody (3E1) (H00000475-M05, Novus Biologicals, Littleton, CO, USA), Anti-ROCK1 antibody [EPR638Y]—BSA and Azide free (ab230799, Abcam, Cambridge, MA, USA), Anti-MCM2 antibody [SP85] (ab95361, Abcam, Cambridge, MA, USA), and Anti-MCM7/PRL antibody [EP1974Y] (ab52489, Abcam, Cambridge, MA, USA).

Techniques: Inhibition, Migration, Transwell Assay

Journal: Journal of translational medicine

Article Title: Atox1 regulates macrophage polarization in intestinal inflammation via ROS-NLRP3 inflammasome pathway.

doi: 10.1186/s12967-024-05314-4

Figure Lengend Snippet: Fig. 1 Atox1 expression was increased in TNBS-induced colitis mice. (A) Uniform Manifold Approximation and Projection (UMAP) plot of cell types in patients with Crohn’s disease (GSE134809). (B) Violin plot showing the expression of Atox1 in macrophages from the inflamed and uninflamed areas of patients with Crohn’s disease. TNBS-induced colitis mice were evaluated by (C) body weight loss, expressed as a percentage of the initial weight, and (D) clinical disease activity index (DAI). (E) Representative images of histological analysis in TNBS-induced colitis mice. (F, G) Western blot analyses of Atox1 expression in colonic tissues of TNBS-induced colitis mice. (H) Biochemical analysis of Cu content in colonic tissues of TNBS-induced colitis mice. (I) Rep resentative images of immunofluorescence analyses of colonic mucosa in control and TNBS-induced colitis mice showing F4/80 + cells and Atox1 + cells. The merged images show co-localization of Atox1 with F4/80. Results of ELISA showing the mucosal production of the pro-inflammatory cytokines (J) IL-6, (K) TNF-α, (L) IFN-γ, and (M) IL-17 in control and TNBS-induced colitis mice. DC, dendritic cell; ILC, innate lymphoid cell; MNP, mononuclear phagocyte; pDC, plasmacytoid dendritic cell. Scale bar, 100 μm. *P < 0.05, ***P < 0.001 vs. inflamed or control

Article Snippet: The Atox1 knockout mouse model (C57BL/6J) was generated by CRISPR/Cas-mediated genome engineering (Cyagen Biosciences) through the insertion of the LoxP site flanking exon 2 of the Atox1 gene.

Techniques: Expressing, Activity Assay, Western Blot, Immunofluorescence, Control, Enzyme-linked Immunosorbent Assay

Journal: Journal of translational medicine

Article Title: Atox1 regulates macrophage polarization in intestinal inflammation via ROS-NLRP3 inflammasome pathway.

doi: 10.1186/s12967-024-05314-4

Figure Lengend Snippet: Fig. 3 DCAC50 reduced CuCl2-induced ROS production and M1 polarization in macrophages. Macrophages isolated from the intestinal mucosa of mice were pre-treated with DCAC50, followed by CuCl2 treatment. (A) Results of flow cytometry to determine ROS production. (B) Results of quantitative RT-PCR and (C) Western blot assay to estimate mRNA and protein expression of p47phox. (D) Luciferase activity and (E) ChIP-PCR assays to measure the binding of p47phox promoter with Atox1. Results of quantitative RT-PCR to determine the mRNA expressions of (F) iNOS, (F) IL-12p40, (G) IL-10 and (G) Arg-1. **P < 0.01, ***P < 0.001 vs. control. ##P < 0.01, ###P < 0.001 vs. CuCl2

Article Snippet: The Atox1 knockout mouse model (C57BL/6J) was generated by CRISPR/Cas-mediated genome engineering (Cyagen Biosciences) through the insertion of the LoxP site flanking exon 2 of the Atox1 gene.

Techniques: Isolation, Flow Cytometry, Quantitative RT-PCR, Western Blot, Expressing, Luciferase, Activity Assay, Binding Assay, Control

Journal: Journal of translational medicine

Article Title: Atox1 regulates macrophage polarization in intestinal inflammation via ROS-NLRP3 inflammasome pathway.

doi: 10.1186/s12967-024-05314-4

Figure Lengend Snippet: Fig. 4 Atox1 knockout decreased the levels of pro-inflammatory cytokines after TNBS-induced colitis. Atox1−/− mice and wild-type (WT) littermates received TNBS. Colitis induction was evaluated by (A) body weight loss, expressed as a percentage of the initial weight, and (B) clinical DAI. (C) Representa tive images of histological analysis. Results of ELISA showing the mucosal production of the pro-inflammatory cytokines (D) IL-6, (E) TNF-α, (F) IFN-γ, and (G) IL-17. Results of quantitative RT-PCR showing the mucosal mRNA expression of (H) iNOS, (H) IL-12p40, (I) IL-10 and (I) Arg-1. (J) Results of quantitative RT-PCR and (K) Western blot assay showing the mucosal expression of p47phox mRNA and protein, respectively. Scale bar, 100 μm. **P < 0.01, ***P < 0.001 vs. WT. #P < 0.05, ###P < 0.001 vs. WT + TNBS

Article Snippet: The Atox1 knockout mouse model (C57BL/6J) was generated by CRISPR/Cas-mediated genome engineering (Cyagen Biosciences) through the insertion of the LoxP site flanking exon 2 of the Atox1 gene.

