1. Progress Report #2 Sha Tim Wai 29/5/2014

2. Infectious Disease BacteriaFungusVirus ProtozoaPrion Rickettsia

3. Infectious Disease Viral Diseases

5. Virus infection Treating Virus replication Immune System HIV: Cocktail therapy Influenza virus: Tamiflu … Antibody therapy (Adaptive) Interferon therapy (Innate) …

6. Plan (provisional) Confirm the ability of DMXAA/8MeXAA to induce interferon in human cells Identify the protein/sensor responsible for DMXAA/8MeXAA induced interferon in human cells Derive mechanism/pathway for DMXAA/8MeXAA induced interferon in human cells Goal OR Ideally: DMXAA/8MeXAA as a potential therapeutic agent for treating virus disease

7. Plan (provisional) Confirm the ability of DMXAA/8MeXAA to induce interferon in human cells Identify the protein/sensor responsible for DMXAA/8MeXAA induced interferon in human cells Derive mechanism/pathway for DMXAA/8MeXAA induced interferon in human cells Goal OR Ideally: DMXAA/8MeXAA as a potential therapeutic agent for treating virus disease

8. 5,6-dimethylxanthenone-4-acetic acid (DMXAA) 8 methylxanthenone-4-acetic acid (8MeXAA)

9. D Hanahan, RA Weinberg (2000) 血管新生

11. DMXAA/8MeXAA ? Endothelial cells damage

12. 5HTCytokines TNF ILs IFNs DMXAA/8MeXAA

14. TLR PAMPs TRIF TRAF3 NAP2 TBK1 P IRF PP PP PP PP PP IFNβ MyD88 dsRNA dsDNA RNA sensor DNA sensor STINGIPS1 Plasma membrane Nucleus Toll Like Receptor pathway Cytosolic Pattern- Recognition Receptor pathway Cytoplasm

15. TBK1 P IRF PP PP PP PP PP IFNβ dsRNA dsDNA RNA sensor DNA sensor STINGIPS1 Plasma membrane Nucleus Cytoplasm

16. TBK1 P IRF PP PP PP PP PP IFNβ dsRNA dsDNA RNA sensor DNA sensor STING IPS1 Plasma membrane Nucleus DMXAA is the potent activator of TBK1-IRF-3 signaling axis (Roberts et al. 2007) Cytoplasm

17. TBK1 P IRF PP PP PP PP PP IFNβ dsRNA dsDNA RNA sensor DNA sensor STING IPS1 Plasma membrane Nucleus DMXAA is the potent activator of TBK1-IRF-3 signaling axis (Roberts et al. 2007) DMXAA STING is a direct sensor for DMXAA (Conlon et al. 2013) Mouse but not human… Cytoplasm

18. Plan (provisional) Confirm the ability of DMXAA/8MeXAA to induce interferon in human cells Identify the protein/sensor responsible for DMXAA/8MeXAA induced interferon in human cells Derive mechanism/pathway for DMXAA/8MeXAA induced interferon in human cells Goal OR Ideally: DMXAA/8MeXAA as a potential therapeutic agent for treating virus disease Recap ✔

19. Plan (provisional) Confirm the ability of DMXAA/8MeXAA to induce interferon in human cells Identify the protein/sensor responsible for DMXAA/8MeXAA induced interferon in human cells Derive mechanism/pathway for DMXAA/8MeXAA induced interferon in human cells Goal OR Ideally: DMXAA/8MeXAA as a potential therapeutic agent for treating virus disease Recap ✔

20. Identify the protein/sensor responsible for DMXAA/8MeXAA induced interferon in human cells In vitro pull down assay Small Interfering RNA (knock down) experiments Recap

21. Identify the protein/sensor responsible for DMXAA/8MeXAA induced interferon in human cells In vitro pull down assay 1 2 3 4 1: DMXAA 2: Bio-DMXAA 3: Biotin 4: DMSO Recap

