The effects of MRP inhibitors on Cd-induced autophagy, apoptosis, and GSK3αβ phosphorylation in RH460 cells. Cells were pretreated 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. S2 Suppl. 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 cytoplasmic compartment (arrows). A representative 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 MKProb Cd Fig. S2B MK Prob PARP-1 MRP1 Fig. S2A Cd β-actin Procaspase-3 Cleaved caspase-3

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 White arrows indicate MRP1 localized in the cytoplasmic compartment. A representative photomicrographs are shown at X 200, original magnifications. Control OA / Cd Vanadate / Cd MRP1 Hoechst Merge Cd → → → → → → → → N MRP N → → → Mortality (% of control) Supplementary Figure S3-S4 For Trypan blue assays, after treating cells with OA and Cd, floating and adherent 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 hemocytometer under a microscope. Cell viabilities are expressed relative to those of untreated 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 experiments. Vector HA β-actin MRP1 GSK3β-HA Endogenous GSK3β → → Endogenous GSK3α → Cd GSK3β 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 immunofluorescence 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 A representative photomicrographs are shown at X 200, original magnifications.