Fig 2: CDK1 reduces ACSL4 protein stability. A) Co‐immunoprecipitation (Co‐IP) assay testing the endogenous interaction between CDK1 and ACSL4. B) His pull‐down assay testing the direct binding of CDK1 to ACSL4 in vitro. C) HCT8‐OR cells transfected with the labeled plasmids for 48 h, followed by IP coupled with immunoblotting (IB) assays using the indicated antibodies. D) IB assay testing the expression of CDK1 and ACSL4 proteins in CDK1 knockout HCT8‐OR cells. E,F) IB assay testing ACSL4 expression in CDK1 knockout HCT8‐OR cells treated with 100 µg mL−1 cycloheximide for the indicated time. G) IB assay testing ACSL4 expression in CDK1‐overexpressing HCT8 cells treated with 5 × 10−6 m MG132 or 10 × 10−6 m chloroquine for 12 h. H) IP assay using anti‐ACSL4 antibody, followed by IB assay with the indicated antibodies in HCT8‐OR cells treated with 20 × 10−6 m MG132 for 6 h. I–K) HEK293T cells were transfected with the indicated vectors for 48 h, followed by IP coupled with IB assays using the indicated antibodies. L,M) IB assays detecting the indicated protein levels in CDK1 knockout HCT8‐OR cells transfected with the indicated vectors or siRNAs for 48 h. N) IP assay using anti‐UBR5 antibody, followed by IB assay with the indicated antibodies in HCT8‐OR cells. O) IB assay detecting the indicated protein levels in CDK1 knockout HCT8‐OR cells transfected with wild‐type or mutant UBR5 plasmid for 48 h. P) HCT8‐OR cells were transfected with the indicated vectors for 48 h, followed by IP coupled with IB assays using the indicated antibodies. The data are shown as the mean ± SD (n = 3). *** p < 0.001, by two‐way ANOVA followed by Tukey's post hoc test (F). NC, negative control; OE, overexpressed; WT, wild‐type; CQ, chloroquine.

Fig 3: ACSL4 is phosphorylated by CDK1 at S447. A) Immunoprecipitation (IP) coupled with immunoblotting (IB) assays using the indicated antibodies testing the effect of CDK1 on ACSL4 phosphorylation. B) Mass spectrometry identifying the phosphorylation of ACSL4 S447. C) Evaluation of the conservation of ACSL4 S447 among different species. D) In vitro phosphorylation assay using the purified proteins, followed by IB assay using the indicated antibodies. E,F) IB assay using the indicated antibodies in HCT8‐OR cells transfected with the indicated vectors for 48 h. G,H) IP coupled with IB assays using the indicated antibodies in HCT8‐OR cells transfected with the indicated vectors for 48 h. I) Immunohistochemical (IHC) staining of CDK1, ACSL4, and p‐ACSL4‐S447 in paraffin‐embedded colorectal cancer (CRC) tissues. Scale bars, 50 µm. J) CCK‐8 assay testing cell viability in CDK1 knockout cells transfected with the indicated vectors after 1 × 10−6 m oxaliplatin treatment for 72 h. The data are shown as the mean ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, by one‐way ANOVA followed by Tukey's post hoc test (J). NC, negative control; OE, overexpressed; WT, wild‐type.

Fig 4: CDK1 represses ACSL4‐mediated ferroptosis. A–D) The indicated vectors or siRNAs were transfected into HCT8‐OR cells treated with 1 × 10−6 m erastin, 100 × 10−9 m RSL3, 2 × 10−6 m RO‐3306, 5 × 10−6 m ferrostatin‐1, 5 × 10−6 m deferoxamine, 5 × 10−3 m N‐acetyl‐cysteine, 10 × 10−6 m Z‐VAD‐FMK or 2 × 10−6 m necrostatin‐1 for 20 h, followed by detection of cell death and lipid peroxidation. E,F) The indicated vectors or siRNAs were transfected into HCT8 cells treated with 2 × 10−6 m erastin, 200 × 10−9 m RSL3 for 20 h, followed by detection of cell death and lipid peroxidation. G,H) Immunoblotting (IB) assay analyzing the indicated protein levels in HCT8 and HT29 cells transfected with the indicated vectors or siRNAs and treated with 2 × 10−6 m erastin or 200 × 10−9 m RSL3 for 20 h. I) Quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) analysis of PTGS2 mRNA levels in HCT8 cells transfected with the indicated vectors or siRNAs and treated with 2 × 10−6 m erastin or 200 × 10−9 m RSL3 for 20 h. J,K) Mass spectrometric analysis of the signal intensities of PE 18:0_20:4 and PE 18:0_22:4 in HCT8 cells transfected with the indicated vectors or siRNAs and treated with 2 × 10−6 m erastin or 200 × 10−9 m RSL3 for 20 h. The data are shown as the mean ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, by one‐way ANOVA followed by Tukey's post hoc test (A–F, I–K). OE, overexpressed; WT, wild‐type. RO, RO‐3306; Fer, ferrostatin‐1; DFO, deferoxamine; NAC, N‐acetyl‐cysteine; ZVF, Z‐VAD‐FMK; Nec, necrostatin‐1.

Fig 5: CDK1 inhibitor restores the sensitivity of oxaliplatin in vivo. A–C) The image, volume, and weight of tumor generated by HCT8‐OR injection in the indicated groups (n = 5). D) Flow cytometry detecting lipid peroxidation levels of HCT8‐OR tumor cells isolated from mice in the indicated groups. E–I) Immunohistochemical (IHC) staining of CDK1, ACSL4, p‐ACSL4‐S447, Ki‐67 and 4‐HNE in tumor tissues generated by HCT8‐OR injection in the indicated groups. Scale bars, 50 µm. J–L) The image, volume, and weight of tumor generated by xenotransplantation of clinical colorectal cancer (CRC) tissues (PDX#1) in the indicated four groups. M) Flow cytometry detecting lipid peroxidation levels of tumor cells isolated from mice in PDX#1 models (n = 5). N–R) IHC staining of CDK1, ACSL4, p‐ACSL4‐S447, Ki‐67 and 4‐HNE in tumor tissues generated by xenotransplantation of clinical CRC tissues (PDX#1) in the indicated groups. Scale bars, 50 µm. *** p < 0.001, by one‐way ANOVA (C,D,F–I,L,M,O–R) or two‐way ANOVA (B,K) followed by Tukey's post hoc test. Oxa, oxaliplatin; Lip‐1, liproxstatin‐1.