Journal: Cancer Research Communications

Article Title: m6A Modification of ATOX1 Inhibits Acute Myeloid Leukemia Progression by Promoting Cuproptosis

doi: 10.1158/2767-9764.CRC-25-0436

Figure Lengend Snippet: ATOX1 is lowly expressed in AML. A, Bioinformatics analysis of ATOX1 expression in bone marrow of normal individuals and patients with AML (data obtained from TCGA, GTEx, and GEO databases, respectively). ***, P < 0.001; *, P < 0.05. B, qRT-PCR and Western blot analysis of ATOX1 expression in NBMMCs, HL-60, THP-1, KG-1, and NOMO-1 cells. Data are shown as mean ± SD. n = 3.

Article Snippet: ATOX1 , 22641-1-AP , 1:2,000 , Proteintech , AB_2879139.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Cancer Research Communications

Article Title: m6A Modification of ATOX1 Inhibits Acute Myeloid Leukemia Progression by Promoting Cuproptosis

doi: 10.1158/2767-9764.CRC-25-0436

Figure Lengend Snippet: ATOX1 overexpression alleviates AML progression. A, qRT-PCR and Western blot analysis of ATOX1 expression in AML cells transfected with sh-NC or sh- ATOX1 . AML cells were transfected with sh- ATOX1 or oe-A TOX1 . B, Western blot analysis of ATOX1 expression in AML cells. C, Cell Counting Kit-8 assay for detecting the viability of AML cells. D, EDU staining assay for detecting the cell proliferation of AML cells. Scale bar, 25 μm. E, Flow cytometry for detecting the cell cycle of AML cells. F, Flow cytometry for detecting the cell death of AML cells. Mortality rate (%) represents the percentage of PI-positive (membrane-compromised necrotic/late apoptotic) cells. Data are shown as mean ± SD. n = 3. SSC, side scatter.

Article Snippet: ATOX1 , 22641-1-AP , 1:2,000 , Proteintech , AB_2879139.

Techniques: Over Expression, Quantitative RT-PCR, Western Blot, Expressing, Transfection, Cell Counting, Staining, Flow Cytometry, Membrane

Journal: Cancer Research Communications

Article Title: m6A Modification of ATOX1 Inhibits Acute Myeloid Leukemia Progression by Promoting Cuproptosis

doi: 10.1158/2767-9764.CRC-25-0436

Figure Lengend Snippet: ATOX1 knockdown inhibits ES-/Cu-induced cuproptosis in AML cells. A, Cell Counting Kit-8 assay for detecting the viability of AML cells transfected with oe-NC or oe-ATOX1 treated with different concentrations of ES/Cu (0, 2, 10, 50, and 200 nmol/L and ES and CuCl 2 combined in a 1:1 ratio). AML cells transfected with sh-NC or sh- ATOX1 were treated with 200 nmol/L ES/Cu for 72 hours. B, Cell Counting Kit-8 assay for detecting the viability of AML cells. C, EDU staining assay for detecting the cell proliferation of AML cells. Scale bar, 25 μm. D, Flow cytometry for detecting the cell death of AML cells. E, Western blot analysis of lipoylated DLAT expression in AML cells. F, Western blot analysis of Fe–S cluster protein (FDX1, LIAS, and ACO2) expression in AML cells. Data are shown as mean ± SD. n = 3. SSC, side scatter.

Article Snippet: ATOX1 , 22641-1-AP , 1:2,000 , Proteintech , AB_2879139.

