press human luad cell lines a549 nci h1975  (ATCC)

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 effect of ferroptosis inhibition, sgTIMELESS and sgCtrl LUAD cells were treated with 2 μmol/l ferrostatin-1 (MedChemExpress, Cat. HY-100579) for 48 h prior to the assay.

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: RBP TIMELESS promotes ferroptosis resistance and accelerates tumor progression in LUAD. (A) KEGG enrichment analysis of the up-regulated DEGs after TIMELESS knockdown. (B) Volcano plots of DEGs following RBPs ( TIMELESS, CENPF, KIF20A , and PRC1 ) knockdown; the cutoffs for differential expression were set at |log 2 FC| > 1 and P < 0.05. Genes were categorized as follows: Sig_Up (significantly up-regulated), Sig_Down (significantly down-regulated), and NoDiff (nonsignificant). (C) Quantitative analysis of cell death rescued by ferrostatin-1 treatment, assessed by costaining with Calcein-AM/PI. (D) Images of lung tumors (white-dashed circle) excised at day 30 post-injection from LLC1 orthotopic models established with control ( shCtrl ) or Timeless -knockdown ( shTimeless ) LLC1 cells. The corresponding quantitative analysis of the weight of tumor-bearing lungs is shown on the right ( n = 5 per group). (E) Representative H&E staining of LLC1-induced lung tumor tissues from the control ( shCtrl ) and Timeless knockdown ( shTimeless ) group. (F) Representative IHC staining images of the ferroptosis marker 4HNE in LLC1-induced lung tumor tissues from shCtrl and shTimeless groups. (G) Representative IF staining and quantification of TIMELESS expression in LUAD and paired normal adjacent tissues from cohort 1 patients (LUAD TMA, n = 87). (H) Kaplan–Meier analysis showing the association between TIMELESS expression with OS in clinical LUAD patient cohort 1 ( n = 87). Patients were dichotomized into high and low TIMELESS expression groups based on IHC staining scores, using the median value as the cutoff. The statistical analysis was performed using a 2-tailed Student’s t test (C, D, and G). The survival analysis of LUAD patients was performed by the Kaplan–Meier method (H). *P < 0.05, **P < 0.01, ***P < 0.001. Abbreviations: 4HNE, 4-hydroxynonenal; DEGs, differentially expressed genes; H&E, hematoxylin and eosin; IHC, immunohistochemistry; KEGG, Kyoto Encyclopedia of Genes and Genomes; LLC, Lewis lung carcinoma; shCtrl , small hairpin RNA for the negative control; shTimeless , small hairpin RNA for Timeless ; siRNA, small interfering RNA.

Article Snippet: To assess the effect of ferroptosis inhibition, sgTIMELESS and sgCtrl LUAD cells were treated with 2 μmol/l ferrostatin-1 (MedChemExpress, Cat. HY-100579) for 48 h prior to the assay.

Techniques: Knockdown, Quantitative Proteomics, Injection, Control, Staining, Immunohistochemistry, Marker, Expressing, Negative Control, Small Interfering RNA

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 effect of ferroptosis inhibition, sgTIMELESS and sgCtrl LUAD cells were treated with 2 μmol/l ferrostatin-1 (MedChemExpress, Cat. HY-100579) for 48 h prior to the assay.

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 effect of ferroptosis inhibition, sgTIMELESS and sgCtrl LUAD cells were treated with 2 μmol/l ferrostatin-1 (MedChemExpress, Cat. HY-100579) for 48 h prior to the assay.

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 effect of ferroptosis inhibition, sgTIMELESS and sgCtrl LUAD cells were treated with 2 μmol/l ferrostatin-1 (MedChemExpress, Cat. HY-100579) for 48 h prior to the assay.

Techniques: Knock-Out, Western Blot, Quantitative RT-PCR, Control, Knockdown, Staining, Fluorescence, In Vivo, 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 recruits CNOT3 to degrade TF mRNA, and their interaction mediates ferroptosis suppression. (A) KEGG pathway enrichment of TIMELESS-interacting proteins identified by IP-MS. (B) Poly(A) tail length of TF mRNA was assessed in H1975 cells overexpressing TIMELESS (TIMELESS-oe) using a quantitative PCR-based assay. GAPDH mRNA served as an internal control. (C) Representative Western blotting showing the protein levels of CNOT3, TIMELESS, and TF in H1975 cells, including control of TIMELESS knockout + control of CNOT3 overexpression ( sgCtrl + vector), TIMELESS knockout + CNOT3 overexpression ( sgTIMELESS + CNOT3 -oe), control of TIMELESS knockout + CNOT3 overexpression ( sgCtrl + CNOT3 -oe), and TIMELESS knockout + control of CNOT3 overexpression ( sgTIMELESS + vector). (D) Predicted structural model of the TIMELESS–CNOT3 complex obtained by molecular docking simulation. (E) Endogenous co-IP of TIMELESS and CNOT3 in H1975 cells. Cell lysates were immunoprecipitated with anti-TIMELESS, anti-CNOT3, or anti-IgG, followed by immunoblotting with the antibodies against TIMELESS and CNOT3. (F) GST pull-down assay of the direct interaction between TIMELESS and CNOT3. Purified His-CNOT3 was incubated with GST or GST-TIMELESS; proteins pulled down were analyzed by immunoblotting with the indicated antibodies. (G) Co-IP validation of the TIMELESS–CNOT3 interaction using flag-tagged wild-type TIMELESS (TIMELESS WT ) and TIMELESS mutants. (H) The poly(A) tail length of TF mRNA was assessed in H1975 cells stably expressing either flag-tagged wild-type TIMELESS (TIMELESS WT ) or the flag-tagged Q898A mutant (TIMELESS Q898A ). (I) TF mRNA half-life was calculated from actinomycin D-based transcription inhibition assays in H1975 cells expressing an empty vector control (Vector), TIMELESS WT , or TIMELESS Q898A . (J) Lipid peroxides were assessed using the Liperfluo probe in H1975 cells expressing vector, TIMELESS WT , or TIMELESS Q898A . (K) Intracellular Fe 2+ measurement by FerroOrange staining in the indicated H1975 cells. The statistical analysis was performed using a 2-tailed Student’s t test (I). ** P < 0.01, *** P < 0.001. Abbreviations: CNOT3, Ccr4-Not transcription complex subunit 3; Co-IP, co-immunoprecipitation; GST, glutathione S -transferase; IP-MS, immunoprecipitation–mass spectrometry; KEGG, Kyoto Encyclopedia of Genes and Genomes; RNA-seq, RNA sequencing.

Article Snippet: To assess the effect of ferroptosis inhibition, sgTIMELESS and sgCtrl LUAD cells were treated with 2 μmol/l ferrostatin-1 (MedChemExpress, Cat. HY-100579) for 48 h prior to the assay.

Techniques: Protein-Protein interactions, Real-time Polymerase Chain Reaction, Control, Western Blot, Knock-Out, Over Expression, Plasmid Preparation, Co-Immunoprecipitation Assay, Immunoprecipitation, Pull Down Assay, Purification, Incubation, Biomarker Discovery, Stable Transfection, Expressing, Mutagenesis, Inhibition, Staining, Mass Spectrometry, RNA Sequencing

