Simultaneous assessment of NF-κB/p65 phosphorylation and nuclear localization using imaging flow cytometry

Abstract

Aberrant activity of Nuclear Factor–kappaB (NF-κB) is associated with many diseases and is therapeutically targeted. Post-translational modifications, particularly phosphorylation of the RELA/p65 sub-unit, are essential for cytoplasmic to nuclear localization of NF-κB/p65 and initiation of transcription of downstream target genes. Immunoblot and phospho-flow cytometry has been used to study the relationship between phosphorylation motifs and NF-κB activation and microscopic analysis of nuclear localization of p65 is also used as a parameter for activation. The labor intensive nature of these approaches commonly limits the number of sampling points or replicates. Recent insights in the relationship between p65 phosphorylation motifs and its nuclear localization indicate that these parameters have different significance and should not be used interchangeably. In this study, we demonstrate feasibility and reproducibility of studying the relationship between p65 phosphorylation and nuclear translocation using imaging flow cytometry (IFC). TNFα- or PMA/Ionomycin-induced phosphorylation of p65 at serine 529 in cell line models and healthy donor lymphocytes served as the experimental model. IFC analysis demonstrated that expression of phosphorylated serine 529 (P-p65s529) increased rapidly following stimulation and that nuclear localization of P-p65s529 followed the nuclear localization pattern of total p65. However, in the presence of tacrolimus, P-p65s529 expression was inhibited without affecting nuclear localization of total p65. The data demonstrate the application of IFC to simultaneously assess phosphorylation of p65 and its cellular localization and the results obtained by this analysis corroborate current insights regarding the specific effect of tacrolimus on serine 529 phosphorylation.

Protein Phosphatase 1α and Cofilin Regulate Nuclear Translocation of NF-κB and Promote Expression of the Anti-Inflammatory Cytokine Interleukin-10 by T Cells

“…It was previously shown that okadaic acid, an inhibitor of PP1 and PP2A, provoked NF-B phosphorylation in MG63 human osteosarcoma cells, but the specific contribution of PP1␣ was not explored (30). In our study, costimulation-related NF-B activity was determined by measuring the phosphorylation (Ser529) of the p65 subunit and its translocation to the nucleus by imaging flow cytometry (31)(32)(33). While costimulation induced the phosphorylation of NF-B (p65) in control siRNA-treated cells, this phosphorylation was completely inhibited in the PP1 KD cells ( Fig.…”

8‐Methoxypsoralen protects bovine mammary epithelial cells against lipopolysaccharide‐induced inflammatory injury via suppressing JAK/STAT and NF‐κB pathway

“…Then NF‐κB migrates into the nucleus and activates gene expression . Following activation, the peak of NF‐κB nuclear localization occurs at 30 min . Therefore, the cells were treated with LPS for 30 min.…”

“…36 Following activation, the peak of NF-κB nuclear localization occurs at 30 min. 37 Therefore, the cells were treated with LPS for 30 min. The results in our study showed that LPS induced NF-κB translocating from cytoplasm to nucleus.…”

Omics-Based Interaction Framework – a systems model to reveal molecular drivers of synergy

“…The spatial relationship between the transcription factors and nuclear images was measured using the ‘Similarity’ feature in the IDEAS software to quantitate the mean similarity score in the cell populations per sample. A similarity score >1 represents nuclear translocation, and the shift in distribution of nuclear translocation between two samples was calculated using the Fisher’s Discriminant ratio (Rd value) (Maguire et al , 2015).…”