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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 1. (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.
Figure 2.
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
rea
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