Zncl2

To analyse PHB2 phosphorylation, phos-tag SDS–PAGE was performed using precast SuperSep gels (50 μM phos-tag acrylamide and 100 μM ZnCl₂, Wako Chemicals, 190–16721), as previously described with some modification45 (link). Molecular size markers for phos-tag SDS–PAGE used the WIDE-VIEW Prestained Protein Size Marker III (Wako Chemicals, 236–02463). The percentage of phosphorylation was calculated according to the formula: (phosphorylated PHB2 band area/total PHB2 band area) × 100.

The cell lines present in this study were obtained from RIKEN BRC CELL BANK. HepG2 cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, GIBCO) supplemented with 10% fetal bovine serum (FBS, Biowest), 100 U mL⁻¹ penicillin, and 100 U mL⁻¹ streptomycin (Nacalai Tesque) at 37°C in a 5% CO₂ atmosphere. The metal exposure was performed as follows: 2 × 10⁴ cells were seeded in a 3.5-cm culture dish and first cultured with normal medium for 48 h; subsequently, they were washed with phosphate-buffered saline (PBS) and then treated with medium including FeCl₃, AlCl₃, CoCl₂, CuSO₄, NiCl₂ and ZnCl₂ (FUJIFILM Wako Pure Chemical Corporation), ranging from 20 to 200 μM, separately. After incubation for 24 h, the cells were fixed using the following steps. First, cells were digested with .25% trypsin/EDTA, followed by PBS washing. Then, the cells were gently suspended in a hypotonic solution (75 mM KCl) and allowed to stand for 8 min at room temperature. After the same volume of Carnoy’s solution (fixative solution, methanol/acetic acid (3/1, v/v)) was added, cells were mixed gently. After centrifugation at 1,500 rpm, the supernatant was discarded, and an equal volume of fresh Carnoy’s solution was added. This procedure was repeated twice. Fixed cells were stored in Carnoy’s solution at −20°C.

Trypsin-like activity was assayed as described previously (Grenier, 1995 (link)). Trypsin-like activity was measured by monitoring the hydrolysis of the chromogenic synthetic peptide benzoyl-dl-arginine-p-nitroanilide (BAPNA; Peptide Institute) in the presence or absence of various compounds: EDTA (Wako Pure Chemical Industries), N-α-p-tosylamide-2-phenylethyl chloromethyl ketone (TLCK; Wako), leupeptin (Peptide Institute), DTT, iodoacetamide (Wako), SDS (Wako), CaCl₂ (Sigma-Aldrich), MgCl₂ (Wako) and ZnCl₂ (Wako). The cells were harvested by centrifugation (10 000 g, 30 min) and suspended in distilled water. The bacterial samples (12.5 µl) were mixed with 125 µl of 150 mM Tris/HCl buffer (pH 7.8), 50 µl of 4 mM BAPNA and 12.5 µl distilled water, and the assay mixtures were incubated at 37 °C for 2 h. The release of p-nitroaniline was determined by measuring the OD₄₀₅ nm using a microplate reader (Bio-Rad).

Each CTF was prepared using essentially the same method described in our previous publications.7 Briefly, 2,6-dicyanopyridine (64.5 mg, Koei Kagaku) and Ketjen Black EC600JD (64.5 mg, Lion Corp.) were mixed with ZnCl₂ (6.82 g, Wako) in a glass vacuum tube and then heated to 400 °C at 3.3 °C min^–1 and held at that temperature for 40 h. The resulting powder was washed with deionized water, tetrahydrofuran (THF, Wako), HCl (1 M, Wako) and aqueous ammonia (1 M, Wako). The obtained CTF was dispersed in a 10 mM aqueous solution of MCl₂ (M = Co, Ni or Cu) and stirred at 80 °C for 3 h. Subsequently, the product was collected by centrifugation and thoroughly washed with deionized water to remove any unbound metal salt, followed by drying at 60 °C in a vacuum oven for 1 day.

An antibody against actin (SC-47778) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against CHOP (#5554) and goat anti-rabbit IgG (horseradish peroxidase- (HRP-) conjugated, #7074) were purchased from Cell Signaling Technology Japan (Tokyo, Japan). Tauroursodeoxycholic acid (TUDCA) was purchased from Tokyo Chemical Industry (Tokyo, Japan). The Zinc Assay Kit was purchased from Metallogenics Co. Ltd. (Chiba, Japan). HRP-conjugated donkey anti-mouse IgG was purchased from GE Healthcare Japan (Tokyo, Japan). Carnosine, ZnCl₂, NiCl₂, and RIPA buffer (20 mmol/L Tris-HCl (pH 7.4), 0.05 w/v% NP-40 substitute, 2.5 mmol/L MgCl₂, and 200 mmol/L NaCl) were purchased from Wako Pure Chemicals (Tokyo, Japan). Protease and phosphatase inhibitors (87786 and 78420), NuPAGE® Novex 4–12% Bis-Tris Protein Gel, iBlot™ Transfer Stack, and SuperSignal™ West Dura Extended Duration Substrate were from Thermo Fisher Scientific K.K. (Tokyo, Japan).

The Nephelostar Plus microplate reader with 2 built-in Reagent injectors and MARS software was obtained from BMG Labtech (Offenburg, Germany) and 96-well plates and 96-well plastic covers were from Corning (Kennebunk, ME, USA). All chemicals (NaCl, Tris, Hepes, CaCl₂, NaH₂PO₄, Na₂HPO₄, NaOH, ZnCl₂) were pro-analysis -grade quality and purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

IMAC analysis was done according to a previous study [38 (link)]. Vanadyl sulfate (VOSO₄·nH₂O, n = 3–4; 99.9%), along with MgCl₂, CaCl₂, MnCl₂, FeCl₃, CoSO₄, CuCl₂, and ZnCl₂, were purchased from Fujifilm Wako Pure Chemical Corporation, Japan. Each metal was dissolved in deionized water (DW) at 1 mM and stored at room temperature.
Chelating Sepharose FF resin (250 μL; Thermo Fisher Scientific, USA) was washed with water and mixed with each metal solution in a 2 mL plastic tube. The resin was washed twice with distilled water and twice with binding buffer (100 mM NaCl, 20 mM Na phosphate, pH 7.4). Protein in binding buffer (∼100 μg mL⁻¹) was mixed with the resin by rotation for 30 min at room temperature. Non-bound proteins were removed by centrifugation, then the resin was washed twice with binding buffer. Bound proteins and metal ions were eluted with 50 mM EDTA (pH 8.0). Non-bound and bound protein fractions were analyzed by SDS-PAGE and CBB staining.