Supporting Information
Kannan et al. 10.1073/pnas.1403813111
a Collagen pre-coated dish 30 n=10
FACS-purified cells and BC i3T3 cells 20 %CFC % 10 SF7 media supplemented with 5% FBS 8-10 days @ 37oC LP
0 Fix, stain and count BC LP
b FACS purified cells 100% matrigel BC LP LC SC
5
in vitro 10
SF7 media supplementedwith 5% FBS D 10-12 days @ 37oC @ 5% O Days 2 15
Fix and count 3D colonies No 3D colonies
Fig. S1. Assays used to detect human mammary cells with clonogenic activity. (A)(Left) The 2D clonogenic assay procedure. Cells were cocultured with a feeder layer of irradiated 3T3 fibroblasts (i3T3) in SF7 medium with 5% FBS in 20% O2 for 8–10 d, and the colonies were stained with Giemsa before counting. − − − + + − (Right) Concentrations of cells with 2D clonogenic activity in the FACS-purified subsets of (DAPI CD45 CD31 CD49f THY1 MUC1 ) basal cells (BCs) and the (DAPI−CD45−CD31−CD49f+MUC1+THY1−) luminal progenitors (LPs) used in the experiments reported in this paper. (Right) Representative photomicrographs of the colonies obtained from each subset, illustrating the different morphologies that allow assessment of clonogenic BCs and LPs in 2D clonogenic assays of unseparated cells. (B)(Left) The 3D clonogenic assay procedure. Each culture was started by suspending 104 cells in 20 μL of 100% Matrigel in one well of an eight-well chamber slide, onto which SF7 medium supplemented with 5% fetal FBS was then added. Cultures were incubated in 5% O2 for 12–14 d before counting of colonies without further staining. (Right) Photomicrographs of representative 5- to 15-d-old cultures (unstained) initiated with different subsets of FACS-purified cells. Photomicrographs were taken at 4× magnification using an inverted light microscope.
Kannan et al. www.pnas.org/cgi/content/short/1403813111 1of6 a ROS eliminating or ROS generating ti genes BC LP 167x higher in GPX2 Affymetrix data GPX7 GCLC GSTO1 GLRX5 SOD1 2.5x higher in TXN Affymetrix data 29x higher in APOE Affymetrix data IDH3G PPP1R15B PRDX2 CAT GPX3 CYBA CYBASC3 CCS NOXA1 XDH HIF1AN IDH3A
Higher in SAGE Lower in SAGE
b BC LP LC SC 123 123123123 HeLa GPX2
TP63
GAPDH
Fig. S2. Glutathione peroxidase 2 (GPX2) expression. (A) Differential expression of reactive oxygen species (ROS)-regulating genes in normal adult human mammary BCs and LPs as identified from published LongSAGE and Affymetrix data (1). (B) Western blot analysis of GPX2 protein expression in purified mammary subpopulations showing its high and selective expression in the BC subset. TP63 expression served as a specificity control for BCs, HeLa cells served as a negative control, and GAPDH served as a loading control.
1. Raouf A, et al. (2008) Transcriptome analysis of the normal human mammary cell commitment and differentiation process. Cell Stem Cell 3(1):109–118.
Kannan et al. www.pnas.org/cgi/content/short/1403813111 2of6 a c FACS-sorted cells CD45/CD31 EpCAM CD49f CD10 H2O2
10 days
Differen a on profiling by qRT-PCR
Unstained 0 μM H2O2
Stained 0 μM H2O2 Unstained 0 μMMHO H22
Unstained 50 μM H2O2
Unstained 100 μM H2O2
Stained 0 μM H2O2
Stained 50 μM H2O2
Stained 100 μM H2O2
b
Fig. S3. Analysis of the differentiation profile of the progeny of clonogenic BCs and LPs generated in the presence of H2O2.(A) Experimental design. (B) Transcript levels determined by quantitative RT-PCR (qRT-PCR). Data are from three independent experiments. Primer sequences are listed in Table S2. (C) Surface marker expression determined by FACS. Data are from one experiment.
Kannan et al. www.pnas.org/cgi/content/short/1403813111 3of6 a OO OO° R/H-OOH R/H-OH
Superoxide dismutases (SOD1-3) Glutathione peroxidases (GPX1-8) Catalase(CAT) Peroxiredoxins (PRDX1-6)
ve damage
b
oxo 5’ dGTP 3’ dGTP dGTP 3’ dCTP 5’
ROS ROS
oxo oxo oxo dGTP 5’ dGTP 3’ 5’ dGTP 3’ 3’ dATP 5’ 3’ dCTP 5’ MTH1 MUTYH OGG1 oxo dGMP oxo 5’ 3’ 5’5 dGTP 33’ 3’ dCTP 5’ 3’ 5’
Fig. S4. Oxidative stress and DNA oxidative damage repair pathways. (A) ROS control mechanisms tested in this study. (B) Oxidative DNA/nucleotide damage repair mechanisms. Deoxyguanosine triphosphate (dGTP) is a preferred ROS target that converts dGTP to 8-oxo-dGTP (1). The 8-oxo-dGTP is mutagenic if incorporated directly into replicating DNA or first modified by pyrophosphatase MTH1 (2). MUTYH facilitates the removal of bases opposite to 8-oxo-dGTP, leading to purine substitutions (1). ROS also targets DNA direct by oxidizing guanine (3). OGG1 catalyzes the excision of oxidized dGTP, also resulting in purine substitutions (1).
