Journal: Redox Biology

Article Title: p53 and fatty acids collaborate to trigger ferroptosis via the FBXO2-FABP5 axis in colorectal cancer

doi: 10.1016/j.redox.2026.104043

Figure Lengend Snippet: FBXO2 is a transcriptional target of p53. Data are represented as mean ± standard deviation (SD), n = 3. ∗ p < 0.05, ∗∗∗ p < 0.001, ns, not significant. See also . (A and B) qPCR analysis of the expression of FBXO2 mRNA in HCT116 p53+/+ (A) and RKO (B) cells treated with or without 5-fluorouracil (5-FU, 40 μM), Etoposide (20 μM), Nutlin-3 (20 μM), or Alrizomadlin (APG-115, 2 μM) for 24 h (C and D) IB analysis of FBXO2 protein expression in HCT116 p53+/+ (C) and RKO (D) cells treated with or without 5-FU, Etoposide, Nutlin-3, or Actinomycin D (Act.D) for 24 h (E and F) qPCR (E) and IB (F) analyses of FBXO2 expression in HCT116 p53−/− cells transfected with the indicated plasmids. (G – J) qPCR (G and I) and IB (H and J) analyses of FBXO2 expression in HCT116 p53+/+ and RKO cells treated with the siRNAs and agents as indicated. (K and L) IB analysis of FBXO2 protein expression in HCT116 p53−/− (K) and HT-29 (with p53-R273H) (L) cells treated with or without the indicated agents. (M) The schematic of the potential p53-responsive element (p53-RE) on the FBXO2 promoter. (N) The luciferase reporter assay verifies the activation of the FBXO2 promoter by p53. (O) ChIP-qPCR analysis confirms the association of p53 with p53-RE on the promoter.

Article Snippet: Nutlin-3, Cisplatin, 5-fluorouracil (5-FU), actinomycin D (Act.D), Etoposide, Alrizomadlin (APG-115), Olaparib, MG132, Liproxstatin-1 (Lipro-1), Ferrostatin-1 (Ferro-1), RSL3, Z-VAD-FMK, Chloroquine (CQ), and Oxaliplatin (Oxa) were purchased from MedChemExpress (Shanghai, China), Erastin and Arachidonic acid (AA) were purchased from Selleck (Shanghai, China).

Techniques: Standard Deviation, Expressing, Transfection, Luciferase, Reporter Assay, Activation Assay, ChIP-qPCR

Journal: Cancer Communications

Article Title: TIMELESS Promotes LUAD Growth via Suppressing Transferrin-Mediated Ferroptosis and Reprograms the Tumor Microenvironment against Anti-PD-1 Immunotherapy

doi: 10.34133/cancomm.0009

Figure Lengend Snippet: Dysregulated RNA-binding proteins in LUAD: from multicohort screening to functional validation. (A to D) Volcano plots showing the DE RBPs in the following 4 LUAD cohorts: The Cancer Genome Atlas Lung Adenocarcinoma (TCGA-LUAD) dataset ( n = 568; A), GSE32863 ( n = 116; B), GSE40419 ( n = 164; C), and GSE75037 ( n = 166; D). The significantly DE RBPs ( P adj < 0.05, |log 2 FC| > 1) are shown in blue, and the nonsignificantly DE RBPs ( P adj > 0.05, |log 2 FC| > 1) are shown in dark gray. The other non-RBP genes are shown in light gray. (E and F) Venn diagram showing the intersection of up-regulated (E) and down-regulated (F) DE RBPs across TCGA-LUAD and the 3 GEO datasets. (G) The volcano plot displayed the hazard ratios for patients stratified by the median expression level of RBP genes in the TCGA-LUAD cohort (red: up-regulated RBPs in tumors; blue: down-regulated RBPs in tumors). (H) Heatmap of significantly dysregulated RBPs identified in (E) and (F). The top 10 highest-expressed RBPs in tumors are highlighted (ranked by average tumor z score). (I) Expression levels of the top 10 LUAD-up-regulated RBPs in LUAD cell lines (A549 and H1975) with or without siRNA-mediated knockdown ( n = 4 each group). (J) Calcein-AM/PI staining showed the percentage of viable cells in the negative control group and following the knockdown of the RBP gene ( n = 4 each group). (K) CCK-8 assay of cell viability in RBP gene knockdown and control cells at the indicated time points. The statistical analysis was performed using a 2-tailed Student’s t test (I and J) or 2-way ANOVA (K). *P < 0.05, **P < 0.01, ***P < 0.001, data without statistically significant differences are not labeled (J and K). Abbreviations: ALDH18A1, aldehyde dehydrogenase 18 family member A1; BZW2, basic leucine zipper and W2 domains 2; CCK-8, cell counting kit-8; CENPF, centromere protein F; DE, differentially expressed; FAM83A, family with sequence similarity 83 member A; FC, fold change; FDR, false discovery rate; GEO, Gene Expression Omnibus; IF, immunofluorescence; IHC, immunohistochemistry; KIF20A, kinesin family member 20A; LUAD, lung adenocarcinoma; OS, overall survival; PDIA4, protein disulfide isomerase family A member 4; PRC1, protein regulator of cytokinesis 1; RBP, RNA-binding protein; SRPK1, SRSF protein kinase 1; TCGA, The Cancer Genome Atlas; TMA, tissue microarray; TOP2A, DNA topoisomerase II alpha.

Article Snippet: To assess the RNA stabilities of TF and Ccr4-Not transcription complex subunit 3 (CNOT3), H1975 and A549 cells were treated with 2.5 μmol/l actinomycin D (MedChemExpress, Cat. HY-17559) to inhibit transcription.

Techniques: RNA Binding Assay, Functional Assay, Biomarker Discovery, Expressing, Knockdown, Staining, Negative Control, CCK-8 Assay, Control, Labeling, Cell Counting, Sequencing, Gene Expression, Immunofluorescence, Immunohistochemistry, Microarray

Journal: Cancer Communications

Article Title: TIMELESS Promotes LUAD Growth via Suppressing Transferrin-Mediated Ferroptosis and Reprograms the Tumor Microenvironment against Anti-PD-1 Immunotherapy

doi: 10.34133/cancomm.0009

Figure Lengend Snippet: TIMELESS deficiency enhances ferroptosis susceptibility in LUAD cells and organoids. (A) Western blotting analysis of TIMELESS protein expression in A549 and H1975 cells transduced with control ( sgCtrl ) or TIMELESS targeting ( sgTIMELESS ) sgRNAs. (B) MMP was assessed by JC-1 flow cytometry in sgCtrl and sgTIMELESS cells following a 48-h treatment with either DMSO or erastin (3 or 5 μmol/l for H1975 or A549 cells, respectively; n = 3 per group). (C) Intracellular total ROS were assessed by DCFH-DA fluorescence in A549 and H1975 cells after 48-h treatments with DMSO or erastin. (D) Lipid peroxidation was monitored using BODIPY 581/591 C11 staining. Experimental groups were as follows: sgCtrl + DMSO, sgCtrl + erastin, sgTIMELESS + DMSO, and sgTIMELESS + erastin. (E) MDA content was detected in sgCtrl and sgTIMELESS cells following DMSO or erastin treatment. (F) Lipid peroxides were assessed using the Liperfluo probe and flow cytometry in sgCtrl and sgTIMELESS cells treated with DMSO or erastin for 48 h. (G) Multiplexed imaging of nuclei (Hoechst 33342, blue), mitochondrial mass (MitoTracker Green FM, green), MMP (TMRE, red), and mitochondrial superoxide (mtSOX deep Red, purple) in sgCtrl and sgTIMELESS H1975 cells treated with either DMSO or erastin. (H) Total iron content was quantified using a colorimetric assay in the indicated groups. (I) Labile Fe 2+ levels were assessed by FerroOrange staining in H1975 cells from the following groups: sgCtrl + DMSO, sgCtrl + erastin, sgTIMELESS + DMSO, and sgTIMELESS + erastin. (J) TEM images of mitochondria in sgCtrl and sgTIMELESS H1975 cells treated with either DMSO or erastin. Blue arrows indicate mitochondria with obvious cristae, while red arrows indicate shrunken mitochondria. (K) Representative micrographs of sgCtrl and sgTIMELESS LUAD organoids treated with DMSO or erastin. The statistical analysis was performed using a 2-tailed Student’s t test (B, D, E, and H). *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant. Abbreviations: DCFH-DA, 2′,7′-dichlorodihydrofluorescein diacetate; DMSO, dimethyl sulfoxide; MDA, malondialdehyde; MMP, mitochondrial membrane potential; mtSOX, mitochondrial superoxide; ROS, reactive oxygen species; TEM, transmission electron microscopy; TMRE, tetramethylrhodamine ethyl ester.

Article Snippet: To assess the RNA stabilities of TF and Ccr4-Not transcription complex subunit 3 (CNOT3), H1975 and A549 cells were treated with 2.5 μmol/l actinomycin D (MedChemExpress, Cat. HY-17559) to inhibit transcription.

Techniques: Western Blot, Expressing, Transduction, Control, Flow Cytometry, Fluorescence, Staining, Imaging, Colorimetric Assay, Membrane, Transmission Assay, Electron Microscopy

Journal: Cancer Communications

Article Title: TIMELESS Promotes LUAD Growth via Suppressing Transferrin-Mediated Ferroptosis and Reprograms the Tumor Microenvironment against Anti-PD-1 Immunotherapy

