ht1080 human fibrosarcoma cell line  (ATCC)

Journal: Nucleic Acids Research

Article Title: Genome-wide mapping of formaldehyde-induced DNA–protein crosslinks reveals unique patterns of formation and transcription-coupled removal in mammalian cells

doi: 10.1093/nar/gkaf720

Figure Lengend Snippet: DPC removal is associated with transcription. ( A ) Heatmap representing the intensity of H3K4me3 signals across five quintiles of genes in human HT1080 cells. ( B ) DPC-seq data for cells immediately following a 2-h exposure to formaldehyde (T), and in formaldehyde-treated cells permitted to recover in drug-free media for 5 h (R). The maps are centered at the transcription start site (TSS) across a 40-kb window. The box above the heatmap depicts the signal intensity for each of the five gene sets, Q0 (gray), Q1 (red), Q2 (blue), Q3 (black), and Q4 (green), described in the text. Data from three independent experiments.

Article Snippet: Human fibrosarcoma cell line (HT1080) was purchased from ATCC (CCL-121) and HT1080-XPA-KO cells were ordered from Synthego.

Techniques:

Journal: Nucleic Acids Research

Article Title: Genome-wide mapping of formaldehyde-induced DNA–protein crosslinks reveals unique patterns of formation and transcription-coupled removal in mammalian cells

doi: 10.1093/nar/gkaf720

Figure Lengend Snippet: Reprogramming transcription alters DPC removal efficiency. ( A ) Representation of experimental design. HT1080 cells were incubated in the presence or absence of 1 μM dexamethasone overnight and subsequently treated with 400 μM formaldehyde for 2 h. Samples were recovered immediately following formaldehyde exposure or after a 5-h drug-free recovery period, and DPC-selective qPCR was performed (see the “Materials and methods” section for details). ( B ). Formaldehyde-induced DPC selective qPCR was performed on cells that had been pre-incubated in the absence (−Dex) or presence (+Dex) of dexamethasone using a primer pair specific for the IL1B locus. See the “Materials and methods” section and . (* P = .004, N = 5). Error bars represent SEM.

Article Snippet: Human fibrosarcoma cell line (HT1080) was purchased from ATCC (CCL-121) and HT1080-XPA-KO cells were ordered from Synthego.

Techniques: Incubation

Journal: Nucleic Acids Research

Article Title: Genome-wide mapping of formaldehyde-induced DNA–protein crosslinks reveals unique patterns of formation and transcription-coupled removal in mammalian cells

doi: 10.1093/nar/gkaf720

Figure Lengend Snippet: Transcription-coupled DPC removal is impaired in NER-deficient cells. ( A ) Heatmap representing the signal intensity of DPC-seq analysis on unmodified HT1080 cells WT and on a clone of HT1080 cells in which the XPA gene was inactivated (XPA-KO, see the “Materials and methods” section for details) exposed to 400 μM formaldehyde and permitted to recover for 5 h. This analysis was performed across five quintiles composed of genes with differing levels of transcriptional activity (see legend to Fig. ), TSS. ( B ) Average signal intensity of DPC-seq signals across a 10 kb region centered on the transcriptional start site for the Q0 (open circles) and Q4 gene sets (filled circles) for WT (blue) and XPA-KO (red) cells. ( C ) − \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} ${\log _{10}}p$\end{document} values comparing WT versus XPA KO cells across the Q0 (gray) and Q4 (green) gene sets. Data from two independent experiments.

Article Snippet: Human fibrosarcoma cell line (HT1080) was purchased from ATCC (CCL-121) and HT1080-XPA-KO cells were ordered from Synthego.

Techniques: Activity Assay

Journal: Nucleic Acids Research

Article Title: Genome-wide mapping of formaldehyde-induced DNA–protein crosslinks reveals unique patterns of formation and transcription-coupled removal in mammalian cells

doi: 10.1093/nar/gkaf720

Figure Lengend Snippet: Removal of DPCs at the IL1B locus is partially deficient in XPA-KO cells. DPC selective qPCR was performed using IL1B-specific primers (see the “Materials and methods” section for details) on material recovered from HT1080 cells WT and XPA-KO cells that had been treated with formaldehyde (400 μM, 2 h) or mechlorethamine (50 μM, 1 h) and permitted to recover for 5 h in drug-free media. ** P = .002, N = 5 (formaldehyde); ** P = .001, N = 3 (mechlorethamine). Error bars represent SEM.

