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ALOX12 is essential for <t>p53-mediated</t> ferroptosis in CC. (A) Western blotting and qRT-PCR analysis of HCT116 and DLD-1 cells incubated with Nutlin-3 (0, 10, 15, 20, 30, 40 µM) for 24 h. Mean ± SD, n=3 independent experiments. (B) Representative phase-contrast images of HCT116 and DLD-1 cells. HCT116 cells were incubated with Nutlin-3 (10 µM), TBH (200 µM), ML355 (2 µM) or Fer-1 (2 µM) for 24 h. DLD-1 cells were incubated with Nutlin-3 (10 µM), TBH (100 µM), ML355 (2 µM) or Fer-1 (2 µM) for 24 h. Scale bars, 100 µm. N=3 independent experiments. (C) Cell death analysis of HCT116 and DLD-1 cells. HCT116 cells incubated with Nutlin-3 (10 µM), TBH (200 µM), ML355 (2 µM), Fer-1 (2 µM), 3-MA (2 mM), Z-VAD (10 µM), or Nec-1 (10 µM) for 24 h, while DLD-1 cells were incubated with Nutlin-3 (10 µM), TBH (100 µM), ML355 (2 µM), Fer-1 (2 µM), 3-MA (2 mM), Z-VAD (10 µM), or Nec-1 (10 µM) for 24 h. Mean ± SD, n=3 independent experiments. (D-F) ROS (D), MDA (E) and Fe 2+ (F) detection of HCT116 and DLD-1 cells. HCT116 cells were incubated with Nutlin-3 (10 µM), TBH (200 µM), ML355 (2 µM) or Fer-1 (2 µM) for 24 h, while DLD-1 cells were incubated with Nutlin-3 (10 µM), TBH (100 µM), ML355 (2 µM) or Fer-1 (2 µM) for 24 h. Mean ± SD, n=3 independent experiments. ** P<0.01; ****, P<0.0001; ns, not significant. CC, colon cancer; MDA, malondialdehyde; qRT-PCR, quantitative reverse transcription polymerase chain reaction; ROS, reactive oxygen species; SD, standard deviation; TBH, tert-Butyl hydroperoxide.
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ALOX12 is essential for <t>p53-mediated</t> ferroptosis in CC. (A) Western blotting and qRT-PCR analysis of HCT116 and DLD-1 cells incubated with Nutlin-3 (0, 10, 15, 20, 30, 40 µM) for 24 h. Mean ± SD, n=3 independent experiments. (B) Representative phase-contrast images of HCT116 and DLD-1 cells. HCT116 cells were incubated with Nutlin-3 (10 µM), TBH (200 µM), ML355 (2 µM) or Fer-1 (2 µM) for 24 h. DLD-1 cells were incubated with Nutlin-3 (10 µM), TBH (100 µM), ML355 (2 µM) or Fer-1 (2 µM) for 24 h. Scale bars, 100 µm. N=3 independent experiments. (C) Cell death analysis of HCT116 and DLD-1 cells. HCT116 cells incubated with Nutlin-3 (10 µM), TBH (200 µM), ML355 (2 µM), Fer-1 (2 µM), 3-MA (2 mM), Z-VAD (10 µM), or Nec-1 (10 µM) for 24 h, while DLD-1 cells were incubated with Nutlin-3 (10 µM), TBH (100 µM), ML355 (2 µM), Fer-1 (2 µM), 3-MA (2 mM), Z-VAD (10 µM), or Nec-1 (10 µM) for 24 h. Mean ± SD, n=3 independent experiments. (D-F) ROS (D), MDA (E) and Fe 2+ (F) detection of HCT116 and DLD-1 cells. HCT116 cells were incubated with Nutlin-3 (10 µM), TBH (200 µM), ML355 (2 µM) or Fer-1 (2 µM) for 24 h, while DLD-1 cells were incubated with Nutlin-3 (10 µM), TBH (100 µM), ML355 (2 µM) or Fer-1 (2 µM) for 24 h. Mean ± SD, n=3 independent experiments. ** P<0.01; ****, P<0.0001; ns, not significant. CC, colon cancer; MDA, malondialdehyde; qRT-PCR, quantitative reverse transcription polymerase chain reaction; ROS, reactive oxygen species; SD, standard deviation; TBH, tert-Butyl hydroperoxide.
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ALOX12 is essential for p53-mediated ferroptosis in CC. (A) Western blotting and qRT-PCR analysis of HCT116 and DLD-1 cells incubated with Nutlin-3 (0, 10, 15, 20, 30, 40 µM) for 24 h. Mean ± SD, n=3 independent experiments. (B) Representative phase-contrast images of HCT116 and DLD-1 cells. HCT116 cells were incubated with Nutlin-3 (10 µM), TBH (200 µM), ML355 (2 µM) or Fer-1 (2 µM) for 24 h. DLD-1 cells were incubated with Nutlin-3 (10 µM), TBH (100 µM), ML355 (2 µM) or Fer-1 (2 µM) for 24 h. Scale bars, 100 µm. N=3 independent experiments. (C) Cell death analysis of HCT116 and DLD-1 cells. HCT116 cells incubated with Nutlin-3 (10 µM), TBH (200 µM), ML355 (2 µM), Fer-1 (2 µM), 3-MA (2 mM), Z-VAD (10 µM), or Nec-1 (10 µM) for 24 h, while DLD-1 cells were incubated with Nutlin-3 (10 µM), TBH (100 µM), ML355 (2 µM), Fer-1 (2 µM), 3-MA (2 mM), Z-VAD (10 µM), or Nec-1 (10 µM) for 24 h. Mean ± SD, n=3 independent experiments. (D-F) ROS (D), MDA (E) and Fe 2+ (F) detection of HCT116 and DLD-1 cells. HCT116 cells were incubated with Nutlin-3 (10 µM), TBH (200 µM), ML355 (2 µM) or Fer-1 (2 µM) for 24 h, while DLD-1 cells were incubated with Nutlin-3 (10 µM), TBH (100 µM), ML355 (2 µM) or Fer-1 (2 µM) for 24 h. Mean ± SD, n=3 independent experiments. ** P<0.01; ****, P<0.0001; ns, not significant. CC, colon cancer; MDA, malondialdehyde; qRT-PCR, quantitative reverse transcription polymerase chain reaction; ROS, reactive oxygen species; SD, standard deviation; TBH, tert-Butyl hydroperoxide.

