1. Probing the Expression Patterns of System x c - in Human Glioma Cells Mazi Condelee Chase Lab Summer 2007 REACH Program

2. Background Research  System x c - is a neurotransmitter transport system important in glutathione homeostasis Exchanges Cystine for Glutamate  Increases amount of glutathione

3. Research Question  As a cell proceeds through the cell cycle, different amounts of reactive oxygen species are produced  Previous work in the lab suggest that System x c - expression patterns change in response to the level of reactive oxygen species  We hypothesize that the expression patterns of System x c - will change as the cell progresses through the cell cycle

4. Protocol  Utilize U138MG (human glioma) cells to study the trafficking of System x c - through the cell cycle  Maintain cell line in MEM media supplemented with Fetal Bovine Serum  To synchronize cell division, we serum starve cells for 24 hours  Addition of MEM media + FBS at T=0 to initiate cell cycle progression  Fix cells at T=0, 2, 4, 6 hours to visualize System x c -

5. Immunocytochemistry  Use immunocytochemistry to examine expressions and cellular localization of xCT and 4F2HC (components of System x c -) during cell cycle progression All signals that are yellow, indicate colocalization of 4F2HC and xCT

6. Results T=0 xCT and 4F2HC appear primarily in endoplasmic reticulum and in vesicles outside of the nucleus Very little transporter is observed on the membrane

7. Results T=2 Expression is more diffuse Some remaining staining in ER, but fewer vesicles are apparent

8. T=4 Less expression in ER And vesicles More expression on the Plasma membrane

9. Results, T=6 Similar expression as T=0 with expression primarily in ER

10. Conclusions and Future Work Expression of System x c - is initially more concentrated in the ER Expression becomes more diffuse Next step: use flow cytometry to better examine expression through the cell cycle. We will also use organelle markers to confirm our hypotheses about transporter location

11. Acknowledgements  Dr. Leah Chase  Lab Members A. Goltz T. Henderson A. Hilbrand L. Jones S. Sherburn M. Wixson  Hope College Departments of Biology and Chemistry  REACH Program  The Campbell Foundation  NSF-MRI