(A) Schematic diagram showing RNA labeling and isolation using CD and 5-FC; (B) Chemical reactions steps involved in the labeling of RNA using CD and 5-FC; (C) Enrichment of mRNAs immunopurified by …
(A) Anti-BrdU antibody detects CD-based 5-fluorouracil (5-FU) derivatives but not 5-fluorocytosine (5-FC) derivatives. CD-expressing or control 293 T cells were treated with either 5-FC or 5-FU for …
(A–D) MDA231 cells expressing CD/UPRT were treated with 5-FC for 30 min prior to TGF-β or SB-505124 (SB, a TGF-β receptor inhibitor) treatment for 150 min. (A) Change in gene expression in …
(A) Schematic diagram of lung colonization xenograft assay used for evaluation of Flura-tagging in vivo. Athymic mice were injected through the tail vein with 50,000 MDA231 cells expressing CD/UPRT …
(A) Representative H and E stained sections of mouse lungs harboring micrometastases (arrows) used in Flura-tagging experiments. Inset shows higher magnification. Scale bar, 2 mm (top), 100 μM …
(A) Schematic diagram of experimental design used to obtain tissue specific transcriptomes of MDA231 cells in mice; (B) Principal component analysis of genes expressed by MDA231 cells in the …
(A) Representative H and E stained sections of mouse lungs (left) and brain (right) harboring micrometastases (arrows) used in Flura-seq experiments. Inset shows higher magnification. Scale bar, 1 …
(A) Gene Ontology (GO) analysis of biological processes (BP) of genes that were upregulated in MDA231 lung micrometastases compared to brain micrometastases or mammary tumors. The top functional …
(A) Genes differentially expressed in lung and brain micrometastases relative to mammary fat pad tumors were identified by Flura-seq, and the overlap of the upregulated genes (left) and …
(A–C) Oxidative stress (A), antioxidant GPX1 (B) and NRF2 (C) in lung and brain tissue sections containing HCC1954 micrometastases were examined by IHC using anti-4-HNE, anti-GPX1 and anti-NRF2 …
(A–D) Expression of nuclear Complex I and antioxidant genes in a gene expression data set of matched primary tumors and lung metastases from patients with breast cancer (Siegel et al., 2018). (A) …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Antibody | anti-BrdU; BrdU antibody (Rat monoclonal) | Abcam | Cat#ab6326 | (1:200) |
Antibody | anti-CD31; CD31 antibody (Rat monoclonal) | Dianova | Cat#DIA-310 | (1:100) |
Antibody | anti-GFP; GFP antibody (Chicken monoclonal) | Aves Labs | Cat#GFP-1020 | (1:500) |
Antibody | anti-4-Hydroxynonenal; 4-HNE antibodyl (Rabbit polyclonal) | Abcam | Cat#ab46545 | (1:75) |
Antibody | anti-NRF2; NRF2 antibody (Rabbit polyclonal) | Abcam | Cat#ab137550 | (1:600) |
Antibody | anti-Glutathione Peroxidase 1; GPX1 antibody (Rabbit polyclonal) | Abcam | Cat#ab22604 | (1:200) |
Antibody | Goat polyclonal anti-chicken | Thermo Fisher | Cat#A-11039 | (1:1000) |
Antibody | Goat polyclonal anti-rat | Thermo Fisher | Cat#A-11006 | (1:1000) |
Antibody | Goat polyclonal anti-mouse | Abcam | Cat#ab150117 | (1:1000) |
Biological sample (Human) | Human breast cancer lung metastases tissue microarray (TMA) | This paper (Section of lung tissue containing cancer cells was surgically removed from breast cancer patients, preserved in paraflim and a small portion of the preserved tumor was used to make the TMA) | N/A | Tissue microarray Available from Edi Brogi |
Chemical compound, drug | Doxycycline | Sigmal-Aldrich | Cat#D9891 | |
Chemical compound, drug | 5-Fluorocytosine; 5-FC | Sigma-Aldrich | Cat#F7129 | |
Chemical compound, drug | 5-Fluorouracil; 5-FU | Sigma-Aldrich | Cat#F6627 | |
Chemical compound, drug | SB-505124 | Sigma-Aldrich | Cat#S4696 | |
Chemical compound, drug | Thymine | Sigma-Aldrich | Cat#T0376 | |