Journal: Translational Cancer Research

Article Title: ALOX12 suppresses colon cancer progression by promoting p53-mediated ferroptosis through upregulating ROS-induced stress

doi: 10.21037/tcr-2026-0503

Figure Lengend Snippet: ALOX12 is essential for p53-mediated ferroptosis in CC. (A) Western blotting and qRT-PCR analysis of HCT116 and DLD-1 cells incubated with Nutlin-3 (0, 10, 15, 20, 30, 40 µM) for 24 h. Mean ± SD, n=3 independent experiments. (B) Representative phase-contrast images of HCT116 and DLD-1 cells. HCT116 cells were incubated with Nutlin-3 (10 µM), TBH (200 µM), ML355 (2 µM) or Fer-1 (2 µM) for 24 h. DLD-1 cells were incubated with Nutlin-3 (10 µM), TBH (100 µM), ML355 (2 µM) or Fer-1 (2 µM) for 24 h. Scale bars, 100 µm. N=3 independent experiments. (C) Cell death analysis of HCT116 and DLD-1 cells. HCT116 cells incubated with Nutlin-3 (10 µM), TBH (200 µM), ML355 (2 µM), Fer-1 (2 µM), 3-MA (2 mM), Z-VAD (10 µM), or Nec-1 (10 µM) for 24 h, while DLD-1 cells were incubated with Nutlin-3 (10 µM), TBH (100 µM), ML355 (2 µM), Fer-1 (2 µM), 3-MA (2 mM), Z-VAD (10 µM), or Nec-1 (10 µM) for 24 h. Mean ± SD, n=3 independent experiments. (D-F) ROS (D), MDA (E) and Fe 2+ (F) detection of HCT116 and DLD-1 cells. HCT116 cells were incubated with Nutlin-3 (10 µM), TBH (200 µM), ML355 (2 µM) or Fer-1 (2 µM) for 24 h, while DLD-1 cells were incubated with Nutlin-3 (10 µM), TBH (100 µM), ML355 (2 µM) or Fer-1 (2 µM) for 24 h. Mean ± SD, n=3 independent experiments. ** P<0.01; ****, P<0.0001; ns, not significant. CC, colon cancer; MDA, malondialdehyde; qRT-PCR, quantitative reverse transcription polymerase chain reaction; ROS, reactive oxygen species; SD, standard deviation; TBH, tert-Butyl hydroperoxide.

