1. Neal et al.Supplementary Figure S1b
14-3-3
Control
siRNA
P-Akt
Control
siRNA
14-3-3ζ
siRNA
C C2 si1 si2 si3
P-Akt
Akt
14-3-3 c d
Control
shRNA
14-3-3
shRNA1
14-3-3
shRNA2
Control
siRNA
14-3-3
siRNA 30 45 30 45 30 45
minutes 20 30 20 30
minutes
P-Akt
P-Akt
Akt
Akt
14-3-3
14-3-3
MDA-MB-435
MDA-MB-231
Supplementary Figure S1. Knockdown of ζ reduces Akt phosphorylation.
a. MDA-MB-435 cells were transiently transfected with control or  siRNA. Forty-eight hours
post-transfection 5 x 106 cells were injected into the mammary fat pad of SCID mice. Tumors were
harvested 6 days post injection, fixed in formalin and embedded into paraffin. Tissue sections
were stained for  and phospho-Akt-S473 (P-Akt). Nuclei were counter-stained blue with hematoxylin.
b. Control siRNA (C#) and ζ siRNA (si#) tumor xenografts treated as in (A) were collected for
western blot analysis  and phospho-Akt -Ser473 (P-Akt) specific antibodies were used for
detection. Dashed line indicates samples were on same gel but not consecutive lanes.
c. MDA-MB-435 cells stably transfected with control or  shRNA were starved for 18 hours (0) and re-stimulated with 8% FBS for the indicated times (minutes). Akt activation was assessed by immunoblot for phospho-Akt -Ser473 (P-Akt).
d. MDA-MB-231 cells transiently transfected with control or  pool siRNA were starved for 18 hours (0) and re-stimulated with 8% FBS for the indicated times (minutes). Akt activation was assessed by immunoblot for phospho-Akt -Ser473 (P-Akt).