Journal: Advanced Science

Article Title: Inhibition of CDK1 Overcomes Oxaliplatin Resistance by Regulating ACSL4‐mediated Ferroptosis in Colorectal Cancer

doi: 10.1002/advs.202301088

Figure Lengend Snippet: 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.

Article Snippet: Human recombinant ACSL4 (#TP305356; OriGene, Nanjing, China) and His‐CDK1 (#ab187447; Abcam, Cambridge, UK) were added to the binding buffer (150 × 10 −3 m NaCl, 100 × 10 −3 m NaF, 50 × 10 −3 m Tris‐HCl, pH 7.6, 0.5% NP‐40, 1 × 10 −3 m PMSF) and incubated with rotation at 4 °C for 2 h. Then, the Ni‐NTA agarose (#30 230; QIAGEN, Dusseldorf, Germany) was added and incubated at 4 °C for another 30 min, followed by washing and elution using buffer with increasing concentrations of imidazole.

Techniques: Co-Immunoprecipitation Assay, Pull Down Assay, Binding Assay, In Vitro, Transfection, Labeling, Western Blot, Expressing, Knock-Out, Mutagenesis, Plasmid Preparation, Negative Control

Journal: Advanced Science

Article Title: Inhibition of CDK1 Overcomes Oxaliplatin Resistance by Regulating ACSL4‐mediated Ferroptosis in Colorectal Cancer

doi: 10.1002/advs.202301088

Figure Lengend Snippet: 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.

Article Snippet: Human recombinant ACSL4 (#TP305356; OriGene, Nanjing, China) and His‐CDK1 (#ab187447; Abcam, Cambridge, UK) were added to the binding buffer (150 × 10 −3 m NaCl, 100 × 10 −3 m NaF, 50 × 10 −3 m Tris‐HCl, pH 7.6, 0.5% NP‐40, 1 × 10 −3 m PMSF) and incubated with rotation at 4 °C for 2 h. Then, the Ni‐NTA agarose (#30 230; QIAGEN, Dusseldorf, Germany) was added and incubated at 4 °C for another 30 min, followed by washing and elution using buffer with increasing concentrations of imidazole.

Techniques: Immunoprecipitation, Western Blot, Phospho-proteomics, Mass Spectrometry, In Vitro, Purification, Transfection, Immunohistochemical staining, Immunohistochemistry, CCK-8 Assay, Knock-Out, Negative Control

Journal: Advanced Science

Article Title: Inhibition of CDK1 Overcomes Oxaliplatin Resistance by Regulating ACSL4‐mediated Ferroptosis in Colorectal Cancer

doi: 10.1002/advs.202301088

Figure Lengend Snippet: 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.

Article Snippet: Human recombinant ACSL4 (#TP305356; OriGene, Nanjing, China) and His‐CDK1 (#ab187447; Abcam, Cambridge, UK) were added to the binding buffer (150 × 10 −3 m NaCl, 100 × 10 −3 m NaF, 50 × 10 −3 m Tris‐HCl, pH 7.6, 0.5% NP‐40, 1 × 10 −3 m PMSF) and incubated with rotation at 4 °C for 2 h. Then, the Ni‐NTA agarose (#30 230; QIAGEN, Dusseldorf, Germany) was added and incubated at 4 °C for another 30 min, followed by washing and elution using buffer with increasing concentrations of imidazole.

Techniques: Transfection, Western Blot, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Advanced Science

Article Title: Inhibition of CDK1 Overcomes Oxaliplatin Resistance by Regulating ACSL4‐mediated Ferroptosis in Colorectal Cancer

doi: 10.1002/advs.202301088

Figure Lengend Snippet: 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.

Article Snippet: Human recombinant ACSL4 (#TP305356; OriGene, Nanjing, China) and His‐CDK1 (#ab187447; Abcam, Cambridge, UK) were added to the binding buffer (150 × 10 −3 m NaCl, 100 × 10 −3 m NaF, 50 × 10 −3 m Tris‐HCl, pH 7.6, 0.5% NP‐40, 1 × 10 −3 m PMSF) and incubated with rotation at 4 °C for 2 h. Then, the Ni‐NTA agarose (#30 230; QIAGEN, Dusseldorf, Germany) was added and incubated at 4 °C for another 30 min, followed by washing and elution using buffer with increasing concentrations of imidazole.