Article Snippet: Human fibrosarcoma cell line (HT1080) was purchased from ATCC (CCL-121) and HT1080-XPA-KO cells were ordered from Synthego.

Techniques:

Journal: Nucleic Acids Research

Article Title: Genome-wide mapping of formaldehyde-induced DNA–protein crosslinks reveals unique patterns of formation and transcription-coupled removal in mammalian cells

doi: 10.1093/nar/gkaf720

Figure Lengend Snippet: Removal of DPCs at the IL1B locus in different cell lines. DPC selective qPCR was performed using IL1B-specific primers on material recovered from XPC-KO, XPA-KO, and CSB-KO HT1080 cells that had been treated with formaldehyde (400 μM, 2 h) and permitted to recover for 5 h in drug-free media. Data plotted as a percent control of WT, XPA-KO-corrected, and WT, respectively. * P = .04, ** P = .01, n.s.; not significant, N = 5 (XPC-KO and XPA-KO), N = 3 (CSB-KO). Error bars represent SEM.

Article Snippet: Human fibrosarcoma cell line (HT1080) was purchased from ATCC (CCL-121) and HT1080-XPA-KO cells were ordered from Synthego.

Techniques: Control

Journal: Nucleic Acids Research

Article Title: Genome-wide mapping of formaldehyde-induced DNA–protein crosslinks reveals unique patterns of formation and transcription-coupled removal in mammalian cells

doi: 10.1093/nar/gkaf720

Figure Lengend Snippet: DPC removal from rDNA. ( A ) HT1080 cells were treated with 400 μM formaldehyde for 2 h and permitted to recover in drug-free media for 5 h, and relative efficiency of formaldehyde-induced DPC removal at the rDNA Q0 and Q4 loci was determined (see text and “Materials and methods” section for details). * P = .02, N = 3. ( B ) WT and XPA-KO HT1080 cells were treated with 400 μM formaldehyde for 2 h, and material was harvested immediately or after a 5-h drug-free recovery period. DPC selective qPCR was performed using three sets of primer pairs specific for the 18S, 28S, and 5S rDNA genes , and the percent removal of formaldehyde-induced DPCs was calculated as described in the “Materials and methods” section. n.s.; not significant, N = 9. Error bars represent SEM.

Article Snippet: Human fibrosarcoma cell line (HT1080) was purchased from ATCC (CCL-121) and HT1080-XPA-KO cells were ordered from Synthego.

Techniques:

Journal: bioRxiv

Article Title: Riboflavin metabolism shapes FSP1-driven ferroptosis resistance

doi: 10.1101/2025.08.05.668651

Figure Lengend Snippet: a, Schematic representation of the FSP1-dependent model used to identify factors supporting FSP1 function. Upper left: the primary protective system against lipid peroxidation (LPO) is the enzyme GPX4, complemented by FSP1. Lower left: cells treated with an FSP1 inhibitor (iFSP1) can rely on GPX4 activity to survive. Upper right: FSP1-dependent HT1080 cells (HT1080 GPX4KO/FSP1OE ). In HT1080 cells, as in many others, knocking out GPX4 induces ferroptosis due to insufficient endogenous FSP1 levels to compensate for GPX4 loss. However, cell survival can be rescued by overexpressing FSP1. Lower right: Upon pharmacological inhibition of FSP1 (iFSP1 treatment), cells undergo ferroptosis as they solely rely on FSP1 function for survival. b , Immunoblot (IB) analysis of ACSL4, FSP1, GPX4, and vinculin in HT1080 parental and HT1080 GPX4KO/FSP1OE cells. c , Dose-dependent toxicity of an FSP1 inhibitor (iFSP1) in HT1080 parental and HT1080 GPX4KO/FSP1OE cell lines. Cell viability was monitored using Alamar blue after 24 h of treatment. Where indicated, cells were treated with the ferroptosis inhibitor Lip-1 (500 nM). d , Schematic representation of the screening strategy used to identify novel factors supporting FSP1 function in the previously-described FSP1-dependent cellular model (see ). HT1080 GPX4KO/FSP1OE cells were transduced with a gRNA library targeting approximately 3000 genes and selected over 7 days in the presence of the ferroptosis inhibitor Lip-1 (500 nM). Subsequently, cells proliferate with or without Lip-1 supplementation for additional 14 days. e, Graphical representation of the results from two independently performed CRISPR screens; plot depicts the score calculated using the MaGeCK package (x-axis) and the –Log false discovery rate (y-axis). Riboflavin kinase (RFK) and stearoyl-CoA desaturase-1 (SCD1) were identified as potentially robust candidates to modulate FSP1 function. f , Immunoblot (IB) analysis of RFK, FSP1, and vinculin in A375 cells transduced with either a non-targeting control (NT) or three different RFK-targeting sgRNAs. g , Dose-dependent toxicity of RSL3 and ML210 in A375 cells transduced with either a non-targeting control (NT) or three different RFK-targeting sgRNAs. Cell viability was monitored after 72 h of treatment.

