Supplementary materials
Primers used in real-time PCR detection of ChIPed DNA and cDNA
To validate the results of NF-κB binding and gene expression obtained by ChIP-Seq and DNA microarray,
several genes were selected to validate by real-time PCR. The primers used in real-time PCR detection
were listed in Table S1.
Table S1 PCR primers for detecting gene transcription and ChIPed genomic DNA
PCR primers for detecting gene transcription with cDNA (5′ to 3′)
WNT5A-cF
GACTATGGCTACCGCTTTGC
WNT5A-cR
CCACCTTGCGGAAGTCTGCC
TRPM7-cF
CTGTTCCAGTAGTTGTGTGT
TRPM7-cR
GTCTCGGAATGGTAAGGAAT
IREB2-cF
TAGTCGGCACAGATTCACACA
IREB2-cR
CAAATCACCCTGTTTTACTGC
TNFAIP2-cF
AGCATCACGCTGGACTTG
TNFAIP2-cR
TTTTCCAGGGTCTCCACA
SLC2A3-cF
TGGCTGCTTTATGGGACTG
SLC2A3-cR
ATGGGCTCTTGAACACCTG
IRAK2-cF
AGCACCTCCATTCCTAAG
IRAK2-cR
TCCTGTAGGGAACCATTT
GAPDH-cF
GTGGCAAAGTGGAGATTGTT
GAPDH-cR
CTCGCTCCTGGAAGATGG
PCR primers for detecting NF-κB p65 binding with ChIPed genomic DNA (5′ to 3′)
WNT5A-gF
AGTAGGGGCCGACTGACA
WNT5A-gR
TGGTTTTGGTGTGGATGT
TRPM7-gF
TCCTGGCTAACATGGTGA
TRPM7-gR
AGGCTTGTCTTGAACTCC
IREB2-gF
CTCCACCTCCTGGGTTCAA
IREB2-gR
AGTCACGGTCGGCAGAAAG
TNFAIP2-gF
TTCGCCAAGTCCAGTCCC
TNFAIP2-gR
TTGCCTGATTATGTCATTAGTA
SLC2A3-gR
AGAAGTCTGGTAGTTTGCC
SLC2A3-gF
GGGATTCTCTTGCTGGTTC
IRAK2-gF
CACGAGGGTGCTTTAGTT
IRAK2-gR
ATCTGAAGAAAGCGAAGC
IL6-gF
AGCGCTAGCCTCAATGACGACCT
IL6-gR
GGCAGTTCCAGGGCTAAGGATTTC
NFKBIA-gF
GACGACCCCAATTCAAATCG
NFKBIA-gR
TCAGGCTCGGGGAATTTCC
IκBε-gF
TTCCTTGACTGCTGGGGTG
IκBε-gR
CTTTGGGGGAGGGCATGTAG
Groγ-gF
CCCAGCTCTTTCCTCCAACC
Groγ-gR
TCGGCGAACCCTTTTTATGC
GO analysis of DTGs of NF-𝛋B RelA in HeLa cells
To find the biological processes involved in by the identified DTGs of NF-κB RelA in
TNFα-stimulated HeLa cells, GO analysis was performed. It was found that 266 activated genes were
involved in 251 biological processes, 24 molecular function and 21 cell component GO terms with P
value over 0.05. 318 repressed genes were involved in 78 biological processes, 29 molecular function and
30 cell component GO terms with P value over 0.05. These significant GO terms were further classified
according to their relationships, such as all GO terms related to immunity. As a result, main biological
processes involved in by NF-κB RelA were obtained, which were displayed in Figure S1.
It was clear that the activated target genes mainly related with 39 GO term key words, including
typical defense response, leukocyte (activation, differentiation and migration), inflammatory response,
chemotaxis (leukocyte, lymphocyte, neutrophils), locomotion (locomotory behavior and positive
regulation), immune (response to, system development and process, signal transduction, T cell activation
and differentiation, and regulation of immune response and system process), lymphocyte (activation and
differentiation), response to various stimuli (endogenous, external, extracellular, abiotic, biotic, chemical,
corticosteroid, cytokine, hormone, mechanical), responses (to starvation, wounding, bacterium, LPS,
molecule of bacterial origin, nutrient levels, organic substance), stress (response to stress and regulation),
apoptosis (apoptosis and anti-apoptosis), cell cycle (regulation), cell death (programmed cell death), cell
differentiation, cell proliferation, cytokine (production and biosynthesis), gene expression (DNA-binding,
transcription, translation), biosynthetic process (macromolecule), metabolic process (phosphate, nucleic
acid，protein, nitrogen compound), development (ectoderm, epidermis, organ, system), signal transduction
(cytokine-mediated, enzyme linked receptor protein, protein kinase cascade, transforming growth factor
beta receptor, intracellular signaling cascade, regulation).
