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Supplementary Figure 1A-B
A
H1299 RH1299
0 15 30 45 60 0 45 80 160 200
LC3-I / II
P-GSK-3 (Ser21/9)
GSK-3α/β
β-actin
β-catenine
P-GSK-3 (Tyr279/216)
B
0 20 40 80 120 160 200 240
0
20
40
60
80
100
120
H1299RH1299
Via
bilit
y (%
of
con
trol
)
Suppl. Fig. 1. Effects of Cd on viability and dual phosphorylation of GSK-3 inH1299 and RH1299 cells. (A) Cells were treated with the indicated concentrations of Cd for 24 h, and viability was determined using an MTT assay. Data are expressed as the mean ± SD of fold-increase compared to the untreated control from three independent experiments performed in triplicate. (B) H1299and RH1299 cells responding to Cd induced p-Ser/Tyr on GSK-3α/β in a dose-dependent manner without changing of total GSK-3α/β, which was higher in H1299 than RH460 cells. H1299 cells showed higher sensitivity for LC3-IIinduction in responding to Cd.
2B 2C
Supplementary Figure 2A-C
2A
Suppl. Fig. 2A. Immunofluorescent staining using p-Tyr GSK-3β antibody revealed that p-Tyr localized to the nuclear membrane of control cells. After 12 h of Cd treatment,it was aggregated in the cytoplasm or dispersed in the nucleus in some cells, which hada condensed or fragmented nucleus (arrows). The number of cells with cytoplasmic and nuclear staining increased at 24 h. Fig. 2B-C. The shuttling of p-Tyr/p-Ser GSK-3Between the nucleus And cytoplasm was investigated in cells treated with increasing Concentrations of Cd. At 12 h, all concentrations of Cd caused nuclear accumulation of p-Tyr/p-Ser GSK-3, but cytoplasmic accumulation was found in cells treated with high concentrations of Cd, which was enhanced at 21 h in treated cells.
p-Tyr-GSK-3 Hochest33342 Merge
Control
12 h
24 h
Nucleus
0 8 16 32 64 80 0 8 16 32 64 80
Cytosol Nucleus
0 8 16 32 64 80 0 8 16 32 64 80
Cytosol
β-actin
HDAC
p-GSK-3 (Ser21/9)
GSK-3α/β
β-catenine
p-GSK-3 (Tyr279/216)
3C 3B
3A
Con
trol
OA
/ Cd
Supplementary Figure 3A-C
Con-trol
Cd OA OA / Cd
0
20
40
60
80
100
120
Via
bilit
y(%
of
cont
rol)
P<0.05
Suppl. Fig. 3A-C. Effects of p-GSK-3 on Cd-induced autophagy in RH460 cells. (A) Cells were treated with 10 nM okadaic acid (OA) for 1 h prior to Cd (60 µM) treatment for 10 h, and determined viability using a MTT assay. (B,C) Cells were treated as described in A, and prepared cytosolic and nuclearextracts (B) or total cell lysates (C),and immunoblotted for indicated antibodies. HDAC and β-actin were used loading controls for nucleus and cytosol, respectively. A representative result from over three experiments is shown.
β-actin
Procaspase-3
LC3-I/II
Cd
OA
p-GSK-3 (Ser21/9)
GSK-3α/β
β-catenine
p-GSK-3 (Tyr279/216)
Nucleus
Con
trol
Cd
OA OA
/ Cd
Con
trol
Cd
OA OA
/ Cd
Cytosol
HDAC
β-actin
p-GSK-3 (Ser21/9)
GSK-3α/β
β-catenine
p-GSK-3 (Tyr279/216)
Supplementary Figure 4
Suppl. Fig. 4. Intracellular redistribution of p-Ser/p-Tyr GSK-3 in Cd-treated H460 cells. Cells were treated with with an increasing concentrations of Cd for 21 h, and prepared for cytosolic and nuclear fractions as described under Materials and Methods, and then immunoblotted for indicated proteins. HDAC and β-actin were used as markers of nuclear and cytosol, respectively. Representative results from four experiments are shown.
Nucleus Cytosol
0 3 6 9 12 0 3 6 9 12
HDAC
β-actin
p-GSK-3 (Ser21/9)
GSK-3α/β
p-GSK-3 (Tyr279/216)
Supplementary Figure 5
PI-
posi
tive
cel
ls
(
% o
f to
tal c
ell n
um
ber)
Effects of blocking of autophagy or apoptosis on Cd-induced
morphological changes in RH460 cells. Massive cytoplasmic
vacuolization (arrows) and many floated cells were observed
by phase-contrast microscopy after 21 hrs treatment with 70
μM Cd. Pretreated with 3MA (10 mM) completely blocked Cd-
induced cytoplasmic vacuolization, and the number of floating
cells markedly reduced. However, zVAD (40 μM) treatment
before addition of Cd did not block Cd-induced cytoplasmic
vacuolization, but resulted in a decrease in number floating
cells. (200).
RH460 cells were transfected with siRNA Atg5 and non-
specific (NS) siRNA as described in the Fig4 A, and treated
with Cd for 21 hrs, and then stained with Hoechst 33342 and
PI; four random fields were then photographed for each condition
under fluorescence microscope. Total cells and PI positive cells
were counted, respectively. Data are expressed as the percent
of total cells that stained with Hoechst 33342. Data are expressed
as mean ± SD (n=4). Cells were transfected with siRNA for
Atg5 as described in Fig. 4I.
Control 3-MA zVAD
Cd 3-MA+Cd zVAD+Cd
NS siRNA
NS siRNA / Cd
si RNA Atg5
siRNA Atg5 /
Cd
0
10
20
30
40
P< 0.01
Supplementary Figure 6