Article Snippet: Human cancer cell lines HT1080, A375, MDA-MB-231, MDA-MB-436, A549 and H460 were purchased from ATCC.

Techniques: Activity Assay, Inhibition, Western Blot, Transduction, CRISPR, Control

Journal: bioRxiv

Article Title: Riboflavin metabolism shapes FSP1-driven ferroptosis resistance

doi: 10.1101/2025.08.05.668651

Figure Lengend Snippet: a , Volcano plot of quantified proteins showing their change in A375 parental cells cultured in riboflavin-deficient medium for 96 h. Proteins are plotted based on their fold change (FC: riboflavin deficient/normal). b , Immunoblot (IB) analysis of FSP1, GPX4, and vinculin in HT1080, A375, MDA-MB-231, and A549 parental cell lines after 96 h of growth in riboflavin-deficient medium or medium supplemented with 1 µM riboflavin. c , Lipid peroxidation evaluated by C11-BODIPY 581/591 staining of A375 parental cell line cultured for 72 h in riboflavin-deficient medium or medium supplemented with 1 µM riboflavin and after treatment with DMSO, RSL3 (200 nM), or RSL3 (200 nM) + Lip-1 (500 nM) for 6 h. d , Dose-dependent toxicity of RSL3 in the absence or presence of an FSP1 inhibitor (iFSP1 3 µM) in HT1080, A375, MDA-MB-231, and A549 parental cell lines pre-cultured in riboflavin-deficient medium or medium supplemented with 1 µM riboflavin for 48 h. Cell viability was monitored using Alamar blue after 96 h of treatment. e , Dose-dependent toxicity of RSL3 in the absence or presence of an FSP1 inhibitor (iFSP1 3 µM) in A375 and MDA-MB-231 cells transduced with either a non-targeting control (NT) or an FSP1-targeting sgRNAs pre-cultured in riboflavin-deficient medium or medium supplemented with 1 µM riboflavin for 48 h. Cell viability was monitored after 96 h of treatment. f , Immunoblot (IB) analysis of ACSL4, FSP1, NQO1, and vinculin in A375 and MDA-MB-231 cells transduced with either a non-targeting control (NT) or an FSP1-targeting sgRNAs cultured in riboflavin-deficient medium or medium supplemented with 1 µM riboflavin for 96 h. g , Epilipidomics analysis of A375 parental cells pre-cultured in riboflavin-deficient medium or medium supplemented with 1 µM riboflavin for 72 h after treatment with DMSO, RSL3 (200 nM), or RSL3 (200 nM) + Lip-1 (500 nM) for 6 h.

Article Snippet: Human cancer cell lines HT1080, A375, MDA-MB-231, MDA-MB-436, A549 and H460 were purchased from ATCC.