Besides these typical NF-κB related typical biological processes, the GO analysis also revealed that
the activated genes also related with muscle (smooth and striated muscle cell development and
differentiation；muscle fiber and tissue development；heart development), bone (regulation of ossification),
blood (circulation, blood vessel development and morphogenesis, angiogenesis, vasculature development,
hemopoietic or lymphoid organ development, hemopoiesis), reproduction (female sex differentiation,
ovulation, development of primary female sexual characteristics), homeostasis (chemical homeostasis,
homeostatic process), enzyme activity (regulation of oxidoreductase, transferase and kinase activity,
positive regulation of catalytic activity), transport (regulation), anatomical structure (development and
morphogenesis), localization (regulation), and hormone secretion, behavior, and others.
The molecular function analysis of activated genes revealed that the most enriched GO terms related
to transcription regulation and DNA-binding. The former contained 7 related Go terms, including
transcription factor activity (40 genes), transcription regulator activity (49 genes), transcription repressor
activity (18 genes), transcription factor binding (22 genes), transcription activator activity (17 genes),
transcription cofactor activity (15 genes), transcription coactivator activity (9 genes), transcription
corepressor activity (7 genes), and RNA polymerase II transcription factor activity (8 genes). The latter
contained 5 related GO terms, including sequence-specific DNA binding (25 genes), DNA binding (56
genes), double-stranded DNA binding (6 genes), nucleic acid binding (64 genes) and structure-specific
DNA binding (7 genes). The most enriched molecular function GO terms of repressed genes related to
nucleic acid binding and transcription repressor activity. These data demonstrated that the key molecular
function of NF-κB DTGs mainly related with nucleic acid binding and transcription regulation.
GO analysis of repressed genes revealed that most GO terms of biological processes related to cell
propagation, such as cell cycle, mitotic cell cycle, cell cycle process, cell cycle phase, M phase of mitotic
cell cycle, mitosis, M phase, nuclear division, organelle fission. Another significant class of biological
process GO terms of repressed genes mainly related to cell metabolism, such as cellular biosynthetic
process and macromolecule biosynthetic process. The GO analysis demonstrated that the molecular
function of repressed genes mainly related to nucleic acid binding, transcription repressor activity
(TBL1XR1, RSF1, ID2, KLF12, RBAK, C19ORF2, PATZ1, PIAS1, ZEB1, IFI16, NRIP1) and enzyme
activity (GTPase, hydrolase, kinase, phosphotransferase and transferase).
NF-κB is a ubiquitous transcription factor that plays a key role in immune, inflammatory responses,
cell growth control and apoptosis. Many studies revealed that NF-κB activity is induced in most cell types
in response to a wide variety of stimuli, with major roles in cell activation, survival, and differentiation
(Ghosh and Hayden 2008, Hoffmann et al. 2006, Vallabhapurapu and Karin 2009). The GO analysis of
DTGs identified in this study agreed with these previously found functions. For example, most GO
categories of biological processes enriched in genes bound by NF-κB in LPS-induced U937 cells
(Schreiber et al. 2006) were also enriched in TNFα-induced HeLa cells, such as responses to stress,
wounding and stimulus, and inflammatory, defense response and immune responses. Especially, the
enriched GO categories of molecular function in two cells were almost identical, including transcription
regulator activity, binding, DNA binding, receptor binding, G-protein-coupled receptor binding,
chemokine activity, chemokine receptor binding.
Figure S1. GO analysis of DTGs of NF-κB. GO analysis was firstly performed with identified DTGs, and
then GO terms with P value over 0.05 were classified according to key words they shared which reflected
their functional relationship. Finally the GO term numbers of each class were counted and plotted into
histogram. FC, fold change of gene transcription in TNFα stimulation.
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