22. Identify the protein/sensor responsible for DMXAA/8MeXAA induced interferon in human cells Small Interfering RNA (Knock Down Experiment) STING IPS-1 STING and IPS-1 (Double knock down) … Recap

23. Identify the protein/sensor responsible for DMXAA/8MeXAA induced interferon in human cells In vitro pull down assay Small Interfering RNA (knock down) experiments

24. Identify the protein/sensor responsible for DMXAA/8MeXAA induced interferon in human cells In vitro pull down assay 1 2 3 4 1: DMXAA 2: Bio-DMXAA 3: Biotin 4: DMSO

25. Coins (Antibodies) Table (Sample)

26. Identify the protein/sensor responsible for DMXAA/8MeXAA induced interferon in human cells Whole cell lysate (HeLa) DMXAA Biotin- DMXAA 1 hr, @ r.t. In vitro pull down assay (The Coin Toss Experiment)

27. Biotin- DMXAA Before pull down (Input) After pull down Identify the protein/sensor responsible for DMXAA/8MeXAA induced interferon in human cells Streptavidin pull down Antibodies binding In vitro pull down assay (The Coin Toss Experiment)

28. Identify the protein/sensor responsible for DMXAA/8MeXAA induced interferon in human cells Anti-RIGI Anti-MDA5 Anti-IPS1 (MAVS) Anti-STING (TMEM173) Anti-IKKε Anti-IRF3 Anti-TBK1

29. Identify the protein/sensor responsible for DMXAA/8MeXAA induced interferon in human cells Before pull down (Input) RIGI 107 kDA 117 kDA MDA5 TBK1 84 kDA IPS170 kDA IRF347 kDA IKKε81 kDA STING 42 kDA β-Actin 42 kDA DMXAA Biotin- DMXAA

30. Identify the protein/sensor responsible for DMXAA/8MeXAA induced interferon in human cells After pull down RIGI 107 kDA 117 kDA MDA5 TBK1 84 kDA IPS170 kDA IRF347 kDA IKKε81 kDA STING 42 kDA DMXAA Biotin- DMXAA

31. Identify the protein/sensor responsible for DMXAA/8MeXAA induced interferon in human cells In vitro pull down assay (The Coin Toss Experiment) Need confirmation… 1.RIG-1, STING input (Increasing cell lysate amount) 2.+ve control for the pull down (Biotin-5’ Triphosphate, pull down for RIGI) 3.One more –ve control for the pull down (Biotin only) 4.Any suggestion from you!

32. Identify the protein/sensor responsible for DMXAA/8MeXAA induced interferon in human cells Knock Down Experiment HeLa SiRNA treatment (48 hours) DMXAA/8MeXAA treatment (300µg, 2hours) hIFNβ mRNA expression Drug Treatment qPCR

33. Identify the protein/sensor responsible for DMXAA/8MeXAA induced interferon in human cells Knock Down Experiment STING IPS-1 STING and IPS-1 (Double knock down)

34. Identify the protein/sensor responsible for DMXAA/8MeXAA induced interferon in human cells Knock Down Experiment STING IPS-1 STING and IPS-1 (Double knock down) TBK1 IKKε (Ongoing) TBK1 and IKKε (Ongoing) IRF3 (Ongoing)

35. Identify the protein/sensor responsible for DMXAA/8MeXAA induced interferon in human cells

36. TBK1

37. P IRF PP PP PP PP PP IFNβ dsRNA dsDNA RNA sensor DNA sensor STING IPS1 Plasma membrane Nucleus DMXAA is the potent activator of TBK1-IRF-3 signaling axis (Roberts et al. 2007) DMXAA STING is a direct sensor for DMXAA (Conlon et al. 2013) Mouse but not human… Cytoplasm

38. Next … Improve and confirm The Coin Toss Experiment results Mass spectrometer analysis of pull down results Other gene knock down. –IKKε –IKKε and TBK1 double knock down –IRF3 –… Other human cell line (THP-1) And… your suggestions!