Collections of siRNAs targeting
key human genes are ideal for studying protein function in
cells. To identify genes involved in a given cellular process,
cells can be transfected with different siRNAs and assayed for
distinct response profiles such as cell cycle arrest.
Cellomics, Inc., who pioneered the field of High Content
Screening (HCS), has built cellular response profiles for
several of Ambion's Silencer
utilizing their automated cell-based screening platform (1).
For example, a Silencer
Validated siRNA targeting the
TNFalpha receptor (TNFR) can be used to identify gene products
associated with TNFalpha-induced activation of the NF-kappaB
pathway. Upon activation, NF-kappaB undergoes a translocation
from its normal localization in the cytoplasm to the nucleus.
Cellomics ArrayScan HCS Reader can be utilized to rapidly
quantitate such nuclear translocation events. In the
experiment detailed below, TNFalpha-triggered activation of
NF-kappaB nuclear translocation was used as a functional HCS
assay of TNFR modulation. When TNFR expression level was
reduced, NF-kappaB nuclear translocation was expected to be
suppressed or reduced. On the other hand, NF-kappaB nuclear
translocation triggered with ligands distinct from TNFalpha
was expected to occur normally. Thus, a functional assay for
the level of TNFR protein in the cell, which did not rely on
direct measurement of TNFR protein content, was created.
In cells treated with TNFalpha, NF-kappaB translocates
from its predominately cytoplasmic localization to become concentrated
in nuclei where it interacts with DNA as a transcription factor (Figure
1). In cells transfected with TNFR siRNA, the translocation of NF-kappaB
in response to TNFalpha treatment was dramatically reduced.
(Figure 4 from the print edition of TechNotes)
Reduction in TNFalpha-induced NF-kappaB Translocation
Caused By an siRNA Targeting the TNFalpha Receptor. HeLa cells were transfected with siRNAs
targeting IL-1 (Control siRNA) and the TNFalpha receptor (TNFR siRNA).
Forty eight hours post-transfection, the cells were recovered from the
24 well plates in which they were transfected and transferred to wells
of a 96 well plate. Following overnight recovery, the cells were processed
for immunofluorescence staining of NF-kappaB.
There are several distinct signaling pathways
that can trigger the nuclear translocation of NF-kappaB. This suggests
that RNAi could be used to distinguish activators of these pathways. As
shown in Figure 2, while the cell population transfected with TNFR siRNA
showed significantly reduced response to TNFalpha treatment (asterisk),
the same cell population still responded robustly to IL1alpha treatment.
This clearly shows the specificity of the siRNA transfection and highlights
the applicability to drug discovery.
Nuclear Translocation of NF-kappaB in Response
to 10 ng/mL TNFalpha or IL1alpha in Untreated or TNFR siRNA Transfected
HeLa Cells. Nuclear translocation was
dily quantitated using Cellomics' Cytoplasm to Nucleus Translocation
BioApplication for the ArrayScan HCS Reader.
1. Giuliano KA, Haskins
JR, Taylor DL. (2003) Advances in High Content Screening for
Drug Discovery. ASSAY Drug Develop Technol (In Press).
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