What a ride it has been!
The 12wdc experimental work is complete. Both GenieUs and Arctoris are closing for the year, so this will be the final results-focussed post. We will be providing a project recap early next year (week 12), where we will dissect the project in-depth, and share the key takeaways.
Let’s get into the final results.
We have matured healthy and ALS motor neurons. Healthy motor neurons were treated with a known EIF2a kinase (compound) and two compound inhibitors (X + Y), to assess the effects of the compound inhibitors on EIF2a phosphorylation levels. Conversely, ALS motor neurons are known to have high EIF2a phosphorylation levels, so there was no need to add the compound to these cells. We only added the two compound inhibitors (X + Y), to observe the effects on the already increased EIF2a phosphorylation levels.
Prior to the experimental work, we had to ensure differentiation of both motor neuron groups via immunofluorescence (IF) imaging. Motor neurons were assessed for the expression of three specific neuronal markers.
1. Blue – Nuclei
2. Red – Beta III tubulin (S100b)
3. Choline acetyltransferase (NDRG2)
For descriptions of each of these markers, please refer to week 6s post.
Once successful differentiation was confirmed, both healthy and ALS cells were treated with the compound inhibitors as follows:
Note: Healthy cells were co-treated with the compound
EIF2a phosphorylation levels were quantified after 24 hours
The healthy motor neurons responded very well to the compound treatment (from 40 to 80% increase). However, the graph shows that the compound inhibitors had no significant effect on EIF2a phosphorylation levels. Unfortunately, on this occasion, ALS motor neurons did not show an increase in EIF2a phosphorylation levels. Therefore, we cannot conclude whether the compound inhibitors had any effects on EIF2a phosphorylation levels in the ALS treated cells. Compared to the results observed in week 2 where ALS motor neurons had EIF2a phosphorylation levels of approximately 50%, this week, the levels were approximately 30%. This demonstrates that our ALS cells are behaving in an inconsistent manner.
As our experimental work is now officially complete, we wanted to thank the 12wdc community for their continued support and encouragement throughout the project. We are excited to share a project recap when our team reconvenes in the new year. We wish everyone happy holidays and a happy new year.