1. Seattle Children’s Hospital & UW lab Ava Torjani
PICS INTERNSHIP 2016
Seattle Children’s Hospital & UW lab
Ava Torjani

2. Summary of techniques learned:
DNA Extraction, Nanodrop, PCR (Mouse colony DNA)
Tissue Culture: Passaging, freezing, thawing, counting cells using hemocytometer, treating cells with EGF/NTS.
IHC (with pictures of slides)
Western blotting (Harvesting cell lysates, running BCA assay, obtaining and interpreting standard curve, observed and assisted with full procedure).
Making cDNA, running rt-PCR (Taqman and Sybr green), and making graphs on GraphPad Prism software
ICC (plating cells on chambered slides, fixing, and BrdU staining).

3. Urea Cycle and FL-HCC
Renay’s rt-PCR results demonstrated lower expression of OTC in human and mouse tumor cells compared to normal ones.

4. Urea Cycle and FL-HCC Follow-up concept for FL-HCC: Plan:
Higher PKA expression
Greater pHNF4-alpha
Lower binding affinity to OTC promoter
Lower OTC gene and protein expression
Lower ornithine, citrulline, arginine
Greater ammonia + lower urea production
Follow-up concept for FL-HCC:
Plan:
rt-PCR for expression of HNF4-alpha and its target genes (HNF1-alpha, Aldolase B, PEPCK, and liver pyruvate kinase).
IHC for OTC expression on normal and tumor cells.
Expected pattern: Down-regulation of all target genes, no change in OTC expression.
Expected pattern: Lower OTC expression in tumor cells compared to normal cells.

5. rt-PCR Results Type Description 9-N Normal adjacent to 10 10-T
FL-HCC tumor adjacent to 9
N-N
Normal adjacent to T
T-T
FL-HCC tumor adjacent to N
4/9-T
Lymph node FL-HCC tumor
4/10-T
Liver FL-HCC tumor
rt-PCR Results

6. rt-PCR Results Conclusion
No coherent results to move forward with – compensatory mechanism?
More effective to compare levels of pHNF4-alpha between normal and tumor cells; however, the antibody for pHNF4-alpha could not be found.

7. IHC for OTC expression
FL-HCC, 400x
FL-HCC, 10x

8. IHC for OTC Expression Normal negative control 10x Normal, 10x
Conclusion
Staining is present in the cytoplasm – none of the nuclei stained.

9. Progenitor Stem Cell Markers
Aim: To establish whether the mutation (chimeric protein) causes cells to de-differentiate.
Conducted rt-PCR for 5 progenitor stem cell markers (GAPDH as housekeeping gene):
ICAM1
KRT19
SOX9
LGR5
CD44

10. rt-PCR results

11. rt-PCR Results Conclusion
Trend in all but Sox9: Tumor has more expression of stem cell marker compared to adjacent normal.

12. Tissue Culture
Throughout internship, maintained the following cells through passaging, freezing, and thawing:
AML-12
Clone 2
Clone 4
Clone 5
Clone 10
Clone 14

13. BrdU Staining
Objective: To see if there is a difference in DNA proliferation among normal media, NTS, and EGF.
Eyeball Experiment (AML-12, Clone 14)
Couldn’t stain because slides were not appropriate for staining.
Results (confluence)
AML-12
7/20/16
7/22/16
7/27/16
Media vs. NTS
50% vs. 50%
60% vs. 75%
100% vs. 100%
Media vs. EGF
70% vs. 70%
Clone 14
7/20/16
7/22/16
7/27/16
Media vs. NTS
50% vs. 50%
85% vs. 95%
100% vs. 100%
Media vs. EGF
60% vs. 60%
Conclusion: Possible trend for greater DNA proliferation for NTS and EGF in clone 14.

14. BrdU Staining
Actual Experiment
Attempted to stain slides for Clone 4 and 10, but no BrdU staining could be found.
Modified protocol – will be staining slides for AML-12, Clone 10, and Clone 14 this week.
Using 2-chambered slides instead of single chambered slides.
Fixing with ethanol (5%), acetic acid (5%), and water (90%) instead of methanol.
Will do an antigen unmasking using HCl (didn’t unmask last time).

15. Lastly, thank you! Dr. Kimberly Riehle Renay Bauer Rigney Turnham
Heidi Kennerson
Kevin Riggle