1 Seattle Children’s Hospital & UW lab Ava TorjaniPICS 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-TFL-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 ConclusionNo 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, 10xConclusion Staining is present in the cytoplasm – none of the nuclei stained.

9 Progenitor Stem Cell MarkersAim: 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 ConclusionTrend 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 TurnhamHeidi Kennerson Kevin Riggle