Article Snippet: Protein extracts were analyzed by western blotting according to standard protocols, using primary antibodies against p53 (A0263, Abclonal, Wuhan, China, 1:1,000), SLC7A11 (CY7046, Abways, Shanghai, China, 1:1,000), ALOX12 (CY8070, Abways, 1:1,000), GAPDH (AB0037, Abways, 1:5,000), and GPX4 (A11243, Abclonal, 1:1,000), followed by an HRP-conjugated goat anti-rabbit secondary antibody (AS014, Abclonal, 1:5,000).

Techniques: Western Blot, Quantitative RT-PCR, Incubation, Reverse Transcription, Polymerase Chain Reaction, Standard Deviation

Mechanisms into the regulation of ALOX12 in p53-mediated ferroptosis. (A) Western blotting analysis of HCT116, DLD-1 cells transfected with ALOX12 lentivirus or si-ALOX12 for 24 h, the expression of ALOX12, SLC7A11 and p53 was detected. Mean ± SD, n=3 independent experiments. (B) Western blotting analysis of HCT116 and DLD-1 cells transfected with si-p53 for 24 h, the expression of ALOX12, SLC7A11 and p53 was detected. Mean ± SD, n=3 independent experiments. (C) Western blotting analysis of HCT116 and DLD-1 cells treated with Nutlin-3 (10 µM) or co-transfected with si-p53 for 24 h, the expression of ALOX12, SLC7A11 and p53 was detected. Mean ± SD, n=3 independent experiments. (D) Cell death analysis of HCT116 and DLD-1 cells. HCT116 cells were incubated with erastin (40 µM) or co-treated with Fer-1 (2 µM) for 24 h, while DLD-1 cells were treated with erastin (60 µM) or co-treated with Fer-1 (2 µM) for 24 h. Mean ± SD, n=3 independent experiments. (E-G) ROS (E), MDA (F) and Fe 2+ (G) detection of HCT116 and DLD-1 cells. HCT116 cells were incubated with erastin (40 µM) or co-treated with Fer-1 (2 µM) for 24 h, while DLD-1 cells were treated with erastin (60 µM) or co-treated with Fer-1 (2 µM) for 24 h. Mean ± SD, n=3 independent experiments. (H) Cell death analysis of HCT116 and DLD-1 cells when considering ALOX12 status. HCT116 cells were incubated with erastin (40 µM) or co-treated with Fer-1 (2 µM) for 24 h, while DLD-1 cells were treated with erastin (60 µM) or co-treated with Fer-1 (2 µM) for 24 h. Mean ± SD, n=3 independent experiments. (I) Co-IP assay of HCT116 and DLD-1 cells using SLC7A11 antibody (see methods). N=3 independent experiments. (J) Co-IP assay of HCT116 and DLD-1 cells using anti-Flag antibody (see Methods). N=3 independent experiments. (K) ALOX12 enzyme activity analysis of HCT116 and DLD-1 cells by ELISA (see Methods), cells were pre-treated with ML355 (2 µM), Nutlin-3 (10 µM) or co-transfected with si-p53 for 24 h. Mean ± SD, n=3 independent experiments. (L,M) MDA (L) and Fe 2+ (M) detection of HCT and DLD-1 cells. HCT116 cells were incubated with Nutlin-3 (10 µM), TBH (200 µM), Lip-1 (2 µM) or transfected with si-p53 for 24 h, while DLD-1 cells were treated with Nutlin-3 (10 µM), TBH (100 µM), Lip-1 (2 µM) or transfected with si-p53 for 24 h. Mean ± SD, n=3 independent experiments. *, P<0.05; ** P<0.01; *** P<0.001; ****, P<0.0001; ns, not significant. Co-IP, co-immunoprecipitation; ELISA, enzyme-linked immunosorbent assay; MDA, malondialdehyde; ROS, reactive oxygen species; SD, standard deviation; TBH, tert-Butyl hydroperoxide.