Techniques: Knock-Out, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Western Blot

Journal: Journal of translational medicine

Article Title: Atox1 regulates macrophage polarization in intestinal inflammation via ROS-NLRP3 inflammasome pathway.

doi: 10.1186/s12967-024-05314-4

Figure Lengend Snippet: Fig. 5 Atox1 knockout inhibited macrophage M1 polarization. (A, B) Flow cytometry analysis of the percentage of macrophages (CD45+CD11b+F4/80+L y6C−) among the colonic lamina propria mononuclear cells isolated from Atox1−/− and WT mice with or without TNBS. Atox1−/− mice and WT littermates received TNBS and the macrophages were isolated. (C) Western blot assay of Atox1 expression. (D) Results of ELISA showing the levels of pro-inflamma tory cytokines TNF-α and IL-6. Results of quantitative RT-PCR showing the mRNA expressions of (E) iNOS, (E) IL-12p40, (F) IL-10 and (F) Arg-1. **P < 0.01, ***P < 0.001 vs. WT. #P < 0.05, ##P < 0.01, ###P < 0.001 vs. WT + TNBS

Article Snippet: The Atox1 knockout mouse model (C57BL/6J) was generated by CRISPR/Cas-mediated genome engineering (Cyagen Biosciences) through the insertion of the LoxP site flanking exon 2 of the Atox1 gene.

Techniques: Knock-Out, Flow Cytometry, Isolation, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: Journal of translational medicine

Article Title: Atox1 regulates macrophage polarization in intestinal inflammation via ROS-NLRP3 inflammasome pathway.

doi: 10.1186/s12967-024-05314-4

Figure Lengend Snippet: Fig. 6 Atox1 knockout inhibited ROS production and NLRP3 inflammasome activation in macrophages. Macrophages isolated from the intestinal mu cosa of control and TNBS-induced mice were treated with NAC or MCC950. (A, B) Quantitative RT-PCR and (C, D) Western blot analyses of the expression of iNOS, IL-12p40, IL-10, and Arg-1. Macrophages were isolated from Atox1−/− mice and WT littermates with or without TNBS treatment. (E) Flow cytometry analysis of ROS production. (F) Western blot assay to estimate the protein expressions of p47phox, NLRP3, and Caspase-1 p20. (G) Results of ELISA show ing the levels of pro-inflammatory cytokines IL-1β and IL-18. ***P < 0.001 vs. control or WT. ###P < 0.001 vs. TNBS or WT + TNBS

Article Snippet: The Atox1 knockout mouse model (C57BL/6J) was generated by CRISPR/Cas-mediated genome engineering (Cyagen Biosciences) through the insertion of the LoxP site flanking exon 2 of the Atox1 gene.

Techniques: Knock-Out, Activation Assay, Isolation, Control, Quantitative RT-PCR, Western Blot, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Journal of translational medicine

Article Title: Atox1 regulates macrophage polarization in intestinal inflammation via ROS-NLRP3 inflammasome pathway.

doi: 10.1186/s12967-024-05314-4

Figure Lengend Snippet: Fig. 7 Atox1 knockout decreased pro-inflammatory cytokines and NLRP3 inflammasome activation in TNBS-induced colitis mice. Atox1−/− and Atox1+/− mice and WT littermates received TNBS with or without DCAC50 treatment. Colitis induction was evaluated by (A) body weight loss, expressed as a per centage of the initial weight, and (B) clinical DAI. (C) Representative images of histological analysis. (D) Results of ELISA showing the mucosal production of the pro-inflammatory cytokines TNF-α and IL-6. Results of quantitative RT-PCR showing the mucosal mRNA expression of (E) iNOS, (E) IL-12p40, (F) IL-10 and (F) Arg-1. (G) Western blot assay to estimate the mucosal expression of p47phox, NLRP3, and Caspase-1 p20 protein; (H) Results of ELISA showing the levels of pro-inflammatory cytokines IL-1β and IL-18. Scale bar, 100 μm. ***P < 0.001 vs. WT. #P < 0.05, ##P < 0.01, ###P < 0.001 vs. WT + TNBS. ΔP < 0.05, ΔΔP < 0.01, ΔΔΔP < 0.001 vs. Atox1+/−+TNBS

Article Snippet: The Atox1 knockout mouse model (C57BL/6J) was generated by CRISPR/Cas-mediated genome engineering (Cyagen Biosciences) through the insertion of the LoxP site flanking exon 2 of the Atox1 gene.

Techniques: Knock-Out, Activation Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Western Blot

Journal: Journal of translational medicine

Article Title: Atox1 regulates macrophage polarization in intestinal inflammation via ROS-NLRP3 inflammasome pathway.

doi: 10.1186/s12967-024-05314-4

Figure Lengend Snippet: Fig. 8 Schematic illustration of Atox1 function in regulating ROS-NLRP3 inflammasome pathway during IBD

Article Snippet: The Atox1 knockout mouse model (C57BL/6J) was generated by CRISPR/Cas-mediated genome engineering (Cyagen Biosciences) through the insertion of the LoxP site flanking exon 2 of the Atox1 gene.

Techniques: