NF-kB and JNK signaling: Segmentation and Tracking in action!
The following video shows NIH3T3 cells (mouse fibroblasts) expressing a nuclear marker in the green channel (H2B-EGFP), the NF-kB transcription factor in the red Channel (p65-DsRed) and a genetically encoded biosensor for JNK activity in the blue channel (JNK KTR-mCerulean3). Upon addition of Interleukin 1Beta, both NF-kB and JNK are activated by the same upstream mechanisms. This results in the nuclear translocation of NF-kB (red) and a cytoplasmic translocation of the JNK KTR (blue) while the nuclear marker H2B (green) stays always nuclear. The video will play 3 times:
- Three channels merged in RGB.
- Starts with the three merged channels, then shows just the nuclear channel (H2B-EGFP) in gray scale and how we transform it into segmented objects that we can track over time. Cool!! We typically use these objects to calculate nuclear intensity.
- Starts with the gray scale movie for the red channel (p65-DsRed) then we overlay the cytoplasmic ring objects that we will use to quantify cytoplasmic mean intensity. Finally it goes back to display the three merged channels. Enjoy!