Journal: Journal of Gastrointestinal Oncology

Article Title: The EZH2-NEAT1 epigenetic axis promotes cuproptosis sensitivity and modulates cancer cell migration in colorectal cancer

doi: 10.21037/jgo-2025-1-1058

Figure Lengend Snippet: Elesclomol-copper complex induces cuproptosis in colorectal cancer cells through proteotoxic stress and lipoylated protein aggregation. (A,B) Cell viability of the HCT116 and RKO cells treated with Elsm alone (A) or Elsm-Cu (B) at indicated concentrations for 48 hours. (C,D) Representative images and quantification of colony formation in the HCT116 and RKO cells treated with vehicle, Elsm, or Elsm-Cu. Images were acquired using a flatbed scanner. The image shows a representative whole well of a 6-well plate. (E,F) Western blot analysis of HSP70 in the HCT116 and RKO cells treated with vehicle (CTL), Elsm, or Elsm-Cu. (G-J) DLAT distribution in soluble and insoluble fractions from the HCT116 (G,I) and RKO (H,J) cells treated with Elsm (G,H) or Elsm-Cu (I,J). β-actin served as the loading control. Data are presented as the mean ± standard deviation from three independent experiments. *, P<0.05; **, P<0.01; ***, P<0.001. CTL, control; DLAT , dihydrolipoamide S-acetyltransferase; Elsm, elesclomol; Elsm-Cu, elesclomol with copper chloride.

Article Snippet: The following primary antibodies were used: anti- EZH2 (Cell Signaling Technology, Danvers, MA, USA, 5246S), anti- HSP70 (Cell Signaling Technology, 4872S), anti- DLAT (Proteintech, Wuhan, China, 16179-1-AP), anti- FDX1 (Abcam, Cambridge, MA, USA, ab206649), anti-LIAS (Proteintech, 14812-1-AP), anti-lipoic acid (Calbiochem, San Diego, CA, USA, 437695), anti-β-actin (Sigma-Aldrich, A2228) and anti-HA tag (Cell Signaling Technology, 3724S).

Techniques: Western Blot, Control, Standard Deviation

Journal: Journal of Gastrointestinal Oncology

Article Title: The EZH2-NEAT1 epigenetic axis promotes cuproptosis sensitivity and modulates cancer cell migration in colorectal cancer

doi: 10.21037/jgo-2025-1-1058

Figure Lengend Snippet: EZH2 expression is correlated with cuproptosis-related genes and is upregulated during copper-induced cell death. (A) Heatmap showing Pearson correlation between EZH2 and cuproptosis-related genes in TCGA colorectal adenocarcinoma dataset. (B) Western blot of EZH2 in the HCT116 and RKO cells treated with indicated concentrations of Elsm or Elsm-Cu. (C,D) RT-qPCR (C) and Western blot (D) validation of EZH2 knockdown in the HCT116 and RKO cells. (E) Cell viability of the control and EZH2 knockdown cells treated with Elsm-Cu over time. (F) Colony formation in the control and EZH2 knockdown cells treated with Elsm-Cu. Images were acquired using a flatbed scanner. The image shows a representative whole well of a 6-well plate. (G) Western blot of HSP70 in the control and EZH2 knockdown cells with or without Elsm-Cu treatment. (H,I) DLAT distribution in soluble and insoluble fractions from the control and EZH2 knockdown HCT116 (H) and RKO (I) cells. β-actin served as the loading control. Data are presented as the mean ± standard deviation from three independent experiments. *, P<0.05; **, P<0.01; ***, P<0.001. DLAT , dihydrolipoamide S-acetyltransferase; Elsm, elesclomol; Elsm-Cu, elesclomol with copper chloride; RT-qPCR, real-time quantitative polymerase chain reaction; TCGA, The Cancer Genome Atlas.

Article Snippet: The following primary antibodies were used: anti- EZH2 (Cell Signaling Technology, Danvers, MA, USA, 5246S), anti- HSP70 (Cell Signaling Technology, 4872S), anti- DLAT (Proteintech, Wuhan, China, 16179-1-AP), anti- FDX1 (Abcam, Cambridge, MA, USA, ab206649), anti-LIAS (Proteintech, 14812-1-AP), anti-lipoic acid (Calbiochem, San Diego, CA, USA, 437695), anti-β-actin (Sigma-Aldrich, A2228) and anti-HA tag (Cell Signaling Technology, 3724S).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Biomarker Discovery, Knockdown, Control, Standard Deviation, Real-time Polymerase Chain Reaction

Journal: Journal of Gastrointestinal Oncology

Article Title: The EZH2-NEAT1 epigenetic axis promotes cuproptosis sensitivity and modulates cancer cell migration in colorectal cancer

doi: 10.21037/jgo-2025-1-1058

Figure Lengend Snippet: EZH2 overexpression enhances cuproptosis sensitivity through increased proteotoxic stress. (A,B) RT-qPCR (A) and Western blot (B) validation of EZH2 overexpression in the HCT116 and RKO cells. (C) Cell viability of the control and EZH2 -overexpressing cells treated with Elsm-Cu over time. (D) Colony formation in the control and EZH2 -overexpressing cells treated with Elsm-Cu. Images were acquired using a flatbed scanner. The image shows a representative whole well of a 6-well plate. (E) Western blot of HSP70 in the control and EZH2 -overexpressing cells with or without Elsm-Cu treatment. (F,G) DLAT distribution in soluble and insoluble fractions from the control and EZH2 -overexpressing HCT116 (F) and RKO (G) cells. β-actin served as the loading control. Data are presented as the mean ± standard deviation from three independent experiments. **, P<0.01; ***, P<0.001. DLAT , dihydrolipoamide S-acetyltransferase; Elsm-Cu, elesclomol with copper chloride; RT-qPCR, real-time quantitative polymerase chain reaction.