Techniques: Cell Culture, Western Blot, Staining, Transduction, Control

Journal: bioRxiv

Article Title: Riboflavin metabolism shapes FSP1-driven ferroptosis resistance

doi: 10.1101/2025.08.05.668651

Figure Lengend Snippet: a , Immunoblot (IB) analysis of FSP1, GPX4 and vinculin in HT1080, A375, MDA-MB-231 and 549 parental cell lines after 24, 48 and 144 h of growth in riboflavin-deficient medium or supplemented with 1 µM of riboflavin. b , Volcano plots of quantified proteins showing their change in A375 parental cells cultured in riboflavin-deficient medium for 24, 48 and 144 h. Proteins are plotted based on their fold change (FC: riboflavin deficient/normal). c , Heatmaps of quantified proteins showing their change in A375 parental cells cultured in riboflavin-deficient medium for 24, 48, 96 and 144 h (FC: riboflavin deficient/normal). d , Volcano plot of quantified flavoproteins showing their change in A375 parental cells cultured in riboflavin-deficient medium for 96 h (FC: riboflavin deficient/normal). e , Heatmap of quantified flavoproteins showing their change in A375 parental cells cultured in riboflavin-deficient medium for 96 h (FC: riboflavin deficient/normal). f , Lipid peroxidation evaluated by C11-BODIPY 581/591 staining of A375 parental cell line cultured for 72 h in riboflavin-deficient medium or supplemented with 1 µM of riboflavin and after treatment with DMSO, RSL3 (200 nM) or RSL3 (200 nM) + Lip-1 (500 nM) for 6 h. g , Dose-dependent toxicity of RSL3 in the presence of Lip-1 (500 nM) and iFSP1 (3 µM, when indicated) in HT1080, A375, MDA-MB-231 and A549 parental cell lines cultured in riboflavin-deficient medium or supplemented with 1 µM of riboflavin for 48 h. Cell viability was monitored using Alamar blue after 96 h of treatment. h , Absorbance at 517 nm corresponding to the radical initiator 2,2-diphenyl-1-picrylhydrazyl (DPPH) co-incubated with ferrostatin-1 (Fer-1), riboflavin (RbF) or roseoflavin (RoF) (n = 3). Data plotted are mean ± SD. Statistical significance was determined by one-way ANOVA followed by Dunnett’s multiple comparison test.

Article Snippet: Human cancer cell lines HT1080, A375, MDA-MB-231, MDA-MB-436, A549 and H460 were purchased from ATCC.

Techniques: Western Blot, Cell Culture, Staining, Incubation, Comparison

Journal: bioRxiv

Article Title: Riboflavin metabolism shapes FSP1-driven ferroptosis resistance

doi: 10.1101/2025.08.05.668651

Figure Lengend Snippet: a , Schematic representation of the metabolism of riboflavin (RbF) and its analog roseoflavin (RoF) by riboflavin kinase (encoded by RFK ) and FAD synthase (encoded by FLAD1 ). b , Dose-dependent toxicity of roseoflavin (RoF) and iFSP1 in the absence or presence of Lip-1 (500 nM) in HT1080 GPX4KO/FSP1OE . Cell viability was monitored using Alamar blue after 48 h of treatment. Cells were cultured in low-riboflavin medium (20 nM). c , Dose-dependent toxicity of the GPX4 inhibitor ML210 in A375 cells transduced with either a non-targeting control (NT) or a FSP1-targeting sgRNAs (FSP1 KO ) that were pre-treated with roseoflavin (RoF, 1, 3, and 10 nM) for 48 h. Cell viability was monitored after 48 h of ML210 treatment. Cells were cultured in low-riboflavin medium (20 nM). d , Immunoblot analysis (IB) of FSP1, NQO1, ACSL4, GPX4, and vinculin treated with roseoflavin (RoF 0, 20, 100, and 1000 nM) for 96 h in the absence or presence of riboflavin (RbF 20 nM). e , Dose-dependent toxicity of the GPX4 inhibitor ML210 in HT1080, MDA-MB-436, PC-9, and H460 parental cell lines pre-treated with roseoflavin (RoF, 1, 3, and 10 nM) for 48 h. Cell viability was monitored after 48 h of ML210 treatment. f , Immunoblot (IB) analysis of FSP1, NQO1, and vinculin in HT1080, MDA-MB-436, PC9, and H460 parental cell lines treated with roseoflavin (RoF, 20 nM) for 48 h in the absence or presence of riboflavin (RbF 20 nM). g , Riboflavin metabolism supports FSP1 function and promotes ferroptosis resistance. This is therefore a process that riboflavin analogs, such as roseoflavin can modulate.

Article Snippet: Human cancer cell lines HT1080, A375, MDA-MB-231, MDA-MB-436, A549 and H460 were purchased from ATCC.

Techniques: Cell Culture, Transduction, Control, Western Blot