Supplementary Figure 1A-B A H1299 RH LC3-I / II P-GSK-3 (Ser21/9) GSK-3α/β β-actin β-catenine P-GSK-3 (Tyr279/216) B H1299 RH1299 Viability (% of control) Suppl. Fig. 1. Effects of Cd on viability and dual phosphorylation of GSK-3 in H1299 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) H1299 and 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-II induction 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 had a 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-3 Between 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 Cytosol Nucleus Cytosol β-actin HDAC p-GSK-3 (Ser21/9) GSK-3α/β β-catenine p-GSK-3 (Tyr279/216)

3C 3B 3A Control OA / Cd Supplementary Figure 3A-C Viability (% of control) 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 nuclear extracts (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 Control Cd OA OA / Cd Control 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 HDAC β-actin p-GSK-3 (Ser21/9) GSK-3α/β p-GSK-3 (Tyr279/216)

Supplementary Figure 5 PI-positive cells (% of total cell number) 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 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 Data are expressed as mean ± SD (n=4). Cells were transfected with siRNA for Atg5 as described in Fig. 4I. Control3-MA zVAD Cd 3-MA+Cd zVAD+Cd P< 0.01 Supplementary Figure 6