NFkB Translocation Analysis

Introduction

The CV8000 nuclear translocation analysis software enables the analysis of changes in the localization of signal molecules that transfer between cytoplasm and nuclei, such as proteins. The following is an example of the translocation analysis of NFκB, a transcription factor.
NFκB plays a key role in immune reactions and is attracting attention as a target of anticancer drugs and anti-inflammatory drugs. Under normal conditions, NFκB is coupled with the inhibitor IκB and localized in the cytoplasm.

However, IκB decomposes when signals are transmitted from receptors existing on the cellular membrane surface due to stimulation by cytokine, etc. Then, the NFκB nuclear localization signal is exposed, and NFκB transfers to the nucleus. The transferred NFκB facilitates the transcription of genes that are involved in the inflammatory response.

Analysis Results

Fig.1: Images captured using the CV8000 and recognition results (blue: nucleus (Hoechst33342), green: NFκB (Alexa Fluor488))

Images were captured using the CV8000 and changes in NFκB localization were analyzed by means of the nuclear translocation analysis software (Fig. 1). As a result, it was possible to quantify the NFκB transfer from the cytoplasm to the nuclei depending on the IL-1β and TNFα concentration and create dose-response curves based on the changes in the mean fluorescence intensity of NFκB in the nuclei and cytoplasm (Fig. 2). The analysis software also allows for the output of numerical data for the fluorescence intensity and area of individual cells. Based on these data, graphs were created using Spotfire® (Fig. 3, 4). In this way, the nuclear translocation analysis software enables detailed analysis of the behaviors of various proteins such as transcription factors and nuclear receptors.

NFκB transfer into the nuclei can be observed in the wells with IL-1β (100ng/ml) or TNFα (100ng/ml) added. In the recognition images, nuclei regions were identified and colored blue, and the regions extending 3μm out from the nuclei boundaries were designated as cytoplasm.

Experiment

1. HeLa cells were cultured in 96-well plates at the ratio of 10,000 cells/well for 24 hours.
2. TNFα and IL-1β were added. (Final concentration: 0~100 ng/ml, reaction time: 20 minutes)
3. The cells were fixed with formaldehyde, and the NFκB was stained using immunostaining, with the nuclei stained using Hoechst33342.
4. Images were captured using the CV8000 under the following conditions:
   • Magnification: 20x
   • Images captured per well: 9 Fields
   • Exposure time: Alexa Fluor 488 (488nm): 500msec, Hoechst33342 (405nm): 500msec
5. The captured images were analyzed using the nuclear translocation analysis software.
   • Nuclei regions were identified from the nuclei images.
   • Cytoplasm regions were identified from the NFκB images. (Regions extending 3μm out from the nuclei boundaries were identified as cytoplasm regions.)
   • NFκB brightnesses in the nuclei regions and cytoplasm regions were obtained, and the nuclei/ cytoplasm mean intensity was calculated.

Fig. 2: Dose-response curves of the ratio of NFκB mean fluorescence intensity in nuclei to that in the cytoplasm	Fig. 3(a): Scatter diagram of fluorescence intensity of individual cells	Fig. 3(b): Histogram of the ratio of NFκB mean fluorescence intensity in nuclei to that in the cytoplasm(nuclei/cytoplasm) of individual cells, at each reagent dosage.

Fig. 4(a): Ratio of cells whose mean NFκB fluorescence intensity ratio (nuclei/cytoplasm) is 2 or higher at each IL-1β concentration (Orange)

Fig. 4(b): Ratio of cells whose mean NFκB fluorescence intensity ratio (nuclei/cytoplasm) is 1.2 or higher at each TNFα concentration (Orange)

Spotfire® is a registered trademark of TIBCO Software Inc.

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