Article Snippet: The following primary antibodies were used: anti- EZH2 (Cell Signaling Technology, Danvers, MA, USA, 5246S), anti- HSP70 (Cell Signaling Technology, 4872S), anti- DLAT (Proteintech, Wuhan, China, 16179-1-AP), anti- FDX1 (Abcam, Cambridge, MA, USA, ab206649), anti-LIAS (Proteintech, 14812-1-AP), anti-lipoic acid (Calbiochem, San Diego, CA, USA, 437695), anti-β-actin (Sigma-Aldrich, A2228) and anti-HA tag (Cell Signaling Technology, 3724S).

Techniques: Over Expression, Quantitative RT-PCR, Western Blot, Biomarker Discovery, Control, Standard Deviation, Real-time Polymerase Chain Reaction

Journal: Journal of Gastrointestinal Oncology

Article Title: The EZH2-NEAT1 epigenetic axis promotes cuproptosis sensitivity and modulates cancer cell migration in colorectal cancer

doi: 10.21037/jgo-2025-1-1058

Figure Lengend Snippet: EZH2 regulation of cuproptosis is independent of FDX1 transcription but involves the modulation of the lipoylation pathway and is correlated with NEAT1 expression. (A,B) Western blot of lipoylated DLAT (Lip- DLAT ), FDX1 , and LIAS in the control and EZH2 knockdown (A) or EZH2 -overexpressing (B) cells. (C,D) RT-qPCR of FDX1 mRNA in the control and EZH2 knockdown (C) or EZH2 -overexpressing (D) cells. (E,F) RT-qPCR of NEAT1 in the control and EZH2 knockdown (E) or EZH2 -overexpressing (F) cells. RT-qPCR of NEAT1 in the HCT116 and RKO cells treated with indicated concentrations of Elsm (G) or Elsm-Cu (H). β-actin served as the loading control. Data are presented as the mean ± standard deviation from three independent experiments. ns, not significant; *, P<0.05; **, P<0.01; ***, P<0.001. DLAT , dihydrolipoamide S-acetyltransferase; Elsm, elesclomol; Elsm-Cu, elesclomol with copper chloride; RT-qPCR, real-time quantitative polymerase chain reaction.

Article Snippet: The following primary antibodies were used: anti- EZH2 (Cell Signaling Technology, Danvers, MA, USA, 5246S), anti- HSP70 (Cell Signaling Technology, 4872S), anti- DLAT (Proteintech, Wuhan, China, 16179-1-AP), anti- FDX1 (Abcam, Cambridge, MA, USA, ab206649), anti-LIAS (Proteintech, 14812-1-AP), anti-lipoic acid (Calbiochem, San Diego, CA, USA, 437695), anti-β-actin (Sigma-Aldrich, A2228) and anti-HA tag (Cell Signaling Technology, 3724S).

Techniques: Expressing, Western Blot, Control, Knockdown, Quantitative RT-PCR, Standard Deviation, Real-time Polymerase Chain Reaction

Journal: Journal of Gastrointestinal Oncology

Article Title: The EZH2-NEAT1 epigenetic axis promotes cuproptosis sensitivity and modulates cancer cell migration in colorectal cancer

doi: 10.21037/jgo-2025-1-1058

Figure Lengend Snippet: NEAT1 mediates EZH2 -dependent cuproptosis sensitivity/promotion and functions as a pro-death factor. (A,B) RT-qPCR of NEAT1 expression in the HCT116 (A) and RKO (B) cells with indicated EZH2 and NEAT1 manipulations. (C) Colony formation in the cells with indicated EZH2 and NEAT1 manipulations treated with Elsm-Cu. Images were acquired using a flatbed scanner. The image shows a representative whole well of a 6-well plate. (D) Western blot of HSP70 in the cells with indicated EZH2 and NEAT1 manipulations. (E,F) DLAT distribution in soluble and insoluble fractions from the HCT116 (E) and RKO (F) cells with indicated manipulations. (G) Western blot of Lip- DLAT and FDX1 in the cells with indicated EZH2 and NEAT1 manipulations. β-actin served as the loading control. Data are presented as the mean ± standard deviation from three independent experiments. **, P<0.01; ***, P<0.001. DLAT , dihydrolipoamide S-acetyltransferase; Elsm-Cu, elesclomol with copper chloride; RT-qPCR, real-time quantitative polymerase chain reaction.

Article Snippet: The following primary antibodies were used: anti- EZH2 (Cell Signaling Technology, Danvers, MA, USA, 5246S), anti- HSP70 (Cell Signaling Technology, 4872S), anti- DLAT (Proteintech, Wuhan, China, 16179-1-AP), anti- FDX1 (Abcam, Cambridge, MA, USA, ab206649), anti-LIAS (Proteintech, 14812-1-AP), anti-lipoic acid (Calbiochem, San Diego, CA, USA, 437695), anti-β-actin (Sigma-Aldrich, A2228) and anti-HA tag (Cell Signaling Technology, 3724S).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Control, Standard Deviation, Real-time Polymerase Chain Reaction