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Macrophage Biology Lab, San Francisco

Macrophage Biology Lab, San Francisco. RNAi II (Continued). Tamara Roach Bob Rebres. Bill Seaman. Thanks. Melissa Kachura David Quan Carrie Wong. Use of RNAi to target signaling molecules. C5a. UDP. IgG2a. P2Y6 P2Y2. Fc g RI. Fc g. C5aR. G a i2 / G a i3.

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Macrophage Biology Lab, San Francisco

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  1. Macrophage Biology Lab, San Francisco RNAi II (Continued) Tamara Roach Bob Rebres Bill Seaman

  2. Thanks Melissa Kachura David Quan Carrie Wong

  3. Use of RNAi to target signaling molecules C5a UDP IgG2a P2Y6 P2Y2 FcgRI Fcg C5aR Gai2 / Gai3 Gaq /Ga11 ITAM ITAM PTKs Gb1, 2, 4, 5 + Gg PI 3-kinases PLCs P - Akt Ca2+ PIP3

  4. Summary of shRNA-transduced RAW264.7 lines • 37 Lines have been studied, covering 31 targets (4 new/week) • Using prescreened shRNA plasmids, target knock-down was • >75% in almost all transduced lines (protein or mRNA) • >90% in two-thirds • >99% in one-third • Apart from receptor knock-downs, alterations in signaling • were observed in: • Half of responses to IgG2a • One-third of responses to C5a • Only one response to UDP by 1.5-fold cutoff, but more by stats • Expected phenotypes were usually detected • Unexpected phenotypes were frequent

  5. Gaq facilitates signaling through P2Y receptors C5a UDP IgG2a P2Y6 P2Y2 FcgRI Fcg C5aR Gai2 / Gai3 Gaq /Ga11 ITAM ITAM PTKs Gb1, 2, 4, 5 + Gg PI 3-kinases PLCs Ca2+ PIP3

  6. shRNA against Gaq reduced the peak [Ca2+]i response to UDPbut increased the response to IgG2a Responses to FXM Ligands Expression of Gq (Western blot) (Peak [Ca2+]i) IB: Gq 75 50 37 Gq Grk2 Vector Mol.Biol.Lab.

  7. shRNA against Gaq reduced the peak [Ca2+]i response to UDP

  8. Gb2 is utilized by C5aR C5a UDP IgG2a P2Y6 P2Y2 FcgRI Fcg C5aR Gai2 / Gai3 Gaq /Ga11 ITAM ITAM PTKs Gb1, 2, 4, 5 + Gg PI 3-kinases PLCs Ca2+ PIP3

  9. shRNA against G2 inhibited the response to both C5a and IgG2a G2 expression Responses to FXM Ligands (Peak [Ca2+]i) (Western blot) IB: G2 49 38 28 PTEN Vector G 2 Mol.Biol.Lab

  10. The C5a signaling defect was observed in each of 3 G2-shRNA lines Line #1 Line #2 Line #3

  11. The IgG2a signaling defect was observed in 2 of 3 G2-shRNA lines Line #1 Line #2 Line #3

  12. The C5a receptor signals through Gai proteins C5a UDP IgG2a P2Y6 P2Y2 FcgRI Fcg C5aR ITAM Gaq /Ga11 ITAM Gai2 / Gai3 PTKs Gb1, 2, 4, 5 + Gg PI 3-kinases PLCs Ca2+ PIP3

  13. shRNA against Gi2 enhancedthe response to C5a Gi2A PTEN Gi3 Vector Gi2 Expression Responses to FXM Ligands (Western blot) (Peak [Ca2+]i) IB: Gi2 Mol.Biol.Lab

  14. The enhanced [Ca2+]i response to C5a was observed in each of 2 Gi2-targeted lines Line #1 Gai2 100nM C5a Vector (HBSS) 100 (nM) Line #2 i ] 2+ 50 [Ca 0 0 50 100 150 Time

  15. Pertussis toxin inhibits both Gai2 and Gai3 Pertussis toxin C5a UDP IgG2a P2Y6 P2Y2 FcgRI Fcg C5aR Gaq /Ga11 Gai2 / Gai3 ITAM ITAM PTKs Gb1, 2, 4, 5 + Gg PLCs PI 3-kinases Ca2+ PIP3

  16. The [Ca2+]i response in Gi2-targeted cells was partially resistant to pertussis toxin 100 nM C5a

  17. High concentrations of pertussis toxin failed to eliminate the [Ca2+]i response to C5a in Gi2-targeted cells (ng/ml)

  18. The next step: targeting both Gai2 and Gai3 C5a UDP IgG2a P2Y6 P2Y2 FcgRI Fcg C5aR Gaq /Ga11 Gai2 / Gai3 ITAM ITAM PTKs Gb1, 2, 4, 5 + Gg PI 3-kinases PLCs Ca2+ PIP3

  19. Overview: RNAi targeting of G proteins and regulators:Both gain of function and loss of function are observed

  20. RNAi targeting G proteins and regulators:Both expected and unexpected results were obtained = “Expected” = “Unexpected”

  21. RNAi targeting InsP and phosphoinositide metabolism:Both expected and unexpected results were obtained = “Expected” = “Unexpected”

  22. RNAi targeting tyrosine kinases and scaffold proteins:Both expected and unexpected results were obtained = Expected = Unexpected

  23. RNA-mediated RNAi: multiple phenotypes for C5a and IgG2a, but not for UDP

  24. C5a, UDP and IgG2a stimulate distinct patterns of intracellular Ca2+ flux UDP C5a IgG2a+ anti-IgG Ligand

  25. RNA-mediated RNAi: multiple phenotypes for C5a and IgG2a, but not for UDP

  26. Summary of shRNA-transduced RAW264.7 lines • 37 Lines have been studied, covering 31 targets (4 new/week) • Using prescreened shRNA plasmids, target knock-down was • >75% in almost all transduced lines (protein or mRNA) • >90% in two-thirds • >99% in one-third • Apart from receptor knock-downs, alterations in signaling • were observed in: • Half of responses to IgG2a • One-third of responses to C5a • Only one response to UDP by 1.5-fold cutoff, but more by stats • Expected phenotypes were usually detected • Unexpected phenotypes were frequent

  27. Approaches to validation of RNAi phenotypes • Replicate lines with different shRNAs • Alternate KD strategy (antisense, siRNA) • Microarrays to look for off-target effects • Knockdown reversal • Test (and confirm) a likely hypothesis (e.g., with a 2nd knockdown)

  28. Conclusions • RNAi can be efficiently used in RAW264.7 cells • Results can be replicated • Lines can be expanded • Expected phenotypes are usually detected • Unexpected and interesting phenotypes are frequent

  29. Thanks Rick Brown, UCSF

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