Techniques: In Vivo, Generated, Injection, Flow Cytometry, Isolation, Immunohistochemical staining, Immunohistochemistry

Journal: Advanced Science

Article Title: Inhibition of CDK1 Overcomes Oxaliplatin Resistance by Regulating ACSL4‐mediated Ferroptosis in Colorectal Cancer

doi: 10.1002/advs.202301088

Figure Lengend Snippet: Summary diagram for the role of CDK1 in oxaliplatin resistance in colorectal cancer (CRC). m 6 A modification on CDK1 mRNA is decreased in oxaliplatin‐resistant CRC cells, resulting in increasing CDK1 mRNA stability. Then, CDK1 binds to and phosphorylates ACSL4, followed by UBR5‐mediated ACSL4 polyubiquitination and degradation, leading to blocking the biosynthesis of polyunsaturated fatty acid containing lipids, ultimately inhibiting lipid peroxidation and ferroptosis. Administration in combination with CDK1 inhibitor can effectively restore the sensitivity of CRC patients to oxaliplatin.

Article Snippet: Human recombinant ACSL4 (#TP305356; OriGene, Nanjing, China) and His‐CDK1 (#ab187447; Abcam, Cambridge, UK) were added to the binding buffer (150 × 10 −3 m NaCl, 100 × 10 −3 m NaF, 50 × 10 −3 m Tris‐HCl, pH 7.6, 0.5% NP‐40, 1 × 10 −3 m PMSF) and incubated with rotation at 4 °C for 2 h. Then, the Ni‐NTA agarose (#30 230; QIAGEN, Dusseldorf, Germany) was added and incubated at 4 °C for another 30 min, followed by washing and elution using buffer with increasing concentrations of imidazole.

Techniques: Modification, Blocking Assay

Journal: Scientific Reports

Article Title: Overcoming radioresistance of breast cancer cells with MAP4K4 inhibitors

doi: 10.1038/s41598-024-57000-6

Figure Lengend Snippet: MAP4K4 functioned as an upstream effector of ACSL4. (A) Immunofluorescence analysis of MAP4K4 and ACSL4 in SR and MR cells indicated that MAP4K4 co-localized with ACSL4 in radioresistant cells. (B) Expressions of ACSL4 in SR cells treated with PF06260933 or GNE-495. (C) Expressions of ACSL4 and MAP4K4 in SR cells treated with triacsin C (ACSL4 inhibitor). (D) Effect of triacsin C on MAP4K4 kinase activity. The protein expressions of MAP4K4 and ACSL4 following siRNA knockdown. MAP4K4 (E) and ACSL4 (F) protein levels are expressed as fold changes versus untreated cells (0 µM). Results are presented as means ± SDs, and * and ** indicate P values of < 0.05 and < 0.01, respectively.

Article Snippet: Primary antibodies for acyl-CoA synthetase-4 (ACSL4), RAD51, and β-actin, and siRNA for MAP4K4 (sc-39243), ACSL4 (sc-60619), and control (sc-37007) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Immunofluorescence, Activity Assay, Knockdown

Journal: Scientific Reports

Article Title: Overcoming radioresistance of breast cancer cells with MAP4K4 inhibitors

doi: 10.1038/s41598-024-57000-6

Figure Lengend Snippet: Schematic diagram of the suggested MAP4K4-ACSL4 signaling axis in radioresistant breast cancer cells. We suggest MAP4K4 positively regulates ACSL4 to mediate radioresistance by enhancing DNA damage response and inhibiting apoptosis.

Article Snippet: Primary antibodies for acyl-CoA synthetase-4 (ACSL4), RAD51, and β-actin, and siRNA for MAP4K4 (sc-39243), ACSL4 (sc-60619), and control (sc-37007) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques:

Journal: Aging (Albany NY)

Article Title: The positive feedback between ACSL4 expression and O-GlcNAcylation contributes to the growth and survival of hepatocellular carcinoma

doi: 10.18632/aging.103092

Figure Lengend Snippet: ACSL4 was overexpressed in HCC tissue samples and cells. ( A ) The Oncomine database was used to retrieve the different expression patterns of ACSL4 in HCC or normal liver. ( B , C ) The mRNA and protein content of ACSL4 in the human normal liver cell line QSG-7701 and the HCC cell lines Huh-7, HLE, SK-HEP-1, BEL-7402 and HCCLM3 were determined by RT-PCR and western blotting, respectively. ( D , E ) The mRNA and protein content of ACSL4 in HCC tissues and normal tissues were detected by RT-PCR and western blotting assays. ( F ) Immunohistochemistry was used to detect ACSL4 protein expression in HCC tissues and normal tissues (Scale bar = 100 μm). ( G ) Kaplan-Meier analysis of the relationship between ACSL4 expression and the overall survival of patients with HCC. ( * P<0.05, ** P<0.01).

