*2.2. Cell Culture*

S1, S2 cells, and hRPTEC were cultured in Dulbecco's modified Eagle's medium/Ham's Nutrient Mixture F12 supplemented with 5% fetal bovine serum (FBS), 1 μg/mL insulin, 10 ng/mL epidermal growth factor, 10 μg/mL transferrin, and penicillin/streptomycin under 5% CO2 at 37 ◦C, as described previously [20]. Cells were used at the passages of 3–10 from the stocked original cells.

#### *2.3. Purification of Recombinant Proteins and Their Fluorescent Labeling*

The cloned mMT-I/pGEX-4T-1 plasmid and mouse β2-MG/pGEX-4T-1 plasmid were transformed into BL21(DE3)pLysS (Promega, Madison, WI, USA). The selected transformed cells were grown in 10 mL SOB medium containing 50 μg/mL ampicillin for 16 h at 37 ◦C until the optical density at 600 nm reached 0.3–0.4. The expression of MT and β2-MG proteins was induced by incubation with 1 mM IPTG for 6 h at 37 ◦C. The cultured cells were harvested by centrifugation at 8000 rpm for 10 min at 4 ◦C, and the GST-fusion proteins were purified by using MagneGSTTM Protein Purification (Promega). After a dialysis against PBS, the GST-fusion MT and GST-fusion β2-MG proteins were digested with thrombin (GE Healthcare, Buckinghamshire, UK). The lysates were loaded onto GST GraviTrapTM gravity-flow columns (GE Healthcare) to remove GST proteins, and the lysates were loaded onto a Benzamidine Sepharose 4 Fast Flow resin (GE Healthcare) to remove thrombin.

The purified recombinant MT and β2-MG proteins were conjugated with fluorescein isothiocyanate (FITC) by Fluorescein Labeling Kit-NH2 (Dojindo, Kumamoto, Japan). For FITC-labeled albumin and Alexa-labeled transferrin, commercially available albumin-fluorescein isothiocyanate conjugate and Alexa Fluor® 488-conjugated ChromPure Mouse Transferrin, respectively, were used.

#### *2.4. Fluorescence Imaging of the Labeled Proteins in S1 Cells*

S1 cells grown in glass-bottom dishes were incubated with each fluorescent-labeled protein and Hoechst33258 for 30 min, washed with phosphate-bu ffered saline (PBS), fixed with 4% paraformaldehyde in PBS for 5 min on ice, and then permeabilized with 0.5% TritonX-100 in PBS for 15 min at room temperature.

For the immunostaining of EEA1 to detect the early endosome, the fixed cells were washed with PBS and incubated with a blocking bu ffer containing bovine serum albumin (BSA) in PBS for 0.5 h at room temperature. The cells were then incubated with an anti-EEA1 antibody at a 1:100 dilution in blocking bu ffer for 1 h at room temperature. After washing with PBS, the cells were incubated with Alexa 555 anti-rabbit IgG at a 1:500 dilution in blocking bu ffer for 1 h at room temperature.

The distributions of FITC-albumin and Alexa-transferrin were visualized by a Nikon A1R-Si HD confocal microscope (Nikon, Tokyo, Japan), and those of FITC-MT and β2-MG were visualized by a BZ-X700 all-in-one fluorescence microscope (Keyence, Osaka, Japan).

#### *2.5. Assay for Sensitivity to Cd*

Cells were plated on 96-well plates at a density of 3 × 103–2 × 10<sup>4</sup> cells per well, incubated for 24 h in Cd-free medium, and then treated with CdCl2 for 1, 3, or 6 days. The media were not changed during the Cd exposure period. The alamarBlue ® assay (Invitrogen) was used to determine cell viability. AlamarBlue solution was premixed with fresh medium and added to the 96-well plates. After incubation for 2 h, the reduction in alamarBlue by active cells was determined by absorbance at 540 nm and expressed as the percentages compared to that of control cells.

#### *2.6. Measurement of Endocytosis E*ffi*ciency by Flow Cytometry*

Endocytosis e fficiency was determined by using flow cytometry (Guava easyCyte 6HT/2L; Millipore, Billerica, MA, USA). S1 and S2 cells (1 × 10<sup>5</sup> cells in 6-well dishes) were cultured with Cd at the concentrations of 10 or 15 μM for 1 day, 1 or 3 μM for 3 days, and 0.1 or 0.5 μM for 6 days. hRPTECs were cultured with Cd at the concentrations of 5 or 25 M for 3 days. After the media were changed to Cd- and serum-free ones, the cells were incubated with each fluorescent-labeled protein for 30 min, washed three times with 0.5 mL ice-cold PBS, and then harvested and subjected to flow cytometry. In order to quantify the percentages of cell populations incorporating fluorescent proteins, the populations were divided into quadrants, and the percentages of the cell populations in the lower-right section were calculated.
