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The effects of MRP inhibitors on Cd-induced autophagy, apoptosis, and GSK3αβ phospho-rylation in RH460 cells. Cells were pre-treated with MK571 (15 µM) and probenecid (500 µM) for 2 h and then treated with Cd (65 µM ) for 21 h, and harvested, lyzed, and immunoblotted for indicated proteins. All immunoblots data are representative of at least three independent experiments.
Suppl. Fig . S2Suppl. Fig . S1
H460 cells cultured on glass coverslips were treated with Cd (8 µM) for 16 h and performed immunofluorecense staining for MRP1, and cells labeled with rhodamine-conjugated secondary antibody. MRP1 immunostaining revealed clustered and strong immunoreactivity in the cytoplas-mic compartment (arrows). A representa-tive photomicrographs are shown at X 200 (original magnifications).
Control Cd
MRP1
Hoechst
Merge
Supplementary Figure S1-S2
LC3-I/II
GSK3αβ
p-Ser GSK3αβ
p-Tyr GSK3αβ
p62
β-actin
MK Prob
Cd - + - + - +
Fig. S2B
MK Prob
PARP-1
MRP1
Fig. S2A
Cd - + - + - +
β-actin
Procaspase-3
Cleaved caspase-3
Control
Cd
OA2.5
OA2.5/CdOA5
OA5/Cd
OA10
OA10/Cd
0
20
40
60
80
100
Suppl. Fig. S3
Suppl. Fig. S4
Modulation of MRP1 by p-Ser or p-Tyr GSK3αβ in Cd-treated RH460 cells. Cells cultured on glass coverslips were treated OA (5 µM), vanadate (300 µM) for 2 h and then further treated with Cd (65 µM) for 18 h, and performed immunofluorescence staining for MRP1 and labeled with FITC- conjugated secondary antibodies. Nucleus was stained with Hoechst 33342. White ar-rows indicate MRP1 localized in the cyto-plasmic compartment. A representative pho-tomicrographs are shown at X 200, original magnifications.
Control
OA / Cd
Vanadate / Cd
MRP1 Hoechst Merge
Cd
→
→→ →
→ →
→
→
N
MRP
N
MRP
→
→→
Mor
talit
y (
% o
f co
ntro
l)
Supplementary Figure S3-S4
For Trypan blue assays, after treating cells with OA and Cd, floating and ad-herent cells were collected, centrifuged, and stained with 0.4% trypan blue for 5 min at room temperature. Numbers of trypan blue-positive (dead) and negative (alive) cells were counted on a hemocy-tometer under a microscope. Cell viabili-ties are expressed relative to those of un-treated controls. Data were statistically analyzed by one-way ANOVA test. P < 0.05
**
*
Suppl. Fig. S5
H460 cells transduced with pcDNA3.1 and GSK3β-HA plasmid DNA (1 ug, each) were cultured for 48 h, and further treated with Cd (8 μM) for 12 h, harvested, lysed, and immunoblotted for indicated proteins. Data shown are representative of two independent experi-ments.
Vect
or
HA
β-actin
MRP1
GSK3β-HAEndogenous GSK3β
→→Endogenous GSK3α
→
Cd - + - + - +
GSK
3β
LC3- I/II
Supplementary Figure S5
MRP1
CathepsinB
Hoechst
Merge
Suppl. Fig. S7
Control Cd
Supplementary Figure S6-S7
Suppl. Fig. S6
Lamp-2
CathepsinD
Hoechst
Merge
Control Cd
Colocalization of cathepsinB and Lamp-2 in Cd-treated RH460 cells. Cells cultured on glass coverslips were treated with Cd (65 µM) for 12 h, and performed im-munofluorescence staining for cathepsinD and Lamp-2 (S6), or cathepsinB and MRP1(S7), and labeled with FITC (cathepsinD, MRP1)- or rhodamine (Lamp-2, cathepsinB) -conjugated secondary antibodies. Nucleus was stained with Hoechst 33342. A representative photomicrographs are shown at X 200, original magnifica-tions.