Electronic Supplementary Material (ESI) for Analyst
This journal is © The Royal Society of Chemistry 2013
Association Rule Mining of Cellular Responses induced by
Metal and Metal Oxide Nanoparticles
Rong Liuab, Bryan Francebc, Saji Georgeb, Robert Rallobd, Haiyuan Zhangb, Tian Xiabe,
Andre E. Nelbe, Kenneth Bradleybc, and Yoram Cohen*abf
a
Institute of the Environment and Sustainability, University of California, Los Angeles,
CA 90095, USA
b
California Nanosystems Institute, University of California, Los Angeles, CA 90095, USA
c
Department of Microbiology, Immunology, and Molecular Genetics, University of California,
Los Angeles, CA 90095, USA
d
Departament d'Enginyeria Informatica i Matematiques, Universitat Rovira i Virgili, Av. Paisos
Catalans 26, 43007 Tarragona, Catalunya, Spain
e
Department of Medicine - Division of NanoMedicine, University of California, Los Angeles,
CA 90095, USA
f
Department of Chemical & Biomolecular Engineering, University of California, Los Angeles,
CA 90095, USA
Supporting Information
1
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
SMAD-T12
SMAD-T24
SRF-T3
SRF-T6
SRF-T12
SRF-T24
Fluo-T1
Fluo-T2
Fluo-T3
Fluo-T4
Fluo-T6
Fluo-T24
JC1-T1
JC1-T2
JC1-T3
JC1-T4
JC1-T6
JC1-T24
Mito-T1
Mito-T2
Mito-T3
Mito-T4
Mito-T6
Mito-T24
PI-T1
PI-T2
PI-T3
PI-T4
PI-T6
PI-T24
SMAD-T24
SRF-T3
SRF-T6
SRF-T12
SRF-T24
Fluo-T1
Fluo-T2
Fluo-T3
Fluo-T4
Fluo-T6
Fluo-T24
JC1-T1
JC1-T2
JC1-T3
JC1-T4
JC1-T6
JC1-T24
Mito-T1
Mito-T2
Mito-T3
Mito-T4
Mito-T6
Mito-T24
PI-T1
PI-T2
PI-T3
PI-T4
PI-T6
PI-T24
SMAD-T6
SMAD-T3
SMAD-T12
p53-T24
p53-T12
p53-T6
p53-T3
NFkB-T24
NFkB-T12
NFkB-T6
NFkB-T3
NFAT-T24
NFAT-T12
NFAT-T6
NFAT-T3
Myc-T24
Myc-T12
Myc-T6
Myc-T3
HIF1A-T24
HIF1A-T12
HIF1A-T6
HIF1A-T3
E2F-T24
E2F-T12
E2F-T6
E2F-T3
CRE-T24
CRE-T12
CRE-T6
CRE-T3
AP1-T24
AP1-T12
AP1-T6
0
AP1-T3
Normalized Control value
Electronic Supplementary Material (ESI) for Analyst
This journal is © The Royal Society of Chemistry 2013
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
SMAD-T6
SMAD-T3
p53-T24
p53-T12
p53-T6
p53-T3
NFkB-T24
NFkB-T12
NFkB-T6
NFkB-T3
NFAT-T24
NFAT-T12
NFAT-T6
NFAT-T3
Myc-T24
Myc-T12
Myc-T6
Myc-T3
HIF1A-T24
HIF1A-T12
HIF1A-T6
HIF1A-T3
E2F-T24
E2F-T12
E2F-T6
E2F-T3
CRE-T24
CRE-T12
CRE-T6
CRE-T3
AP1-T24
AP1-T12
AP1-T6
0
AP1-T3
Normalized Control value
RAW264.7
BEAS-2B
Figure S1. Distribution of the control values of each HTS plate (the control values of each HTS
plate were normalized over [0, 1] so that they can be displayed in the same plot). The exposure
time is identified by the “-Tx” appended to a cellular response. The upper and lower boundaries
of a solid bar identify the first quartile (Q1) and the third quartile (Q3) of the data, the symbol
“ͼ” inside the solid bar represents the median of the data; ends of the whiskers are the smallest
and largest observations within the range defined by [Q1-1.5(Q3-Q1), Q3+1.5(Q3-Q1)]. Control
values outside the range are considered as outliers (marked by “○”) since they are abnormally
deviated from the majorities of control populations. It is noted that only a small percent of
outliers were identified in control data (1.5% and 2.8% for RAW264.7 and BEAS-2B cell lines,
respectively) via the box-plots.
