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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). - PowerPoint PPT Presentation
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Sample Graphs for torc2 Paper
T450A
S473A
D292A
K179M
E17K
R25C
K8R
K14R
T308A
AA107
AA131
PH Domain
Akt Crystal Structure
- Akt crystal structure PDB #3O96- AAs 430-480 modeled using I-TASSER- Image generated using CHIMERA (UCSF)
No rescue Akt1 Akt2 K179M D292A T308A
T450A T308D S473D myrΔ1-107S473A T308A/S473A
myrΔ1-107K179M
E17K R25C Δ1-131 Δ1-107
Mutational Data(HCT-116)
G0 ASY G0 ASY G0 ASY G0 ASY G0 ASY G0 ASY
G0 ASY G0 ASY G0 ASY G0 ASY G0 ASY G0 ASY
G0 ASY G0 ASY G0 ASY G0 ASY G0 ASY
Control (HCT-116 wt)Experimental (HCT-116 Akt1/2 k.o.)
Control (HCT-116 wt)Experimental (HCT-116 Akt1/2 k.o.)
G0
Asymmetry
K8R K14R
G0 ASY G0 ASY
K8/14R
G0 ASY
Mutational Analysis(HCT-116)
G0 Count
AKT1/2 k.o.AKT rescueΔKinase domain ΔCanonical phosphorylation sitesΔUbiquitination domainMyristylationΔPlekstrin homology
Akt1/2 k.o.
AKt1 rescu
e
AKt2 rescu
e
K179MD292A
T308AS4
73A
T308A/S473A
T450AT308D
S473D
K8RK14R
K8R/K14R
Myr-delPH107
Myr-delPH107(K179M)
E17K
R25C
delPH107
delPH131
% o
f Wild
type
Phe
noty
pe
Genetic Construct
Asymmetry Count
Akt1/2 k.o.
AKt1 rescu
e
AKt2 rescu
e
K179MD292A
T308AS4
73A
T308A/S473A
T450AT308D
S473D
K8RK14R
K8R/K14R
Myr-delPH107
E17K
R25C
delPH107
delPH131
Myr-delPH107(K179M)
AKT1/2 k.o.AKT rescueΔKinase domain ΔCanonical phosphorylation sitesΔUbiquitination domainMyristylationΔPlekstrin homology
% o
f Wild
type
Phe
noty
pe
Genetic Construct
AKt1/2i (20uM)
MK2206 (10uM)
TORIN 1 (0.5uM)
Palomid529 (10uM)
INK128 (0.05uM)
AZD8055 (0.7uM)
Rapamycin (20uM)
Rad001 (10uM)
Pharmacologic Data (HCT-116)
G0 ASY G0 ASY
G0 ASY G0 ASY G0 ASY G0 ASYG0 ASY G0 ASY
Control (HCT-116)Experimental (HCT-116 + drug.)G0
Asymmetry Control (HCT-116)Experimental (HCT-116+ drug.)
MG132 1d (1uM)
G0 ASY
Velcade 1d(0.4uM)
G0 ASY
GDC0068(50uM)
AZD5363(50uM)
G0 ASY G0 ASY
AKT PH domain inhibitors AKT kinase inhibitors
Torc1 inhibitors Torc1/2 inhibitors
Proteasomal inhibitors
Small Molecule Treatment (HCT-116)
G0 Count
AKT PHD inhibitorAKT kinase domain inhibitorTorc1 inhibitorTorc1/2 inhibitorProteasomal inhibitor
% o
f Wild
type
Phe
noty
pe
(Log
arith
mic
Axi
s)
Small Molecule
Asymmetry Count
Akt1/2i
MK2206
GDC0063
AZD5363
Rapamycin
Rad001Torin
1
Palomid529Ink128
AZD8055
MG132
Velcade
AKT PHD inhibitorAKT kinase domain inhibitorTorc1 inhibitorTorc1/2 inhibitorProteasomal inhibitor
% o
f Wild
type
Phe
noty
pe
(Log
arith
mic
Axi
s)
Small Molecule
Akt1/2i
MK2206
GDC0063
AZD5363
Rapamycin
Rad001Torin
1
Palomid529Ink128
AZD8055
MG132
Velcade
Rapamycin
Rad001Torin
1
Palomid529Ink128
AZD8055
MG132
Velcade
Torc1 inhibitorTorc1/2 inhibitorProteasomal inhibitor
% o
f Wild
type
Phe
noty
pe
(With
out A
KT in
hibi
tors
)
Rapamycin
Rad001Torin
1
Palomid529Ink128
AZD8055
MG132
Velcade
Torc1 inhibitorTorc1/2 inhibitorProteasomal inhibitor
% o
f Wild
type
Phe
noty
pe
(With
out A
KT in
hibi
tors
)
Small Molecule
Small Molecule
AKt1/2i (2uM)
MK2206 (3uM)
TORIN 1 (0.25uM)
Palomid529 (20uM)
INK128 (0.01uM)
AZD8055 (0.1uM)
TTC3 K.D.
Rapamycin (20uM)
Rad001 (5uM)
Pharmacologic Data (MCF-7)
G0 ASY G0 ASY
G0 ASY G0 ASY G0 ASY G0 ASYG0 ASY G0 ASY
G0 ASY
Control (MCF-7)Experimental (MCF-7 + drug.)G0
Asymmetry Control (MCF-7)Experimental (MCF-7 + drug.)
TTC3hp3 K.D.
G0 ASY
MG132 1d (10uM)
G0 ASY
Velcade 1d (4uM)
G0 ASY
GDC0068(5uM)
AZD5363(5uM)
G0 ASY G0 ASY
AKT PH domain inhibitors AKT kinase inhibitors
Torc1 inhibitors Torc1/2 inhibitors
Proteasomal inhibitors
Small Molecule Treatment (MCF7)
G0 Count
AKT PHD inhibitorAKT kinase domain inhibitorTorc1 inhibitorTorc1/2 inhibitorProteasomal inhibitor
% o
f Wild
type
Phe
noty
pe
(Log
arith
mic
Axi
s)
Small Molecule
Asymmetry Count
Akt1/2i
MK2206
GDC0063
AZD5363
Rapamycin
Rad001Torin
1
Palomid529Ink128
AZD8055
MG132
Velcade
AKT PHD inhibitorAKT kinase domain inhibitorTorc1 inhibitorTorc1/2 inhibitorProteasomal inhibitor
% o
f Wild
type
Phe
noty
pe
(Log
arith
mic
Axi
s)
Small Molecule
Akt1/2i
MK2206
GDC0063
AZD5363
Rapamycin
Rad001Torin
1
Palomid529Ink128
AZD8055
MG132
Velcade
Rapamycin
Rad001Torin
1
Palomid529Ink128
AZD8055
MG132
Velcade
Torc1 inhibitorTorc1/2 inhibitorProteasomal inhibitor
% o
f Wild
type
Phe
noty
pe
(With
out A
KT in
hibi
tors
)
Rapamycin
Rad001Torin
1
Palomid529Ink128
AZD8055
MG132
Velcade
Torc1 inhibitorTorc1/2 inhibitorProteasomal inhibitor
% o
f Wild
type
Phe
noty
pe
(With
out A
KT in
hibi
tors
)
Small Molecule
Small Molecule
Empty Vector shRNA-225915
shRNA-120392 shRNA-120389 shRNA-38014
shRNA Rictor Data (HCT-116)
G0 ASY G0 ASY G0 ASY G0 ASY G0 ASY
Control (HCT-116 + non-silencing shRNA)Experimental (HCT-116 + hairpin)G0
Asymmetry Control (HCT-116 + non-silencing shRNA)Experimental (HCT-116 + hairpin)
(Control = non-silencing)
Non-Silencing
shRNA-225915
shRNA-120392 shRNA-120389 shRNA-38014
G0 ASY G0 ASY G0 ASY G0 ASY G0 ASY
(Control = empty-vector)
Live-cell Imaging Results2/18/12
• HCT-116 shRictor variants– +/- 1ug/mL doxy
Boxplot of Cell Cycle Time(HCT116 shRNA-Rictor 225915)
No doxy 1ug/mL doxy
Alpha Plot comparison
No doxy1ug/mL doxy
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)
Cell Fate – Overall Comparison
shRNA225915 No Rx
shRNA225915 2 ug/mL Doxy
Mitosis (55%)Lost (24%)
Death (20%)Other (1%)
Mitosis (55%)Lost (26%)
Death (18%)
Other (1%)
Non-silencing 2 ug/mL Doxy
Mitosis (52%)Lost (28%)
Death (20%)
Alpha Plot comparison
225915 + No doxy225915 + doxy
Non-silencing + doxy
Boxplot of Cell Cycle Time(HCT116 shRNA-Rictor 225915)
225915 No doxy 225915 2ug/mL doxy
Non-silencing 2ug/mL doxy
225915 2ug/mL doxy
Beta Plot comparison
225915 + No doxy225915 + doxy
Non-silencing + doxy
Boxplot of Sibling-sibling Cell Cycle Time
(HCT116 shRNA-Rictor 225915)
225915 No doxy 225915 2ug/mL doxy
Non-silencing 2ug/mL doxy
225915 2ug/mL doxy
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