1. David SS, O’Shea VL, Kundu S (2007) Base-excision repair of oxidative DNA damage. Nature 447(7147):941–950. 2. Yoshimura D, et al. (2003) An oxidized purine nucleoside triphosphatase, MTH1, suppresses cell death caused by oxidative stress. J Biol Chem 278(39):37965–37973. 3. Neeley WL, Essigmann JM (2006) Mechanisms of formation, genotoxicity, and mutation of guanine oxidation products. Chem Res Toxicol 19(4):491–505.
Kannan et al. www.pnas.org/cgi/content/short/1403813111 4of6 Table S1. Antibodies used for FACS and Western blot analysis Target human protein Antibody Company
FACS CD49f (clone GoH3) Rat monoclonal BD EpCAM (clone 9C4) Mouse monoclonal BioLegend MUC1 (clone 214D4) Mouse monoclonal Stem Cell Technologies THY1 (clone 5E10) Mouse monoclonal Gift from P. Lansdorp (BC CancerAgency, Vancouver, BC, Canada) CD31 (WM59) Mouse monoclonal Biolegend CD45 (clone HI30) Mouse monoclonal Biolegend Western blot SOD1 Rabbit polyclonal Abgent/MJS BioLynx SOD2 Rabbit polyclonal Abgent/MJS BioLynx SOD3 Rabbit polyclonal Abgent/MJS BioLynx PRDX1 Rabbit polyclonal Acris/Medicorp PRDX2 Rabbit polyclonal Abgent/MJS BioLynx PRDX3 Mouse polyclonal Abnova PRDX4 (clone 9A3) Mouse monoclonal Origene Technologies/Cedarlane PRDX5 Mouse polyclonal Abnova/Cedarlane PRDX6 (clone 477068) Mouse monoclonal R&D Systems CAT Rabbit polyclonal Acris Antibodies GPX1 (clone 13B2AF) Novus Biologicals Mouse monoclonal GPX2 (C1C3) Rabbit polyclonal Genetex GPX3 (clone 23B1) Mouse monoclonal Abnova GPX7 Goat polyclonal Abnova β-F1-ATPase (clone 11/21–1-C7) QED Biosciences Mouse monoclonal OGG1 Rabbit monoclonal Epitomics MTH1 (NUDT1) Rabbit polyclonal Novus Biologicals MUTYH (MYH) Rabbit polyclonal Cell Signaling/New England Biolabs TP63 (clone 4A4) Mouse monoclonal Genetex HISTONE H3 (clone D1H2) XP Cell Signaling Rabbit monoclonal GAPDH (clone GAPDH-71.1) Sigma-Aldrich Mouse monoclonal
Western blot analysis was performed using X-ray film imaging (1) or digital imaging (VersaDoc; Bio-Rad) to capture volumetric light signals displayed spectrally.
1. Eirew P, et al. (2012) Aldehyde dehydrogenase activity is a biomarker of primitive normal human mammary luminal cells. Stem Cells 30(2):344–348.
Kannan et al. www.pnas.org/cgi/content/short/1403813111 5of6 Table S2. Primer sequences used for qRT-PCR Target human gene Forward Reverse
GCLC TTGTCCTTTCCCCCTTCTTCTCT CAAGGACGTTCTCAAGTGGG GCLM TCCTGAAGAGCTTCTTGGAAA TGTGTGATGCCACCAGATTT GPX2 AGACAGGATGCTCGTTCTGC TTGCAACCAATTTGGACATC NRF2 GCTCATACTCTTTCCGTCGC ATCATGATGGACTTGGAGCTG ΔN-TP63 TGCCCAGACTCAATTTAGTGAG TCTGGATGGGGCATGTCTTTGC GATA3 TGTAGTAGAGCCCACAGGCA CTCATTAAGCCCAAGCGAAG NOTCH3 TGAACTCTGGCAGACACCAC GTGTGCAAATGGAGGTCGTT PGR CGATGCAGTCATTTCTTCCA AATCTGTGGGGATGAAGCAT ESR1 CAGGATCTCTAGCCAGGCAC ATGATCAACTGGGCGAAGAG ESR2 ATCACCCAAACCAAAGCATC CCATGATCCTGCTCAATTCC KRT4 TTCCATTTGGTCTCCAGGAC CCTGAGATCCAGAAAGTCCG KRT5 CTGGTCCAACTCCTTCTCCA CTGCGTGAGTACCAGGAGC KRT8 CTCCACTTGGTCTCCAGCAT GGAGCAGATCAAGACCCTCA KRT18 CGGGCATTGTCCACAGTATT GGGAGCACTTGGAGAAGAAG KRT19 CCTGGAGTTCTCAATGGTGG CTAGAGGTGAAGATCCGCGA GAPDH ACGTACTCAGCGCCAGCATC ACCGTCAAGGCTGAGAACGG mtF3212 CACCCAAGAACAGGGTTTGT —— mtR3319 —– TGGCCATGGGATTGTTGTTAA 18S1546F TAGAGGGACAAG TGGCGTTC —— 18S1650R —— CGCTGAGCCAGTCAGTGT
Primer sequences were obtained using qPrimerDepot (primerdepot.nci.nih.gov). Primers used for measure- ment of relative mitochondrial DNA content are described elsewhere (1).
1. Bai R-K, Perng C-L, Hsu C-H, Wong, L-JC (2004) Quantitative PCR analysis of mitochondrial DNA content in patients with mitochondrial disease. Ann NY Acad Sci 1011:304–309.
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