Techniques: Knockdown, Cell Counting, Transfection, Staining, Flow Cytometry, Western Blot, Expressing

Journal: Cancer Research Communications

Article Title: m6A Modification of ATOX1 Inhibits Acute Myeloid Leukemia Progression by Promoting Cuproptosis

doi: 10.1158/2767-9764.CRC-25-0436

Figure Lengend Snippet: ATOX1 overexpression promotes ES-/Cu-induced cuproptosis in AML cells. AML cells transfected with oe-NC or oe- ATOX1 were treated with 200 nmol/L ES/Cu for 72 hours. A, Cell Counting Kit-8 assay for detecting the viability of AML cells. B, EDU staining assay for detecting the cell proliferation of AML cells. Scale bar, 25 μm. C, Flow cytometry for detecting the cell death of AML cells. D, Western blot analysis of lipoylated DLAT expression in AML cells. E, Western blot analysis of Fe–S cluster protein (FDX1, LIAS, and ACO2) expression in AML cells. F, Cell Counting Kit-8 assay for detecting the viability of AML cells transfected with oe-NC or oe-ATOX1 and sh-NC or sh- FDX1 treated with different concentrations of ES/Cu (0, 2, 10, 50, and 200 nmol/L) for 72 hours. Data are shown as mean ± SD. n = 3. SSC, side scatter.

Article Snippet: ATOX1 , 22641-1-AP , 1:2,000 , Proteintech , AB_2879139.

Techniques: Over Expression, Transfection, Cell Counting, Staining, Flow Cytometry, Western Blot, Expressing

Journal: Cancer Research Communications

Article Title: m6A Modification of ATOX1 Inhibits Acute Myeloid Leukemia Progression by Promoting Cuproptosis

doi: 10.1158/2767-9764.CRC-25-0436

Figure Lengend Snippet: ATOX1 expression is affected by ALKBH5-mediated modification of m6A methylation. A, RNA pull-down assay for identifying the interaction of ATOX1 RNA with METTL3, ALKBH5, ELAVL1, and HNRNPC. AML cells were transfected with oe-NC or oe- ALKBH5 . B, RIP-qPCR assay for detecting the m6A methylation level of ATOX1 in AML cells. C, qRT-PCR of ATOX1 transcripts in ActD-treated AML cells. D, Western blot analysis of ATOX1 expression in AML cells. AML cells were transfected with sh-NC or sh- ALKBH5 . E, Western blot analysis of ATOX1 expression in AML cells. Data are shown as mean ± SD. n = 3.

Article Snippet: ATOX1 , 22641-1-AP , 1:2,000 , Proteintech , AB_2879139.

Techniques: Expressing, Modification, Methylation, Pull Down Assay, Transfection, Quantitative RT-PCR, Western Blot

Journal: Cancer Research Communications

Article Title: m6A Modification of ATOX1 Inhibits Acute Myeloid Leukemia Progression by Promoting Cuproptosis

doi: 10.1158/2767-9764.CRC-25-0436

Figure Lengend Snippet: ALKBH5-mediated m6A modification regulating ATOX1 expression affects cuproptosis in AML cells. AML cells transfected with oe- ATOX1 and/or oe- ALKBH5 were treated with 200 nmol/L ES/Cu for 72 hours. A, Western blot analysis of ATOX1 and ALKBH5 expression in AML cells. B, CCK-8 assay for detecting the viability of AML cells. C, EDU staining assay for detecting the cell proliferation of AML cells. Scale bar, 25 μm. D, Flow cytometry for detecting the cell death of AML cells. E, Western blot analysis of lipoylated DLAT expression in AML cells. F, Western blot analysis of Fe–S cluster protein (FDX1, LIAS, and ACO2) expression in AML cells. Data are shown as mean ± SD. n = 3. SSC, side scatter.

Article Snippet: ATOX1 , 22641-1-AP , 1:2,000 , Proteintech , AB_2879139.

Techniques: Modification, Expressing, Transfection, Western Blot, CCK-8 Assay, Staining, Flow Cytometry

Journal: Cancer Research Communications

Article Title: m6A Modification of ATOX1 Inhibits Acute Myeloid Leukemia Progression by Promoting Cuproptosis

doi: 10.1158/2767-9764.CRC-25-0436

Figure Lengend Snippet: Animal experiments validate the therapeutic potential of the ALKBH5–ATOX1 axis for AML. Female BALB/c nude mice were subcutaneously injected with 1 × 10 7 NOMO-1 cells in the right axilla. A, Western blot analysis of ATOX1 and ALKBH5 expression in tumor tissues of AML model mice. B, The growth curve of tumor volume in AML model mice. C, Representative images and weight of tumors in AML. D and E, IHC assay for detecting Ki67 expression. Scale bar, 25 and 100 μm. F, Western blot analysis of lipoylated DLAT expression in tumor tissues of AML model mice. G, Western blot analysis of Fe–S cluster protein (FDX1 and LIAS) expression in tumor tissues of AML model mice. Data are shown as mean ± SD. n = 5.

