Figure 1.
NF-κB oscillation is regulated by an auto-regulatory negative feedback loop.
Simplified schematic overview of the TNFα-induced canonical NF-κB response. TNFα binding to the TNF receptor (TNFR) activates the inhibitor of kappa-B kinase (IKK) complex, leading to phosphorylation of the inhibitor of NF-κB, IκB, upon which NF-κB is free to enter the nucleus to activate transcription of its target genes. One of the primary NF-κB target genes is IκB, which may retrieve NF-κB from the nucleus to maintain inactive IκB::NF-κB complex in the cytoplasm. Ongoing TNFR signaling can re-initiate the induction-inhibition cycle.
Figure 2.
Image-based NF-κB nuclear translocation analysis.
Time series images of GFP-p65 expressing HepG2 cells stimulated with 10 ng/mL TNFα. (A') Nuclear channel. (A'') GFP-p65 channel. Examples of multiple nuclear translocations at 30, 150 and 270 minutes (white arrow) and at 30, 120, 210 and 330 minutes (yellow arrow). Example of only one, long, nuclear translocation event (red arrow). (B) Flowchart of the individual cell NF-κB nuclear translocation analysis. 1. Splitting of the two-channel image time series of the NF-κB response 2. Nuclear image preprocessing and segmentation. 3. Tracking of nuclear mask throughout the time series. 4. Segmentation of cell locations. 5. Definition of the best ellipse fitting within a Voronoi cell (BEVC) as the cytoplasmic mask. 6. Quantification of the ratio of the nuclear and cytoplasmic GFP intensity per time-point, per cell. 7. Analysis of the nuclear translocation profile of individual cells. 8. Categorization of responses to perform population analyses.
Figure 3.
Stepwise demonstration of the image analysis method.
The original nuclear Hoechst channel (A) is pre-processed by image sharpening and background subtraction (B), followed by WMC and nuclear mask definition (C). Subsequently, the Voronoi diagram (D) is generated based on the disjointed nuclear masks. For the GFP-p65 channel, the original image (E) is preprocessed by a smoothing filter (F) for global cell location definition (G). By multiplication of the global cell masks (G) with the Voronoi diagram (D), the Voronoi mask is defined for the each cell (H). Within each Voronoi masks the cytoplasmic areas are redefined as the best-fit ellipse in each Voronoi cell (I). Figure (J) shows the composite view of original Hoechst channel, GFP-p65 channel and the BEVC segmentation result.
Figure 4.
Statistical validation of the automated image segmentation and NF-κB translocation quantification.
(A) Comparison of 3 cytoplasmic segmentation methods based on the criterion of error rate. The error rate of the Dilation method is 14.5%±3.2; of Voronoi it is 11.8%±1.4; and of BEVC it is 10.3%±2.2.* p-value<0.05; ** p-value <0.005; Paired t test (B) Example translocation profiles of (i) cells without translocation and cells with translocation, (ii) cells with and without a synchronized first round of NF-κB translocation, (iii) cells with NF-κB translocation occurring only once and cells with more than one NF-κB translocation event. (C) Bias assessment of our quantification method by comparison of the computational results with the benchmark for different subpopulation. No significant differences (P-value >0.1) were found between the computational results and the benchmark for the different cell subpopulations within a 6 hour imaging timeframe.
Figure 5.
Population analysis of NF-κB nuclear translocation perturbation by the IKKβ inhibitor BMS-345541.
Cells were pre-treated for 2 hours with increasing concentrations of BMS-345541 before TNFα stimulation. (A) Average nuclear translocation response graphs, calculated from the translocation profiles of individual cells. (B) Average nuclear translocation response graphs with standard error bars for cells with one, two or three translocation peaks. The total number of cells, the number (n) and percentage of cells which show responding number of peaks are presented (C) Analysis of the time distribution of the median of 1st, 2nd and 3rd nuclear translocation maximum in TNFα stimulated and TNFα stimulated plus 0.5 µM BMS pre-treated cells. ns: No significant difference; *** p-value <0.001; **** p-value <0.0001.
Figure 6.
Application of the individual cell NF-κB nuclear translocation analysis in siRNA screening assays.
(A) The average nuclear translocation response graphs for negative controls siCASP8, siCntrl#1, transfection reagent without siRNA (mock), and positive control siNFKBIA. Inset: representative images of mock and siNFKBIA treated GFP-p65 cells, at 0 and 30 minutes after TNFα stimulation (B) Table showing the univariate Z'-factors of all 32 individual parameters. The definitions of the 26 analogue parameters are given in Table S2. Absolute Curve Difference: the absolute point-by-point difference between control and treatment averages. (C) Multivariate Z'-factor calculation based on top-scoring univariate Z'-factors. Both the conventional as well as the robust multivariate Z'-factors exceed the confidence threshold of 0.5 by combining ≥5 top-scoring univariate Z'-factors by linear projection.