Journal: Translational Cancer Research

Article Title: ALOX12 suppresses colon cancer progression by promoting p53-mediated ferroptosis through upregulating ROS-induced stress

doi: 10.21037/tcr-2026-0503

Figure Lengend Snippet: Mechanisms into the regulation of ALOX12 in p53-mediated ferroptosis. (A) Western blotting analysis of HCT116, DLD-1 cells transfected with ALOX12 lentivirus or si-ALOX12 for 24 h, the expression of ALOX12, SLC7A11 and p53 was detected. Mean ± SD, n=3 independent experiments. (B) Western blotting analysis of HCT116 and DLD-1 cells transfected with si-p53 for 24 h, the expression of ALOX12, SLC7A11 and p53 was detected. Mean ± SD, n=3 independent experiments. (C) Western blotting analysis of HCT116 and DLD-1 cells treated with Nutlin-3 (10 µM) or co-transfected with si-p53 for 24 h, the expression of ALOX12, SLC7A11 and p53 was detected. Mean ± SD, n=3 independent experiments. (D) Cell death analysis of HCT116 and DLD-1 cells. HCT116 cells were incubated with erastin (40 µM) or co-treated with Fer-1 (2 µM) for 24 h, while DLD-1 cells were treated with erastin (60 µM) or co-treated with Fer-1 (2 µM) for 24 h. Mean ± SD, n=3 independent experiments. (E-G) ROS (E), MDA (F) and Fe 2+ (G) detection of HCT116 and DLD-1 cells. HCT116 cells were incubated with erastin (40 µM) or co-treated with Fer-1 (2 µM) for 24 h, while DLD-1 cells were treated with erastin (60 µM) or co-treated with Fer-1 (2 µM) for 24 h. Mean ± SD, n=3 independent experiments. (H) Cell death analysis of HCT116 and DLD-1 cells when considering ALOX12 status. HCT116 cells were incubated with erastin (40 µM) or co-treated with Fer-1 (2 µM) for 24 h, while DLD-1 cells were treated with erastin (60 µM) or co-treated with Fer-1 (2 µM) for 24 h. Mean ± SD, n=3 independent experiments. (I) Co-IP assay of HCT116 and DLD-1 cells using SLC7A11 antibody (see methods). N=3 independent experiments. (J) Co-IP assay of HCT116 and DLD-1 cells using anti-Flag antibody (see Methods). N=3 independent experiments. (K) ALOX12 enzyme activity analysis of HCT116 and DLD-1 cells by ELISA (see Methods), cells were pre-treated with ML355 (2 µM), Nutlin-3 (10 µM) or co-transfected with si-p53 for 24 h. Mean ± SD, n=3 independent experiments. (L,M) MDA (L) and Fe 2+ (M) detection of HCT and DLD-1 cells. HCT116 cells were incubated with Nutlin-3 (10 µM), TBH (200 µM), Lip-1 (2 µM) or transfected with si-p53 for 24 h, while DLD-1 cells were treated with Nutlin-3 (10 µM), TBH (100 µM), Lip-1 (2 µM) or transfected with si-p53 for 24 h. Mean ± SD, n=3 independent experiments. *, P<0.05; ** P<0.01; *** P<0.001; ****, P<0.0001; ns, not significant. Co-IP, co-immunoprecipitation; ELISA, enzyme-linked immunosorbent assay; MDA, malondialdehyde; ROS, reactive oxygen species; SD, standard deviation; TBH, tert-Butyl hydroperoxide.

Article Snippet: Protein extracts were analyzed by western blotting according to standard protocols, using primary antibodies against p53 (A0263, Abclonal, Wuhan, China, 1:1,000), SLC7A11 (CY7046, Abways, Shanghai, China, 1:1,000), ALOX12 (CY8070, Abways, 1:1,000), GAPDH (AB0037, Abways, 1:5,000), and GPX4 (A11243, Abclonal, 1:1,000), followed by an HRP-conjugated goat anti-rabbit secondary antibody (AS014, Abclonal, 1:5,000).

Techniques: Western Blot, Transfection, Expressing, Incubation, Co-Immunoprecipitation Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, Standard Deviation