Article Snippet: ATOX1 , 22641-1-AP , 1:2,000 , Proteintech , AB_2879139.

Techniques: Injection, Western Blot, Expressing

Journal: Cancer Research Communications

Article Title: m6A Modification of ATOX1 Inhibits Acute Myeloid Leukemia Progression by Promoting Cuproptosis

doi: 10.1158/2767-9764.CRC-25-0436

Figure Lengend Snippet: Schematic illustration of the proposed mechanisms: As depicted, ATOX1 undergoes m6A modification mediated by ALKBH5, which is upregulated in AML. This posttranscriptional modification destabilizes ATOX1 mRNA, leading to reduced ATOX1 expression. Consequently, the suppression of cuproptosis in AML cells is alleviated, thereby promoting AML progression.

Article Snippet: ATOX1 , 22641-1-AP , 1:2,000 , Proteintech , AB_2879139.

Techniques: Modification, Expressing

Journal: Molecular and cellular biology

Article Title: The Copper Chaperone ATOX1 Exhibits Differential Protein–Protein Interactions and Contributes to Skeletal Myoblast Differentiation

doi: 10.1080/10985549.2026.2621941

Figure Lengend Snippet: Generation of APEX2-ATOX1 constructs. (A) The APEX2-ATOX1 construct contains 3× Flag and 3× Myc tags flanking the full-length APEX2 sequence at the amino terminal end of the human ATOX1 protein. All tags are separated by glycine-serine hinges. Constructs were subcloned into a pcDNA 3.1 plasmid under the control of a CMV promoter. (B) Immunoblot of lysates from C2C12 myoblasts transfected with APEX2-ATOX1 constructs showing detection of APEX2-ATOX1 fusion constructs by antibodies to Flag, human (h) or mouse (m) ATOX1. Antibodies to HSP-90 and total proteins detected using stain-free gel imaging technology (Bio-Rad) were used as loading controls. Labeling efficiency was determined by probing blots with streptavidin-HRP in cells titrated with biotin phenol (C), hydrogen peroxide (H 2 O 2 ) (D), or at variable time points in seconds (s) (E). For all, shown are representative images of three-to-five replicates of mixed populations of stable cells.

Article Snippet: The sequence encoding the APEX2-ATOX1 construct was synthesized by Azenta/Genewiz and subcloned from commercial vector into pcDNA 3.1 (ThermoFisher V79020) and then into the lentiviral vector pCW57.1 using EcoRI restriction enzyme recognition sites.

Techniques: Construct, Sequencing, Plasmid Preparation, Control, Western Blot, Transfection, Staining, Imaging, Labeling

Journal: Molecular and cellular biology

Article Title: The Copper Chaperone ATOX1 Exhibits Differential Protein–Protein Interactions and Contributes to Skeletal Myoblast Differentiation

