Quantifying nuclear p65 as a parameter for NF‐κB activation: Correlation between ImageStream cytometry, microscopy, and Western blot

Abstract

The nuclear factor kappa B (NF-κB) pathway, which regulates many cellular processes including proliferation, apoptosis, and survival, has emerged as an important therapeutic target in cancer. Activation of the NF-κB transcription factor is associated with nuclear translocation of the p65 component of the complex. Conventional methods employed to determine nuclear translocation of NF-κB either lack statistical robustness (microscopy) or the ability to discern heterogeneity within the sampled populations (Western blotting and Gel Shift assays). The ImageStream platform combines the high image content information of microscopy with the high throughput and multiparameter analysis of flow cytometry which overcomes the aforementioned limitations of conventional assays. It is demonstrated that ImageStream assessment of receptor-mediated (TNFα) and drug (Daunorubicin, DNR)-induced NF-κB translocation in leukemic cell lines correlates well with microscopy analysis and Western blot analysis. It is further demonstrated that ImageStream cytometry enables quantitative assessment of p65 translocation in immunophenotypically-defined subpopulations; and that this assessment is highly reproducible. It is also demonstrated that, quantitatively, the DNR-induced nuclear translocation of NF-κB correlates well with a biological response (apoptosis). We conclude that the ImageStream has the potential to be a powerful tool to evaluate NF-κB /p65 activity as a determinant of response to therapies designed to target aberrant NF-κB signaling activities.

Quantitate nuclear images for clinical diagnosis

“…Minderman and colleagues from Buffalo, New York, and Seattle, Washington, USA (this issue, page 776) applied the image flow cytometry approach in order to analyze cell nuclei labeled by multiple different chromosome fluorescence in situ hybridization (FISH) probes to enumerate abnormal chromosome numbers. This work continues previous work by the authors using image stream to analyze cell nuclei in suspension (4). In their present work, the authors demonstrate the high level of accuracy obtained by their method to determine aneuploidy by multiple FISH in flow taking advantage of the multispectral capabilities of the Image Flow cytometer.…”

Differential induction of plasma cells by isoforms of human TACI

“…25 To compare NF-kB activation in transduced cells, A20 cells expressing either the short or long isoform, GFP alone, or the S194X mutant were starved for 6 hours to achieve basal signaling ( Figure 3A). Baseline cytoplasmic p65 staining was noted for cells containing both short and long TACI isoforms, reflecting a basal activation state noted previously for TACI-expressing cells in the absence of ligands.…”

Pevonedistat, a NEDD8-activating enzyme inhibitor, is active in mantle cell lymphoma and enhances rituximab activity in vivo

“…NF-kB activity was determined using a nuclear translocation assay as previously described. 24 Changes in Ikka and phosphoIkBa were determined by western blot analysis.…”