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Electronic Supplementary Material (ESI) for Analyst
This journal is © The Royal Society of Chemistry 2013
Figure S2. Identified significant cellular responses (nanoparticle concentration: 0.39-200 mg/L,
exposure period: 3-24 h or 1-24 h (denoted by the wedges)).
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Electronic Supplementary Material (ESI) for Analyst
This journal is © The Royal Society of Chemistry 2013
Table S1. Summary of the ten luciferase-reporter based and the four fluorescence-based cell
response assays
TF
Pathway
CRE
cAMP/PKA
E2F/
DP1
Cell Cycle
Myc/
Max
c-myc
NFkB
NFkB
SMAD2/
SMAD3/
SMAD4
TGF-beta
p53
p53/DNA Damage
Significance
cAMP response element is a point of convergence for many extracellular and
intracellular signals, including cAMP, calcium, G-protein coupled receptors
(GPCR) and neurotrophins
Regulator of cell-cycle checkpoints in mammalian cells-major target of the
retinoblastoma gene product (Rb) and the activity of E2F/pRb is intimately
connected with the G1-S transition of the cell cycle
Transcription factor that heterodimerizes with an obligatory partner, Max, and
regulates the transcription of genes important for cell proliferation,
differentiation, and apoptosis.
Nuclear factor-kappaB plays a key role in inflammation, immune response, cell
proliferation and protection against apoptosis
Transforming growth factor β (TGFβ) signaling pathway is involved in many
cellular processes, including cell cycle arrest, differentiation, homeostasis, and
immunosuppression. TGFβ signaling induces phosphorylation and activation
of the SMAD2 and SMAD3 proteins, which then form complexes with the
mediator SMAD4. These SMAD complexes then translocate to the nucleus,
where they activate the expression of TGFβ-responsive genes
Role in DNA repair, cell cycle arrest, and apoptosis.
TCR and Elk-1, form a complex with the SRF over the serum response element
(SRE), and activate gene expression. The Elk-1 protein is phosphorylated by
mitogen-activated protein kinase (MAPK), causing increased DNA binding,
ternary complex formation, and transcriptional activation of target genes
Hypoxia-inducible factor-1 protein is a key regulator of oxygen homeostasis
and plays significant roles in cancer progression as well as in cardiovascular
diseases
Activator protein-1 (AP1) transcription factor is a hetero- or homo-dimeric
complex that comprises members of the proto-oncogene Jun protein family (cJun, JunB and JunD) and Fos protein family (c-Fos, Fos B, Fra-1 and Fra-2).
The stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK) signal
transduction pathway is responsible for the phosphorylation and activation of
Jun, which in turn activates AP1
NFAT family of transcription factors plays a role in the transcriptional
regulation of cytokine genes and other genes critical for the immune response.
Several pathways are associated with activation of the NFAT enhancer
element, including calcineurin and protein kinase C
Elk-1/
SRF
MAPK/ERK
HIF1A
Hypoxia
AP1
MAPK/JNK
NFAT
PKC/Ca++
Probe
pathway
Significance
Fluo-4
Intracellular calcium
Detect intracellular calcium influx. Calcium ions in cellular cytoplasm are
detected by increased fluorescence of Fluo-4.
JC1
MitoSox
PI
Mitochondrial
membrane potential
Mitochondrial
superoxide
generation
Cell membrane
Detect the level of mitochondria membrane depolarization.
Detect the superoxide radicals in mitochondria.
Propidium Iodide (PI) Detects the cell membrane integrity.
4