doi: 10.1080/10985549.2026.2621941

Figure Lengend Snippet: Stable cells express inducible APEX2-ATOX1. (A) Stable C2C12 cells were generated that express the APEX2-ATOX1 fusion construct under the control of a tetracycline responsive element (TRE). (B) Doxycycline (DOX) titration revealed the optimal concentration to be 2.5 μg/mL as determined by immunoblot using an antibody to ATOX1. DOX at 2.5 μg/mL was used to induce APEX2-ATOX1 expression in myocytes (C) and myotubes (D). Localization of APEX2-ATOX1 construct (A2A1) as determined by fractionation in myoblasts (E), myocytes (F), and myotubes (G) as analyzed by immunoblot using antibodies to GAPDH to indicate the non-nuclear fraction and histone H3 to indicate the nuclear fraction. Total protein as detected by Stain-free gel imaging technology (Bio-Rad) was used as a loading control. (H) Portion of APEX2-ATOX1 in the nuclear fraction as measured by densitometry. Shown for all is mean ± standard deviation for n = 2–3 experiments. Statistical significance was determined using one-way ANOVA with Dunnett’s post hoc correction for multiple comparisons. None of the comparisons were statistically significant. (I) Immunofluorescence staining using an antibody to the Flag tag in the APEX2-ATOX1 construct in transfected myoblasts showing its cytosolic localization in myoblasts. Myoblasts transfected with empty pcDNA 3.1 plasmid (empty vector) were used as a control and nuclei were visualized with DAPI. Images are representative of n = 3 replicates. Bar = 200 μm. For C to G, all immunoblots are representative of two-to-three replicates.

Article Snippet: The sequence encoding the APEX2-ATOX1 construct was synthesized by Azenta/Genewiz and subcloned from commercial vector into pcDNA 3.1 (ThermoFisher V79020) and then into the lentiviral vector pCW57.1 using EcoRI restriction enzyme recognition sites.

Techniques: Generated, Construct, Control, Titration, Concentration Assay, Western Blot, Expressing, Fractionation, Staining, Imaging, Standard Deviation, Immunofluorescence, FLAG-tag, Transfection, Plasmid Preparation

Journal: Molecular and cellular biology

Article Title: The Copper Chaperone ATOX1 Exhibits Differential Protein–Protein Interactions and Contributes to Skeletal Myoblast Differentiation

doi: 10.1080/10985549.2026.2621941

Figure Lengend Snippet: Comparative proteomic analysis and label-free quantification reveal differential ATOX1 proximal proteins detected during myoblast differentiation. (A) Expression of APEX2-ATOX1 was induced in myoblasts (MB), myocytes (MC), and myotubes (MT). After labeling, lysates were isolated with streptavidin beads and analyzed by comparative proteomics. Cells without biotin phenol were used as negative controls and three independent replicates per differentiation condition were used for proteomic analysis. (B) Venn diagram of proteins detected in all three replicates of MB, MC, and MT that were not detected in negative control cells without biotin phenol. These hits were used for gene ontology and pathway analysis. The top three hits each for GO_Biological Process (BP), GO_Molecular Function (MF), and the Kyoto Encyclopedia of Genes and Genome (KEGG) pathways are shown for MB (C), MC (D), and MT (E).

Article Snippet: The sequence encoding the APEX2-ATOX1 construct was synthesized by Azenta/Genewiz and subcloned from commercial vector into pcDNA 3.1 (ThermoFisher V79020) and then into the lentiviral vector pCW57.1 using EcoRI restriction enzyme recognition sites.

Techniques: Quantitative Proteomics, Expressing, Labeling, Isolation, Negative Control

Journal: Molecular and cellular biology

Article Title: The Copper Chaperone ATOX1 Exhibits Differential Protein–Protein Interactions and Contributes to Skeletal Myoblast Differentiation

doi: 10.1080/10985549.2026.2621941

Figure Lengend Snippet: Shifting ATOX1 binding partners during myoblast differentiation detected by immunoblot. (A) Representative blots showing streptavidin bead elutions from myoblasts (MB), myocytes (MC), and myotubes (MT) expressing APEX2-ATOX1 and treated with biotin phenol (BP). Cells without BP (-BP) were used as negative controls. Total protein as detected by Ponceau stain was used as loading control. (B to F) Densitometric quantification of candidate ATOX1 interacting proteins from blots shown in (A). Shown are mean ± standard deviation for n = 2–5 experiments. Statistical significance was determined using two-way ANOVA with Sidak post hoc correction for multiple comparisons. * P < 0.05, ** P < 0.01.

Article Snippet: The sequence encoding the APEX2-ATOX1 construct was synthesized by Azenta/Genewiz and subcloned from commercial vector into pcDNA 3.1 (ThermoFisher V79020) and then into the lentiviral vector pCW57.1 using EcoRI restriction enzyme recognition sites.

