Journal: Apoptosis

Article Title: Copper modulates cell fate through the PLK1–FOXO3a–β-catenin signaling pathway by differentially regulating cuproptosis and EMT

doi: 10.1007/s10495-025-02211-z

Figure Lengend Snippet: Transcriptomic profiling identified differential gene expression and polo-like kinase 1–forkhead box O3a (FOXO3a) signaling pathway modulation in response to CuCl₂ and Cu-elesclomol (ES) treatments. A mRNA sequencing analysis is performed on LoVo cells treated for 12 h with CuCl₂ (10 μM) alone, elesclomol (10 nM) alone, or the Cu-ES combination (10 μM + 10 nM). The Venn diagram (left) illustrates the number of differentially expressed genes across the treatment groups. Differentially expressed gene expression profiles are further categorized into 12 distinct clusters based on their expression patterns (right), with red and green indicating upregulation and downregulation, respectively. B Gene Ontology analysis is performed for differentially expressed genes modulated by Cu-ES treatment, demonstrating enriched biological processes for upregulated (top) and downregulated (bottom) genes, plotted as -log₁₀( p -value). C Functional pathway enrichment analysis using DAVID identified significantly altered pathways in response to Cu-ES treatment, with pathway significance presented as -log₁₀(p-value) and corresponding gene counts. D Heatmap of selected genes associated with the FOXO signaling pathway (FOXO3, growth arrest and DNA damage-inducible beta, polo-like kinase 1, cyclin D2, growth arrest and DNA damage-inducible gamma, and gamma-aminobutyric acid receptor-associated protein-like 1). Expression alterations are normalized using Z-scores across treatment groups. Red indicates high expression, and blue indicates low expression. E mRNA expression levels of polo-like kinase 1 and FOXO3a in colorectal cancer are analyzed using the UALCAN database. Box plots illustrate expression in normal (blue) vs. primary tumor tissues (red), across cancer stages (Stage 1–4). Data are presented as median with interquartile range

Article Snippet: The following primary antibodies were used: poly(ADP-ribose) polymerase 1, cleaved caspase-3, caspase-9, receptor-interacting protein kinase 1, receptor-interacting protein kinase 3, solute carrier family 7 member 11, vimentin, and cellular myelocytomatosis oncogene (Cell Signaling Technology, Danvers, MA, USA); heme oxygenase 1, glutathione peroxidase 4, and DLAT (Abcam, Cambridge, UK); E-cadherin and β-catenin (BD Biosciences, San Jose, CA, USA); cyclin D1, cyclin D3, FOXO3a, and β-actin (Santa Cruz Biotechnology, Dallas, TX, USA); PLK1 (ABclonal, Woburn, MA, USA); and ferredoxin 1 (FDX1) (Proteintech, Rosemont, IL, USA).

Techniques: Gene Expression, Sequencing, Expressing, Functional Assay

Journal: Apoptosis

Article Title: Copper modulates cell fate through the PLK1–FOXO3a–β-catenin signaling pathway by differentially regulating cuproptosis and EMT

doi: 10.1007/s10495-025-02211-z

Figure Lengend Snippet: Cu-elesclomol (ES) combination differentially regulates the polo-like kinase 1 (PLK1)–forkhead box O3a (FOXO3a) pathway and disrupts FOXO3a–β-catenin transcriptional activity. A Quantitative reverse transcription-polymerase chain reaction is performed to measure PLK1 and FOXO3a mRNA expression in SW480 and LoVo cells following 12 h of treatment with Cu (5, 10, 25, and 50 μM) or Cu-ES combination (same Cu concentrations + matched elesclomol doses in nM). mRNA levels are normalized to β-actin. *ns: not significant; * P < 0.05, ** P < 0.01, and * **P < 0.001 vs. untreated control. B Western blotting is used to assess PLK1 and FOXO3a protein levels under similar treatment conditions. β-actin is used as a loading control. C Subcellular fractionation followed by immunoblotting is used to analyze the nuclear and cytoplasmic localization of FOXO3a and β-catenin in cells treated with CuCl₂ (25 μM) or Cu-ES (25 μM + 25 nM) for 12 h. Lamin B1 and β-actin confirmed the nuclear and cytoplasmic fractions, respectively. D Immunofluorescence staining visualized FOXO3a localization under similar conditions. FOXO3a (red) and 4',6-diamidino-2-phenylindole (blue) are merged to assess nuclear translocation. Scale bar = 20 μm. E Co-immunoprecipitation is performed to assess the interaction between FOXO3a and β-catenin. β-catenin antibody is used for pulldown, with lamin B1 confirming nuclear input and IgG as a negative control. F Western blotting is used to assess the expression of β-catenin target genes (cyclin D1, cyclin D3, and cellular myelocytomatosis oncogene) under similar treatment conditions. β-actin is used as the loading control

Article Snippet: The following primary antibodies were used: poly(ADP-ribose) polymerase 1, cleaved caspase-3, caspase-9, receptor-interacting protein kinase 1, receptor-interacting protein kinase 3, solute carrier family 7 member 11, vimentin, and cellular myelocytomatosis oncogene (Cell Signaling Technology, Danvers, MA, USA); heme oxygenase 1, glutathione peroxidase 4, and DLAT (Abcam, Cambridge, UK); E-cadherin and β-catenin (BD Biosciences, San Jose, CA, USA); cyclin D1, cyclin D3, FOXO3a, and β-actin (Santa Cruz Biotechnology, Dallas, TX, USA); PLK1 (ABclonal, Woburn, MA, USA); and ferredoxin 1 (FDX1) (Proteintech, Rosemont, IL, USA).