Article Snippet: The overexpressing (OE) lentivirus vector of ACSL4 (OE-ACSL4) and its negative control (OE-NC) were purchased from Origene (no. RC221413L4) and were used to upregulate ACSL4 expression.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunohistochemistry

Journal: Aging (Albany NY)

Article Title: The positive feedback between ACSL4 expression and O-GlcNAcylation contributes to the growth and survival of hepatocellular carcinoma

doi: 10.18632/aging.103092

Figure Lengend Snippet: Association between ACSL4 expressions with the clinical process of patients with HCC.

Article Snippet: The overexpressing (OE) lentivirus vector of ACSL4 (OE-ACSL4) and its negative control (OE-NC) were purchased from Origene (no. RC221413L4) and were used to upregulate ACSL4 expression.

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: The positive feedback between ACSL4 expression and O-GlcNAcylation contributes to the growth and survival of hepatocellular carcinoma

doi: 10.18632/aging.103092

Figure Lengend Snippet: ACSL4 promoted HCC cell proliferation and repressed cell apoptosis via activating mTOR signalling. Huh-7 and SK-HEP-1 cells were transfected with si-NC, si-ACSL4, OE-NC or OE-ACSL4, with or without rapamycin treatment, and then the following assays were carried out. ( A , B ) RT-PCR and western blotting assays were carried out to assess the expression levels of ACSL4 at the mRNA and protein levels, respectively ( * P<0.05, ** P<0.01, si-ACSL4 group compared with si-NC group; # P<0.05, ## P<0.01, OE-ACSL4 group compared with OE-NC group). ( C , D ) The expression levels of mTOR and p-mTOR were detected by using a western blotting assay. ( E , F ) CCK-8 assay was used to detect cell proliferation ( * P<0.05, compared with the control group; # P<0.05, compared with the OE-ACSL4 group). ( G ) Flow cytometry assay was used to test cell apoptosis ( * P<0.05, compared with the control group; # P<0.05, compared with the OE-ACSL4 group).

Article Snippet: The overexpressing (OE) lentivirus vector of ACSL4 (OE-ACSL4) and its negative control (OE-NC) were purchased from Origene (no. RC221413L4) and were used to upregulate ACSL4 expression.

Techniques: Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, CCK-8 Assay, Flow Cytometry

Journal: Aging (Albany NY)

Article Title: The positive feedback between ACSL4 expression and O-GlcNAcylation contributes to the growth and survival of hepatocellular carcinoma

doi: 10.18632/aging.103092

Figure Lengend Snippet: Evaluation of the effects of ACSL4 on O-GlcNAc-mediated HCC growth. Huh-7 and SK-HEP-1 cells were transfected with OE-ACSL4 or si-ACSL4, and then the cells were harvested for the western blotting assay to detect the expression of the following proteins. ( A ) OGT and O-GlcNAc in Huh-7 cells. ( B ) UAP1, GLUT1, GLUT2, GUCY1A3, CANT1 and SGLT1 in Huh-7 cells. ( C , D ) OGT, O-GlcNAc, UAP1, GLUT1, GLUT2, GUCY1A3, CANT1 and SGLT1 in SK-HEP-1 cells. ( E ) IP assay used to detect the interaction between O-GlcNAc and ACSL4 with an antibody against O-GlcNAc or ACSL4. IgG served as a negative control. Then, the si-ACSL4-transfected or untransfected Huh-7 and SK-HEP-1 cells were treated with PUGNAc, GlcNAc or nothing, and the following assays were carried out. ( F , G ) The levels of ACSL4, O-GlcNAc, mTOR and p-mTOR were determined by using a western blotting assay. ( H , I ) The protein stability was determined by western blotting after incubation with CHX (100 μg/ml) for 0, 1, 2, 4, 8 or 24 hours. ( J , K ) Cell proliferation was detected by CCK-8 assay. ( L ) Cell apoptosis was assessed by flow cytometry assay. ( A – D ) * P<0.05, si-ACSL4/OE-ACSL4 group compared with control group; ( E – L ) * P<0.05, PUGNAc/GlcNAc group compared with control group; # P<0.05, PUGNAc + si-ACSL4/GlcNAc + si-ACSL4 group compared with PUGNAc/GlcNAc group).

