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: