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Sample Graphs for torc2 Paper

Sample Graphs for torc2 Paper. Akt Crystal Structure. S473A. T450A. D292A. T308A. K179M. R25C. K8R. AA131. E17K. AA107. K14R. PH Domain. - Akt crystal structure PDB #3O96 - AAs 430-480 modeled using I-TASSER - Image generated using CHIMERA (UCSF). Mutational Data (HCT-116).

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Sample Graphs for torc2 Paper

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  1. Sample Graphs for torc2 Paper

  2. Akt Crystal Structure S473A T450A D292A T308A K179M R25C K8R AA131 E17K AA107 K14R PH Domain - Akt crystal structure PDB #3O96 - AAs 430-480 modeled using I-TASSER - Image generated using CHIMERA (UCSF)

  3. Mutational Data (HCT-116) Control (HCT-116 wt) G0 Experimental (HCT-116 Akt1/2 k.o.) Control (HCT-116 wt) Asymmetry Experimental (HCT-116 Akt1/2 k.o.) Akt1 Akt2 No rescue D292A T308A K179M G0 ASY G0 ASY G0 ASY G0 ASY G0 ASY G0 ASY S473A T308A/S473A T450A T308D S473D myrΔ1-107 G0 ASY G0 ASY G0 ASY G0 ASY G0 ASY G0 ASY E17K R25C myrΔ1-107 K179M Δ1-131 Δ1-107 G0 ASY G0 ASY G0 ASY G0 ASY G0 ASY K8R K14R K8/14R G0 ASY G0 ASY G0 ASY

  4. Mutational Analysis(HCT-116) G0 Count AKT1/2 k.o. AKT rescue ΔKinase domain ΔCanonical phosphorylation sites ΔUbiquitination domain Myristylation ΔPlekstrin homology % of Wildtype Phenotype E17K R25C delPH107 delPH131 Akt1/2 k.o. AKt1 rescue AKt2 rescue K179M D292A T308A S473A T308A/S473A T450A T308D S473D K8R K14R K8R/K14R Myr-delPH107 Myr-delPH107(K179M) Genetic Construct Asymmetry Count AKT1/2 k.o. AKT rescue ΔKinase domain ΔCanonical phosphorylation sites ΔUbiquitination domain Myristylation ΔPlekstrin homology % of Wildtype Phenotype E17K R25C delPH107 delPH131 Akt1/2 k.o. AKt1 rescue AKt2 rescue K179M D292A T308A S473A T308A/S473A T450A T308D S473D K8R K14R K8R/K14R Myr-delPH107 Myr-delPH107(K179M) Genetic Construct

  5. Pharmacologic Data (HCT-116) Control (HCT-116) G0 Experimental (HCT-116 + drug.) Control (HCT-116) Asymmetry Experimental (HCT-116+ drug.) AKT PH domain inhibitors AKT kinase inhibitors AKt1/2i (20uM) MK2206 (10uM) AZD5363 (50uM) GDC0068 (50uM) G0 ASY G0 ASY G0 ASY G0 ASY Torc1 inhibitors Torc1/2 inhibitors Rapamycin (20uM) Rad001 (10uM) INK128 (0.05uM) AZD8055 (0.7uM) Palomid529 (10uM) TORIN 1 (0.5uM) G0 ASY G0 ASY G0 ASY G0 ASY G0 ASY G0 ASY Proteasomal inhibitors MG132 1d (1uM) Velcade 1d (0.4uM) G0 ASY G0 ASY

  6. Small Molecule Treatment (HCT-116) G0 Count AKT PHD inhibitor Torc1 inhibitor AKT kinase domain inhibitor Torc1/2 inhibitor Torc1 inhibitor Proteasomal inhibitor Torc1/2 inhibitor Proteasomal inhibitor % of Wildtype Phenotype (Logarithmic Axis) % of Wildtype Phenotype (Without AKT inhibitors) Akt1/2i MK2206 GDC0063 AZD5363 Rapamycin Rad001 Torin1 Palomid529 Ink128 AZD8055 MG132 Velcade Rapamycin Rad001 Torin1 Palomid529 Ink128 AZD8055 MG132 Velcade Small Molecule Small Molecule Asymmetry Count AKT PHD inhibitor Torc1 inhibitor AKT kinase domain inhibitor Torc1/2 inhibitor Torc1 inhibitor Proteasomal inhibitor Torc1/2 inhibitor Proteasomal inhibitor % of Wildtype Phenotype (Logarithmic Axis) % of Wildtype Phenotype (Without AKT inhibitors) Rapamycin Rad001 Torin1 Palomid529 Ink128 AZD8055 MG132 Velcade Akt1/2i MK2206 GDC0063 AZD5363 Rapamycin Rad001 Torin1 Palomid529 Ink128 AZD8055 MG132 Velcade Small Molecule Small Molecule