doi: 10.34133/cancomm.0009

Figure Lengend Snippet: TIMELESS inhibits ferroptosis through TF down-regulation in LUAD. (A) Distribution of TIMELESS PAR-CLIP binding sites across genomic features. (B) Overlap between TIMELESS PAR-CLIP targets and up-regulated mRNAs upon its knockdown in H1975 cells. (C) RT-qPCR validation of the 21 overlapping genes (B) in siCtrl and siTIMELESS H1975 cells. (D) TIMELESS PAR-CLIP peaks in the TF transcript in H1975 cells. Mapping of the TIMELESS-binding site to the GAGATGG motif located in TF 3′UTR. TIMELESS IP: immunoprecipitated with an anti-TIMELESS antibody. Input: whole cell lysate control. (E) The de novo discovered RNA-binding motif for TIMELESS was identified through PAR-CLIP-seq and its significance was analyzed using the MEME suite. (F) Luciferase activity of wild-type (WT) or mutant (MUT) TF 3′UTR reporter plasmid was measured in A549 and H1975 cells cotransfected with a TIMELESS-overexpression plasmid (TIMELESS-oe) or an empty vector control (Vector). (G) Localization of TIMELESS protein (red) and TF mRNA (green) in A549 and H1975 cells, as detected by IF and RNA-FISH assays, respectively. (H) RIP verified the binding of TF mRNA to TIMELESS in A549 and H1975 cells. IgG antibody served as the negative control. (I) The decay of TF mRNA was measured after actinomycin D (2.5 μmol/l) treatment in siCtrl and siTIMELESS A549 and H1975 cells. The mRNA half-life ( t 1/2 ) was derived from the best-fit curve ( R 2 values shown). The statistical analysis was performed using a 2-tailed Student’s t test (C, F, and H) and one-phase decay modeling for half-life calculation (I). *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant. Abbreviations: FISH, fluorescence in situ hybridization; GO, Gene Ontology; IF, immunofluorescence; IP, immunoprecipitation; PAR-CLIP, photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation; RT-qPCR, reverse transcription quantitative real-time PCR; RIP, RNA immunoprecipitation; TF, transferrin; UTR, untranslated region.

Article Snippet: To assess the RNA stabilities of TF and Ccr4-Not transcription complex subunit 3 (CNOT3), H1975 and A549 cells were treated with 2.5 μmol/l actinomycin D (MedChemExpress, Cat. HY-17559) to inhibit transcription.

Techniques: Binding Assay, Knockdown, Quantitative RT-PCR, Biomarker Discovery, Immunoprecipitation, Control, RNA Binding Assay, Luciferase, Activity Assay, Mutagenesis, Plasmid Preparation, Over Expression, Negative Control, Derivative Assay, Fluorescence, In Situ Hybridization, Immunofluorescence, Reverse Transcription, Real-time Polymerase Chain Reaction, RNA Immunoprecipitation

Journal: Cancer Communications

Article Title: TIMELESS Promotes LUAD Growth via Suppressing Transferrin-Mediated Ferroptosis and Reprograms the Tumor Microenvironment against Anti-PD-1 Immunotherapy

doi: 10.34133/cancomm.0009

Figure Lengend Snippet: TF silencing partially alleviates ferroptosis and restores tumor growth in TIMELESS knockout models. (A and B) Western blotting (A) and RT-qPCR (B) were performed in control ( sgCtrl ), TIMELESS -knockout ( sgTIMELESS ), and TIMELESS -knockout followed by TF -knockdown ( sgTIMELESS + shTF ) A549 and H1975 cells. (C) Flow cytometric analysis of MMP using JC-1 in sgCtrl , sgTIMELESS , and sgTIMELESS + shTF A549 and H1975 cells. (D) ROS production in sgCtrl , sgTIMELESS , and sgTIMELESS + shTF cells. (E) Lipid peroxidation accumulation was assessed in sgCtrl , sgTIMELESS , and sgTIMELESS + shTF A549 and H1975 cells using BODIPY 581/591 C11 staining. (F) Lipid peroxides were monitored with Liperfluo fluorescence probe in the indicated groups. (G) MDA levels were measured in A549 and H1975 cells from the following groups: sgCtrl , sgTIMELESS , and sgTIMELESS + shTF . (H) Nuclei (Hoechst 33342, blue), mitochondrial mass (MitoTracker Green FM, green), MMP (TMRE, red), and mitochondrial superoxide (mtSOX deep Red, purple) were simultaneously stained in the indicated cell groups. (I) Representative TEM images of mitochondria in H1975 cells from the sgCtrl , sgTIMELESS , and sgTIMELESS + shTF groups. (J) Intracellular Fe 2+ was detected using the FerroOrange probe in the indicated groups. (K) Total iron content was quantified in A549 and H1975 cells from the sgCtrl , sgTIMELESS , and sgTIMELESS + shTF groups. (L and M) In vivo tumor growth was shown by representative images (L) and tumor weight quantification (M) from xenograft models. The statistical analysis was performed using a 2-tailed Student’s t test (B, C, E, G, K, and M). * P < 0.05, **P < 0.01, ***P < 0.001. Abbreviations: MDA, malondialdehyde; MMP, mitochondrial membrane potential; mtSOX, mitochondrial superoxide; ROS, reactive oxygen species; TEM, transmission electron microscopy; TMRE, tetramethylrhodamine ethyl ester.

Article Snippet: To assess the RNA stabilities of TF and Ccr4-Not transcription complex subunit 3 (CNOT3), H1975 and A549 cells were treated with 2.5 μmol/l actinomycin D (MedChemExpress, Cat. HY-17559) to inhibit transcription.

Techniques: Knock-Out, Western Blot, Quantitative RT-PCR, Control, Knockdown, Staining, Fluorescence, In Vivo, Membrane, Transmission Assay, Electron Microscopy