Chemical compound, drug | 4-Thiouracil; TU | Sigma-Aldrich | Cat#440736 | |
Other | Oligo (dT)25 magnetic beads | New England Biolabs | Cat#S1419S | |
Other | Protein G Dynabeads | Thermo Fisher Scientific | Cat#10004D | |
Commercial assay or kit | Tissue digestion C-tube | Miltenyi | Cat#130-096-334 | |
Commercial assay or kit | Mouse Tumor Dissociation Kit | Miltenyi | Cat#130-096-730 | |
Commercial assay or kit | TruSeq RNA Library Prep Kit v2 | Illumina | RS-122–2001 | |
Commercial assay or kit | SMARTer PCR cDNA synthesis kit | Clontech | Cat#634926 | |
Commercial assay or kit | Nextera XT DNA library Preparation Kit | Illumina | FC-131–1024 | |
Commercial assay or kit | RNeasy MinElute Cleanup kit | Qiagen | Cat#74204 | |
Commercial assay or kit | cDNA kit-First Strand Transcriptor | Roche | Cat#043790–12001 | |
Cell line (Human) | MDA231 | Laboratory of Joan Massague | PMID: 19421193 | Expresses TGL |
Cell line (Human) | MDA231-CD | This paper (MDA231 cells were transduced with rtTA3 and TRE-CD-IRES-RFP) | N/A | Available from Massague lab |
Cell line (Human) | MDA231-CD/UPRT | This paper (MDA231 cells were transduced with rtTA3 and TRE-UPRT-T2A-RFP-IRES-CD) | N/A | Available from Massague lab |
Cell line (Human) | 293T | Laboratory of Joan Massague | N/A | |
Strain, strain background (Mus musculus) | Hsd:Athymic Nude- Foxn1nu | Envigo | Cat#069 | |
Sequence-based reagents | Oligonucleotides | This paper | N/A | Oligonucleotide sequences are provided in Supplementary file 6 |
Recombinant DNA reagent | CMV Tight RFP-IRES-CD | This paper (RFP-IRES-CD was subcloned into CMV Tight EGFP Puro (Addgene: Plasmid #26431) vector by removing EGFP). | N/A | Available from Massague lab |
Recombinant DNA reagent | CMV Tight UPRT- T2A-RFP-IRES-CD | This paper (UPRT-T2A-RFP-IRES-CD was subcloned into CMV Tight EGFP Puro (Addgene: Plasmid #26431) vector by removing EGFP). | N/A | Available from Massague lab |
Recombinant DNA reagent | rtTA3 | Addgene | Plasmid #26730 | |
Software and Algorithms | STAR2.5.2b | PMID: 23104886 | https://github.com/alexdobin/STAR | |
Software and Algorithms | HTSeq v0.6.1p1 | PMID: 20979621 | https://htseq.readthedocs.io/en/release_0.10.0/ | |
Software and Algorithms | DESeq2 v3.4 | PMID: 25516281 | https://bioconductor.org/packages/release/bioc/html/DESeq2.html | |
Software and Algorithms | GSVA v3.4 | PMID: 23323831 | https://bioconductor.org/packages/release/bioc/html/GSVA.html |
Genes that are differentially expressed in MDA231 cells expressing CD/UPRT and treated with indicated concentration of 5-FC for 4 hr compared to control cells were obtained by DESEQ2 analysis.
Genes that are differentially expressed in TGF-β treated cells compared to SB-505124 by more than twofold are shown as identified by RNA-seq and Flura-seq.
Genes commonly identified by RNA-seq 6 hr post TGF-β, but not 2.5 hr post treatment, and Flura-seq 2.5 hr post TGF-β treatment are also shown.
Genes that are differentially expressed in MDA231 cells in different organs in situ as determined by Flura-seq or in vitro after isolation from the organs as determined by RNA-seq are shown.
Top 100 NRF2 target genes identified by two independent ChIP-seq experiments in Hela cells (ENCODE Project Consortium, 2012), and the genes that were common in both experiments were used as NRF2-responsive signature genes.
Genes identified to be up-regulated by more than two-fold in lung metastases compared to the corresponding primary tumors in breast cancer patients described in Siegel et al. (2018) for each patients are shown.
Complex I genes are highlighted in red color and the total number of upregulated Complex I genes in each patient is shown.
Oligonucleotide sequences used in the experiments described in the manuscript are shown.