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.