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 is correlated with ferroptosis, immune infiltration, and disease progression. (A and B) Representative mIF images from 2 independent 6-plex panels in matched normal and tumor tissues of LUAD patient cohort 2 ( n = 38). Panel 1 stained for TIMELESS (red), 4HNE (green), PD-L1 (orange), CK7 (cyan), CD4 (yellow), and CD8 (white) (A). Panel 2 stained for TIMELESS (red), 4HNE (green), PD-L1 (orange), CK7 (cyan), CD86 (white), and CD206 (yellow) (B). Nuclei were counterstained with DAPI (blue), and TIMELESS, 4HNE, PD-L1, and CK7 served as internal spatial anchors for image alignment and comparative analysis in both panels. (C) Heatmap depicting standardized IHC scores for marker proteins in tumor tissues from LUAD patient cohort 2 ( n = 42). Each cell in the matrix represents the expression level of a single protein (rows) in an individual sample (columns). (D to G) Correlation analysis of cancer cell TIMELESS expression across the tumor tissues from LUAD cohort 2 ( n = 42). TIMELESS expression was assessed for correlation with 4HNE expression (D), the extent of CD8 + T cell infiltration (E), PD-L1 expression on cancer cells (F), and the density of CD206 + M2 macrophage infiltration (G). All markers were assessed by mIF and quantified as the percentage of positive cells. (H) Schematic of the hypothesized mechanism. The model proposes that TIMELESS knockdown, through disrupting CNOT3 binding and subsequently increasing TF expression, may drive ferroptosis and immune microenvironment remodeling to suppress tumor growth. The statistical analysis was performed using Spearman rank correlation (D to G). Abbreviations: 4HNE, 4-hydroxynonenal; CD, cluster of differentiation; CNOT3, Ccr4-Not transcription complex subunit 3; CK7, cytokeratin 7; IHC, immunohistochemistry; LUAD, lung adenocarcinoma; mIF, multiplex immunofluorescence; PD-L1, programmed cell death ligand 1; TF, transferrin.

Article Snippet: To assess the effect of ferroptosis inhibition, sgTIMELESS and sgCtrl LUAD cells were treated with 2 μmol/l ferrostatin-1 (MedChemExpress, Cat. HY-100579) for 48 h prior to the assay.

Techniques: Biomarker Discovery, Staining, Marker, Expressing, Knockdown, Binding Assay, Immunohistochemistry, Multiplex Assay, Immunofluorescence

Journal: Cancer Cell International

Article Title: Circulating extracellular vesicle PTPRO methylation: an exploratory biomarker for minimally invasive diagnosis of early-stage lung adenocarcinoma

doi: 10.1186/s12935-025-04127-9

Figure Lengend Snippet: Methylation analysis of PTPRO in LUAD cell lines and LUAD cells derived sEVs. (A) The mRNA expression levels of PTPRO were determined by RT-qPCR in the normal lung epithelial cell line HBE and LUAD cell lines A549, NCI-H460, and PC-9. (B-C) PTPRO methylation status in HBE and the LUAD cell lines A549, NCI-H460, and PC-9 was assessed using MSP and q-MSP assays. (D) Representative TEM images of sEVs purified from the conditioned media of HBE and LUAD cell lines (A549, NCI-H460, and PC-9). Scale bar: 100 nm. (E) The concentration and size distribution of sEVs from HBE and LUAD cell lines (A549, NCI-H460, and PC-9) were evaluated using NTA. (F) Expression of sEV markers (TSG101, Alix, CD63, and Calnexin) and PTPRO in sEVs purified from HBE and LUAD cell lines (A549, NCI-H460, and PC-9) was detected by immunoblotting. (G) The mRNA expression levels of PTPRO in sEVs purified from the conditioned media of HBE and LUAD cell lines (A549, NCI-H460, and PC-9) were measured by RT-qPCR. (H-I) MSP and q-MSP analyses of purified sEVs from HBE and LUAD cell lines (A549, NCI-H460, and PC-9). H 2 O was used as a negative control. Error bars indicate SEM. *** P < 0.001 by one-way ANOVA with Dunnett's multiple comparisons test.

Article Snippet: Human LUAD cell lines (A549, PC-9, and NCI-H460) and normal human bronchial epithelial cells (HBE) were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Methylation, Derivative Assay, Expressing, Quantitative RT-PCR, Purification, Concentration Assay, Western Blot, Negative Control