Article Snippet: The overexpressing (OE) lentivirus vector of ACSL4 (OE-ACSL4) and its negative control (OE-NC) were purchased from Origene (no. RC221413L4) and were used to upregulate ACSL4 expression.

Techniques: Transfection, Western Blot, Expressing, Negative Control, Incubation, CCK-8 Assay, Flow Cytometry

Journal: Aging (Albany NY)

Article Title: The positive feedback between ACSL4 expression and O-GlcNAcylation contributes to the growth and survival of hepatocellular carcinoma

doi: 10.18632/aging.103092

Figure Lengend Snippet: ACSL4 upregulation enhanced the stability of GLUT1 protein and reduced its ubiquitination. ( A ) After 12 hours of cell transfection with si-ACSL4 or OE-ACSL4, Huh-7 cells were treated with CHX (100 μg/ml) for 0, 1, 2, 4, 8 or 24 hours, and the western blotting assay was performed to detect GLUT1 expression. ( B ) An IP assay was used to detect the interaction between Ub and GLUT1 proteins after Huh-7 cells were transfected with si-ACSL4 or OE-ACSL4. ( C ) After 12 hours of cell transfection with si-ACSL4 or OE-ACSL4, SK-HEP-1 cells were treated with CHX (100 μg/ml) for 0, 1, 2, 4, 8 or 24 hours, and the western blotting assay was performed to detect GLUT1 expression. ( D ) IP assay was used to detect the interaction between Ub and GLUT1 protein in SK-HEP-1 cells. ( * P<0.05, si-ACSL4/OE-ACSL4 group compared with control group).

Article Snippet: The overexpressing (OE) lentivirus vector of ACSL4 (OE-ACSL4) and its negative control (OE-NC) were purchased from Origene (no. RC221413L4) and were used to upregulate ACSL4 expression.

Techniques: Transfection, Western Blot, Expressing

Journal: Aging (Albany NY)

Article Title: The positive feedback between ACSL4 expression and O-GlcNAcylation contributes to the growth and survival of hepatocellular carcinoma

doi: 10.18632/aging.103092

Figure Lengend Snippet: Evaluation of the effects of the ACSL4/GLUT1 axis on cell proliferation, apoptosis and tumorigenesis in Huh-7 and SK-HEP-1 cells. ( A , B ) The mRNA and protein expression levels of GLUT1 were determined by RT-PCR and western blotting assays after cells were transfected with sh-GLUT1 or sh-NC, respectively ( * P<0.05, ** P<0.01, compared with the sh-NC group). Next, Huh-7 and SK-HEP-1 cells were transfected with OE-ACSL4 and/or sh-GLUT1 and subjected to the following assays. ( C – E ) Western blotting assays were used to assess the levels of O-GlcNAc, ACSL4 and GLUT1. ( F , G ) CCK-8 assay was carried out to test cell proliferation. ( H ) Flow cytometry assay was used to determine cell apoptosis. ( I ) An in vivo xenotransplantation assay was used to assess the effects of the ACSL4/GLUT1 axis on the tumour formation ability of Huh-7 and SK-HEP-1 cells. ( C – I : * P<0.05, compared with control group; # P<0.05, compared with OE-ACSL4 group).

Article Snippet: The overexpressing (OE) lentivirus vector of ACSL4 (OE-ACSL4) and its negative control (OE-NC) were purchased from Origene (no. RC221413L4) and were used to upregulate ACSL4 expression.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, CCK-8 Assay, Flow Cytometry, In Vivo

Journal: Aging (Albany NY)

Article Title: The positive feedback between ACSL4 expression and O-GlcNAcylation contributes to the growth and survival of hepatocellular carcinoma

doi: 10.18632/aging.103092

Figure Lengend Snippet: Evaluation of the levels of GLUT1 and O-GlcNAc in HCC and their clinical significance. ( A ) Immunohistochemistry technology was used to assess the protein levels of ACSL4, O-GlcNAc and GLUT1 in HCC tissues and adjacent normal tissues (Scale bar = 100 μm). ( B – D ) Pearson correlation analysis of the correlations between the levels of ACSL4, O-GlcNAc and GLUT1 in HCC tissues. ( E , F ) Kaplan-Meier analysis of the relationship between GLUT1/O-GlcNAc levels and the overall survival of patients with HCC.

Article Snippet: The overexpressing (OE) lentivirus vector of ACSL4 (OE-ACSL4) and its negative control (OE-NC) were purchased from Origene (no. RC221413L4) and were used to upregulate ACSL4 expression.

Techniques: Immunohistochemistry