A
B
LY294002 20 µM
─
─
+
+
ATO 1 µM
─
+
─
+
ATO 1 µM
─
+
─
+
─
+
U0126 5 µM
─
─
+
+
─
─
Mcl-1
PD184352 1 µM
─
─
─
─
+
+
PARP
Mcl-1
GSK-3β
GSK-3β
p-GSK-3β
(Ser9)
p-GSK-3β
(Ser9)
β-actin
β-actin
Suppl. Fig 1. (A) AKT inhibitor LY294002 plus ATO are synergistic in decreasing Mcl-1 protein levels and
in inducing apoptosis. NB4 cells were pretreated with 20 µM LY294002 for 2 h and then treated with 1 µM
ATO for another 24 h. The relative levels of Mcl-1, PARP, GSK-3β, p-GSK-3β(Ser9), and β-actin were
determined using specific antibodies with Western blot analysis. (B) MEK/ERK inhibitors, U0126 and
PD184352, decrease p-GSK-3β(Ser9) levels. NB4 cells were pretreated with 5 µM U0126 or 1 µM
PD184352 for 2 h and then treated with 1 µM ATO for another 24 h. The levels of Mcl-1, GSK-3β, p-GSK3β(Ser9), and β-actin were determined using specific antibodies with Western blot analysis.
GSH Content (nmol/106 cells)
20
Control
siRNA (Con)
+ATO 2 µM
siRNA (Con)
16
42.3%
47.4%
12
8
siRNA (Mcl-1)
51.6%
4
siRNA (Mcl-1)
62.4%
0
Con
ATO 2µM
siRNA (Con)
Con
ATO 2µM
siRNA(Mcl-1)
Suppl. Fig 2. Silencing Mcl-1 decreases GSH levels and enhances ATO-induced ROS production in HL60 cells. HL-60 cells transfected with Mcl-1 siRNA or control siRNA (Con) were treated with or without 2
µM ATO for 16 h. The levels of GSH and ROS were determined as described in Material and Methods.