Techniques: Activity Assay, Reverse Transcription, Polymerase Chain Reaction, Expressing, Control, Western Blot, Fractionation, Immunofluorescence, Staining, Translocation Assay, Immunoprecipitation, Negative Control

Journal: Apoptosis

Article Title: Copper modulates cell fate through the PLK1–FOXO3a–β-catenin signaling pathway by differentially regulating cuproptosis and EMT

doi: 10.1007/s10495-025-02211-z

Figure Lengend Snippet: Polo-like kinase 1 inhibition by BI-2536 mimics the cellular and molecular effects of Cu-elesclomol (ES) combination treatment. A Wound healing assays are performed to assess the migratory capacity of SW480 and LoVo cells treated with the vehicle control or BI-2536 (10 nM) for 24 h. Representative images at 0, 24, and 48 h are indicated with yellow dashed lines, indicating wound margins (left panels), and quantification of wound closure percentages is provided (right panels). Data are presented as the mean ± standard deviation (n = 3). * P < 0.05 and *** P < 0.001 vs. untreated control. B Transwell migration assay is performed under similar conditions. Migrated cells are stained with crystal violet (left panels), and cell migration is quantified (right panels). Data are presented as the mean ± standard deviation (n = 3). * P < 0.05 and *** P < 0.001 vs. untreated control. (C) Quantitative reverse transcription-polymerase chain reaction analysis is used to measure the mRNA expression levels of E-cadherin, vimentin, and FOXO3a in cells treated with vehicle control or BI-2536 (10 nM) for 24 h. Expression levels are normalized to β-actin. Data are presented as the mean ± standard deviation (n = 3). ** P < 0.01 and *** P < 0.001 vs. untreated control. D Western blotting is performed to assess protein levels of E-cadherin, vimentin, forkhead box O3a (FOXO3a), cyclin B1, cyclin D1 and cyclin D3 under similar treatment conditions, with β-actin as a loading control. E Subcellular localization of β-catenin and FOXO3a is analyzed using cytoplasmic and nuclear fractionation, followed by western blotting. Fraction purity is confirmed using β-actin (cytoplasmic marker) and lamin B1 (nuclear marker). F Immunofluorescence staining is performed to visualize FOXO3a subcellular localization after BI-2536 treatment. Red fluorescence indicates FOXO3a, blue indicates 4',6-diamidino-2-phenylindole nuclear staining, and merged images are shown. Scale bar = 20 μm. G Co-immunoprecipitation is used to assess the interaction between FOXO3a and β-catenin. β-catenin antibody is used for pulldown, and protein levels in total lysates are presented below. IgG served as the negative control, and lamin B1 is used as the nuclear marker

Article Snippet: The following primary antibodies were used: poly(ADP-ribose) polymerase 1, cleaved caspase-3, caspase-9, receptor-interacting protein kinase 1, receptor-interacting protein kinase 3, solute carrier family 7 member 11, vimentin, and cellular myelocytomatosis oncogene (Cell Signaling Technology, Danvers, MA, USA); heme oxygenase 1, glutathione peroxidase 4, and DLAT (Abcam, Cambridge, UK); E-cadherin and β-catenin (BD Biosciences, San Jose, CA, USA); cyclin D1, cyclin D3, FOXO3a, and β-actin (Santa Cruz Biotechnology, Dallas, TX, USA); PLK1 (ABclonal, Woburn, MA, USA); and ferredoxin 1 (FDX1) (Proteintech, Rosemont, IL, USA).

Techniques: Inhibition, Control, Standard Deviation, Transwell Migration Assay, Staining, Migration, Reverse Transcription, Polymerase Chain Reaction, Expressing, Western Blot, Fractionation, Marker, Immunofluorescence, Fluorescence, Immunoprecipitation, Negative Control