  7. Pharmacologic Data (MCF-7) Control (MCF-7) G0 Experimental (MCF-7 + drug.) Control (MCF-7) Asymmetry Experimental (MCF-7 + drug.) AKT PH domain inhibitors AKT kinase inhibitors MK2206 (3uM) AZD5363 (5uM) AKt1/2i (2uM) GDC0068 (5uM) G0 ASY G0 ASY G0 ASY G0 ASY Torc1 inhibitors Torc1/2 inhibitors Rapamycin (20uM) Rad001 (5uM) INK128 (0.01uM) AZD8055 (0.1uM) Palomid529 (20uM) TORIN 1 (0.25uM) G0 ASY G0 ASY G0 ASY G0 ASY G0 ASY G0 ASY Proteasomal inhibitors MG132 1d (10uM) Velcade 1d (4uM) TTC3 K.D. TTC3hp3 K.D. G0 ASY G0 ASY G0 ASY G0 ASY

  8. Small Molecule Treatment (MCF7) G0 Count AKT PHD inhibitor Torc1 inhibitor AKT kinase domain inhibitor Torc1/2 inhibitor Torc1 inhibitor Proteasomal inhibitor Torc1/2 inhibitor Proteasomal inhibitor % of Wildtype Phenotype (Logarithmic Axis) % of Wildtype Phenotype (Without AKT inhibitors) Akt1/2i MK2206 GDC0063 AZD5363 Rapamycin Rad001 Torin1 Palomid529 Ink128 AZD8055 MG132 Velcade Rapamycin Rad001 Torin1 Palomid529 Ink128 AZD8055 MG132 Velcade Small Molecule Small Molecule Asymmetry Count AKT PHD inhibitor Torc1 inhibitor AKT kinase domain inhibitor Torc1/2 inhibitor Torc1 inhibitor Proteasomal inhibitor Torc1/2 inhibitor Proteasomal inhibitor % of Wildtype Phenotype (Logarithmic Axis) % of Wildtype Phenotype (Without AKT inhibitors) Rapamycin Rad001 Torin1 Palomid529 Ink128 AZD8055 MG132 Velcade Akt1/2i MK2206 GDC0063 AZD5363 Rapamycin Rad001 Torin1 Palomid529 Ink128 AZD8055 MG132 Velcade Small Molecule Small Molecule

  9. shRNA Rictor Data (HCT-116) Control (HCT-116 + non-silencing shRNA) G0 Experimental (HCT-116 + hairpin) Control (HCT-116 + non-silencing shRNA) Asymmetry Experimental (HCT-116 + hairpin) (Control = non-silencing) Empty Vector shRNA-225915 shRNA-120392 shRNA-120389 shRNA-38014 G0 ASY G0 ASY G0 ASY G0 ASY G0 ASY (Control = empty-vector) Non-Silencing shRNA-225915 shRNA-120392 shRNA-120389 shRNA-38014 G0 ASY G0 ASY G0 ASY G0 ASY G0 ASY

  10. Live-cell Imaging Results2/18/12 • HCT-116 shRictor variants • +/- 1ug/mL doxy

  11. Boxplot of Cell Cycle Time(HCT116 shRNA-Rictor 225915) No doxy 1ug/mL doxy

  12. Alpha Plot comparison No doxy 1ug/mL doxy

  13. Live-cell Imaging Results3/12/12 • 3 conditions: • HCT-116 shNon-silencing + 2ug/mL doxy • (~700 events) • HCT-116 shRictor225915 No doxy • (~1300 events) • HCT-116 shRictor225915 No +2ug/mL doxy • (~1300 events)

  14. Cell Fate – Overall Comparison Death (20%) Non-silencing 2 ug/mL Doxy Mitosis (52%) Lost (28%) Other (1%) Death (20%) shRNA225915 No Rx Mitosis (55%) Lost (24%) Other (1%) Death (18%) Mitosis (55%) shRNA225915 2 ug/mL Doxy Lost (26%)

  15. Alpha Plot comparison Non-silencing + doxy 225915 + No doxy 225915 + doxy

  16. Boxplot of Cell Cycle Time(HCT116 shRNA-Rictor 225915) Non-silencing 2ug/mL doxy 225915 2ug/mL doxy 225915 2ug/mL doxy 225915 No doxy

  17. Beta Plot comparison Non-silencing + doxy 225915 + No doxy 225915 + doxy

  18. Boxplot of Sibling-sibling Cell Cycle Time(HCT116 shRNA-Rictor 225915) Non-silencing 2ug/mL doxy 225915 2ug/mL doxy 225915 2ug/mL doxy 225915 No doxy

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