The last post highlighted some of the results of using chloroquine to investigate the localisation and functionality of the SmartFlares. This post will deal solely with the interpretation of those results.
With the Undergraduate project reports now marked, Gemma has kindly agreed to post her project report on the blog. You can find a pdf of the report here.
As most of the methods and results are already on the blog, a slightly abridged and edited version of the discussion section is replicated below:
This is a final repeat of the basic SmartFlare experiment. HeLa cells are loaded with the three variants of SmartFlares and imaged 18h later.
The purpose of these experiments is to be able to visualise the SmartFlares (SFs) in the cell after having followed the loading protocol provided by the manufacturers. After 16-20h the SFs should be in a position to interact with mRNA in the cell and this means a cytosolic localisation. We will use DMOG to stimulate VEGF mRNA production in the cell.
The SmartFlare that we have chosen for this project is specific to vascular endothelial growth factor (VEGF) mRNA.
VEGF was selected as the target because it’s transcript becomes increased in a cell as part of a characterized response to hypoxia with the aid of hypoxia inducible factor-1 (HIF-1). Active HIF-1 is a heterodimer of α and β subunits, levels of HIF-1α are kept low by oxygen-dependent prolyl hydroxylases (PHDs). When a cell becomes hypoxic, PHD levels are reduced so more HIF-1α is present to dimerise with HIF-1β forming the active heterodimer that can act as the VEGF transcription factor.