Techniques: Binding Assay, Western Blot, Expressing, Staining, Control, Standard Deviation

Journal: Molecular and cellular biology

Article Title: The Copper Chaperone ATOX1 Exhibits Differential Protein–Protein Interactions and Contributes to Skeletal Myoblast Differentiation

doi: 10.1080/10985549.2026.2621941

Figure Lengend Snippet: ATOX1 deficiency impacts proliferation but not ERK phosphorylation in myoblasts. (A) Immunoblot representative of n = 3 separate experiments showing phosphorylated ERK1/2 (pERK) and total ERK1/2 (ERK) in wild-type or APEX2-ATOX1 expressing myoblasts transfected with negative control siRNA (siScr) or Atox1 targeting siRNA (siAtox1). Blots probed with an antibody to ATOX1 were used to show expression of APEX2-ATOX1 and endogenous ATOX1 knockdown (mATOX1). Total protein as detected by Ponceau stain was used as a loading control. (B) Ratio of pERK/ERK bands in wild-type (WT) and APEX2-ATOX1 (A2A1) expressing myoblasts with (siAtox1) and without (siScr) Atox1 knockdown as measured by densitometry. Shown is mean ± standard deviation for quantification of n= 3 immunoblots. Statistical significance was determined using one-way ANOVA with Dunnett’s post hoc correction for multiple comparisons. None of the comparisons were statistically significant. (C) Immunoblot representative of n = 3 separate experiments showing immunoprecipitation (IP) using an antibody to MEK1/2 in wild-type myoblasts with (+Ins) or without (NT) insulin treatment to stimulate MEK signaling. The IP was probed with an antibody to ERK1/2 (ERK) showing interaction between MEK1/2 and ERK1/2 and an antibody to ATOX1 showing no interaction. Total protein as detected by Ponceau stain was used as a loading control. (D) Immunoblot representative of n = 4 separate experiments probed with an antibody to ATOX1 and an antibody to CCS in siScr and siAtox1 myoblasts. Quantifications showing reduced ATOX1 and CCS protein show mean ± standard deviation for n = 4 experiments. Statistical significance was determined by paired t test. **** P < 0.0001, * P < 0.05. (E) Proliferation as determined by cell counting in siScr and siAtox1 myoblasts as counted at 0, 16, 24 and 48 h after transfection. Shown is mean ± standard deviation for n = 3 experiments. Statistical significance was determined using two-way ANOVA with Sidak post hoc correction for multiple comparisons. * P < 0.05. (F) Quantitative PCR of Atox1 and Ccs transcript levels in siScr and siAtox1 myoblasts showing no increase in Ccs levels. Shown is mean ± standard deviation for n = 3 experiments. Statistical significance was determined using two-way ANOVA with Sidak post-hoc correction for multiple comparisons. ** P < 0.01, *** P < 0.0001. (G) Total Cu as measured by ICP-MS and normalized to total phosphorus (P) showing increased Cu in siAtox1 compared to siScr myoblasts. Shown is mean ± standard deviation for n = 8 experiments. Statistical significance was determined using paired t test. ** P < 0.01. (H) Wet weights of myoblasts showing increased cell mass in siAtox1 compared to siScr myoblasts. Shown is mean ± standard deviation for n = 8 experiments. Statistical significance was determined using paired t test. *** P < 0.001. (I) ICP-MS measurements showing total Cu (μg) normalized to total wet weight of cells (g). Shown is mean ± standard deviation for n = 8 experiments. Statistical significance was determined using paired t test. ** P < 0.01.

Article Snippet: The sequence encoding the APEX2-ATOX1 construct was synthesized by Azenta/Genewiz and subcloned from commercial vector into pcDNA 3.1 (ThermoFisher V79020) and then into the lentiviral vector pCW57.1 using EcoRI restriction enzyme recognition sites.

Techniques: Phospho-proteomics, Western Blot, Expressing, Transfection, Negative Control, Knockdown, Staining, Control, Standard Deviation, Immunoprecipitation, Cell Counting, Real-time Polymerase Chain Reaction