Journal: Apoptosis

Article Title: Copper modulates cell fate through the PLK1–FOXO3a–β-catenin signaling pathway by differentially regulating cuproptosis and EMT

doi: 10.1007/s10495-025-02211-z

Figure Lengend Snippet: BI-2536 enhances Cu-elesclomol (ES)–induced cell death and modulates forkhead box O3a, epithelial-mesenchymal transition, and cuproptosis-related pathways. A Annexin V-fluorescein isothiocyanate/propidium iodide dual staining followed by flow cytometry is performed to analyze cell death in SW480 and LoVo cells treated with vehicle control, BI-2536 (10 nM), Cu-ES (10 μM + 10 nM), or BI-2536 (10 nM) pre-treatment followed by Cu-ES (10 μM + 10 nM) for 12 h. Representative dot plots (left) and quantification of dead cell percentages (right) are presented. B Cell cycle profiles are assessed using flow cytometry after propidium iodide staining of cells treated under similar conditions. Representative histograms (left) and quantitative analysis of cell cycle distribution (right) are shown. * P < 0.05, ** P < 0.01, and *** P < 0.001 vs. untreated control. C Quantitative reverse transcription-polymerase chain reaction is conducted to assess mRNA expression levels of epithelial-mesenchymal transition markers (E-cadherin and vimentin) and FOXO3a in cells treated with vehicle control, Cu-ES (10 μM + 10 nM), or BI-2536 pre-treatment (10 nM) followed by Cu-ES. Expression levels are normalized to β-actin. Data are presented as the mean ± standard deviation (n = 3). * P < 0.05, ** P < 0.01, and * **P < 0.001 vs. untreated control. D Western blotting is used to examine the protein expression of E-cadherin, vimentin, and FOXO3a. E Immunofluorescence staining is performed to determine FOXO3a subcellular localization in treated cells. FOXO3a is stained red, nuclei are counterstained with 4',6-diamidino-2-phenylindole (blue), and merged images are shown. Scale bar = 20 μm. F Co-immunoprecipitation is conducted to assess the interaction between β-catenin and FOXO3a. The upper panel represents proteins pulled down with anti-β-catenin antibody, whereas the lower panel represents total protein levels in the lysates. IgG served as the negative control and lamin B1 is used as the nuclear marker. G Western blotting is performed to assess dihydrolipoamide S-acetyltransferase aggregation and ferredoxin 1 expression. Aggregated and total DLAT protein levels are presented in the upper and middle panels, respectively, and ferredoxin 1 expression is presented in the lower panel. β-actin is used as a loading control. H Immunofluorescence staining is used to assess the subcellular localization of dihydrolipoamide S-acetyltransferase in cells. Dihydrolipoamide S-acetyltransferase (red), 4',6-diamidino-2-phenylindole (blue), and merged images are shown. Scale bar = 20 μm

Article Snippet: The following primary antibodies were used: poly(ADP-ribose) polymerase 1, cleaved caspase-3, caspase-9, receptor-interacting protein kinase 1, receptor-interacting protein kinase 3, solute carrier family 7 member 11, vimentin, and cellular myelocytomatosis oncogene (Cell Signaling Technology, Danvers, MA, USA); heme oxygenase 1, glutathione peroxidase 4, and DLAT (Abcam, Cambridge, UK); E-cadherin and β-catenin (BD Biosciences, San Jose, CA, USA); cyclin D1, cyclin D3, FOXO3a, and β-actin (Santa Cruz Biotechnology, Dallas, TX, USA); PLK1 (ABclonal, Woburn, MA, USA); and ferredoxin 1 (FDX1) (Proteintech, Rosemont, IL, USA).

Techniques: Staining, Flow Cytometry, Control, Reverse Transcription, Polymerase Chain Reaction, Expressing, Standard Deviation, Western Blot, Immunofluorescence, Immunoprecipitation, Negative Control, Marker

Journal: Apoptosis

Article Title: Copper modulates cell fate through the PLK1–FOXO3a–β-catenin signaling pathway by differentially regulating cuproptosis and EMT

doi: 10.1007/s10495-025-02211-z

Figure Lengend Snippet: Schematic model illustrating copper-induced regulation of cuproptosis and epithelial-mesenchymal transition through the polo-like kinase 1-forkhead box O3a-beta-catenin pathway. Copper treatment increases polo-like kinase 1 expression, resulting in forkhead box O3a inhibition and β-catenin nuclear translocation that enhances epithelial-mesenchymal transition-related gene expression. In contrast, co-treatment with CuCl₂ and elesclomol (Cu-ES) facilitates cuproptosis through dihydrolipoamide S-acetyltransferase aggregation and mitochondrial dysfunction while simultaneously suppressing polo-like kinase 1 and activating forkhead box O3a. Nuclear forkhead box O3a interacts with β-catenin to inhibit its transcriptional activity, thereby repressing epithelial-mesenchymal transition

Article Snippet: The following primary antibodies were used: poly(ADP-ribose) polymerase 1, cleaved caspase-3, caspase-9, receptor-interacting protein kinase 1, receptor-interacting protein kinase 3, solute carrier family 7 member 11, vimentin, and cellular myelocytomatosis oncogene (Cell Signaling Technology, Danvers, MA, USA); heme oxygenase 1, glutathione peroxidase 4, and DLAT (Abcam, Cambridge, UK); E-cadherin and β-catenin (BD Biosciences, San Jose, CA, USA); cyclin D1, cyclin D3, FOXO3a, and β-actin (Santa Cruz Biotechnology, Dallas, TX, USA); PLK1 (ABclonal, Woburn, MA, USA); and ferredoxin 1 (FDX1) (Proteintech, Rosemont, IL, USA).

Techniques: Expressing, Inhibition, Translocation Assay, Gene Expression, Activity Assay