1. A Single Cell PIP 3 Assay Based on Akt PH domain Translocation Nancy O’Rourke Microscopy Lab, Stanford University Jim Whalen, Takako Mukai, Wei Sun Park, Liz Gehrig Grischa Chandy, Tobias Meyer

2. Macrophage Lab, UCSF Tamara Roach Robert Rebres Bioinformatics Lab, UCSD Ilango Vadivelu Bob Sinkovits Molecular Biology Lab, Caltech Iain Fraser Joelle Zavzavadjian Leah Santat Jamie Liu Estelle Wall Kavitha Dhandapani

3. A Single Cell PIP 3 Assay Based on Akt PH domain Translocation Assay Protocol and Analysis Testing the FXM Ligands Perturbation of the System with RNAi and Chemical Reagents

4. Assay Protocol Transfect YFP Akt PH domain and CFP KRAS CAAX Cells in 8-well chamber slide Serum Deprive 1 hour Run at 37 o Image on confocal microscope 40 scans w/ 10 second interval Ligand added at 50 seconds

5. -0.32380.4148 Correlation Coefficient Image Analysis Time0 secs.140 secs. C5a

6. Individual and Average Responses Akt PH Domain Translocation after 100 nM C5a Stimulation

7. Diverse Patterns of Akt PH Domain Translocation

8. Responses to C5a, UTP and Fc  R1 C5a responses are consistent and robust. C5a stimulation used for current perturbation studies. Translocation does not occur following UDP/UTP stimulation. Responses to Fc  R1stimulation vary with cell batch.

9. 100  M UTP 0.44  M F(ab’) 2 100 nM C5a Akt PH Domain Translocation with UTP, FcgR1 and C5a Stimulation

10. Variability in the Fc  R1 Response

11. Perturbation Experiments C5a stimulation utilized for perturbation studies. Lentiviral cell lines expressing GFP interfere with the YFP-Akt PH fluorescence signal. Three CD4 lentiviral cell lines: SHIP1, G  4 and G  i3. Chemical perturbation studies initiated with LY

12. Knockdown of SHIP1 Increases Translocation

13. Testing of lentiviral lines for knockdown efficacy IB: G alpha i3 UCIP SHIP1 Gi3 49 38 SHIP1 Gi3 IB: SHIP1 UCIP 188 98 99% Knockdown in UC lines. Caltech Lab

14. Knockdown of G  4 does not alter Translocation

15. UCSF lab Results are Consistent with Population Ca 2+ Results

16. LY Pretreatment Decreases the Translocation 10 minute pretreatment with100  M LY

17. GRK2 (4,5,6) Arrestin 2,3 G  q, G  11 P2Y2,6 G  1,2 (4,5) G  i2,3 CD64 Lyn, Hck Syk PTEN SHIP-2 (1) PLC  2,3 (4) PLC  1,2 Akt1,2,3 PI3K p85 , p110  p101, p110  IP3R1,2,3 IP3KTB(C) SERCA2,3 PMCA1,3 (4) Btk, Tec C5aR Lentiviral Lines with CD4 marker LY PI(3,4,5)P 3 Gil Sambrano Lily Jiang

18. FXM Translocation Data Display

19. N - 8 cells % cells with Translocation – 88% Treatment Summary

20. Conclusions The Akt PH domain translocation assay can be employed to probe PIP3 signaling pathway C5a provides a consistent and robust response in the translocation assay Results from preliminary studies confirm the roles of PI3K and SHIP1 in PIP3 signaling. FXM Translocation Display Available

21. Future Directions Increase throughput of assay. Screen Additional Non-fluorescent Lentiviral Cell Lines Develop Methods for Using Duplex RNAi with Translocation Assay Refine FXM Display