Conference Presentation: Dynamic live-cell visualization and quantification of Akt activity скачать в хорошем качестве

Conference Presentation: Dynamic live-cell visualization and quantification of Akt activity

Studying dynamic changes in kinase activity can be difficult, and assays to measure these changes in live cells within a physiologically relevant environment are lacking. Standard approaches to monitoring Akt kinase activity are limited to end-point assays, which lack the ability to monitor the effects of treatments over time. During this presentation we demonstrate the utility of the Incucyte® Kinase Akt Lentivirus Reagent, encoding a kinase translocation reporter based on a green fluorescent protein-tagged Akt substrate whose subcellular localization is phosphorylation-dependent, and a red fluorescent nuclear protein to denote the nuclear/cytoplasmic boundary. Learn more: https://www.sartorius.com/en/products... 00:30 Dysregulation of signal transduction pathways is associated with cancer initiation, progression, and recurrence. The PI3K/Akt signaling pathway has been extensively investigated as a therapeutic target due to its mechanistic association with several hallmarks of cancer Studying dynamic changes in kinase activity can be difficult, and assays to measure these changes in live cells in a physiologically relevant environment are lacking. Standard approaches to monitoring Akt kinase activity are limited to end point assays which lack the ability to monitor the effects of treatments over time Standard approaches to monitoring Akt kinase activity are limited to end point assays which lack the ability to monitor the effects of treatments over time. The Incucyte® Kinase Akt Green/Red Lentivirus encodes a kinase translocation reporter based on a green fluorescent protein-tagged Akt substrate whose subcellular localization is phosphorylation-dependent, and a red fluorescent nuclear protein to denote the nuclear/cytoplasmic boundary. This enables analysis of dynamic changes in Akt kinase activity via a nuclear translocation ratio metric. By using the Incucyte® Live-Cell Analysis System to monitor Akt activity, we can observe variable time- and concentration-dependent responses to treatments targeting the PI3K/Akt pathway. 02:05 Live-cell imaging of Akt kinase activity 03:34 Concentration and time-dependent effects of compounds on Akt activity 06:02 Effects of serum starvation and Akt activation over time 08:31 Kinetics of Akt activation by Epidermal Growth Factor 11:37 Monitoring Akt activity in PTEN knockout mutant cells 14:12 Concurrent measurements of Akt activity and proliferation 17:05 By using the Incucyte® Live-Cell Analysis System combined with the Kinase Akt reagent to monitor Akt activity, we can observe variable time- and concentration-dependent responses to treatment with inhibitors targeting the PI3K/Akt pathway. The Kinase Akt reagent can be also be used to study activation of Akt. After 4 hours of incubation in serum-free conditions, cells treated with growth factors displayed variable Akt activation profiles over time. HeLa cells with a PTEN knockout mutation maintained high levels of Akt activity in serum free conditions, in contrast to wild type HeLa cells which exhibit a decrease in Akt activation upon removal of serum. This was confirmed with representative live cell images. Concurrent measurements of Akt activity and cell proliferation can be made by monitoring the NTR and Red Object Counts over time. While both MDA-MB-231 and T-47D cell lines exhibited Akt inhibition by MK2206, differential effects on proliferation were observed The Incucyte® Kinase Akt Lentivirus Reagent provides valuable kinetic measurements of Akt activity using live cells within a physiologically relevant environment. Follow us on LinkedIn:   / incucyte-live-cell-analysis-systems   Be sure to subscribe to have better access to our videos! #cancerresearch #livecellimaging #labelfree