Zinc acetate

HA with a mean molecular weight of 30,000 (polydispersity index of 1.212) was purchased from MRC polysaccharide Corp., Ltd (Toyama, Japan). RPMI 1640 medium, 4-dimethylaminopyridine (DMAP) isoflurane, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Tween 20, sodium dodecyl sulfate (SDS) and zinc acetate were purchased from Wako pure chemical (Osaka, Japan). Protoporphyrin IX (PP) and cetyltrimethylammonium bromide (CTA) were from Sigma-Aldrich Chemical (St. Louis, MO, USA). Water-soluble carbodiimide (WSC) was purchased from Dojindo Laboratory, Kumamoto, Japan. Additionally, 2,2,6,6-Tetramethyl-4-piperidone (4-oxo-TEMP) was purchased from Tokyo Chemical Industry (Tokyo, Japan). Esterase from porcine liver was purchased from Roche Diagnostics GmbH (Mannheim, Germany). The other reagents and solvents of a reagent grade were from commercial sources and were used without further purification.

Sulfane sulfur probe 4 (SSP4) was a kind gift from Dr. Ming Xian at Washington State University, USA. Cell counting kit-8 (WST-8), sodium sulfide, and Diethylenetriamine-N,N,N′,N′′,N′′-pentaacetic acid (DTPA) were purchased from DOJINDO chemical laboratory, Japan. Human serum albumin and N,N-dimethyl-p-phenylenediamine (DPDA) were obtained from Sigma Aldrich, St. Louis, MO, USA. Zinc acetate, 2,2′-azobis(2-amidinopropane), dihydrochloride (AAPH), ascorbic acid, dimethyl sulfoxide (DMSO), hydrochloric acid (HCl), potassium hydroxide (KOH), iron (III), chloride 5,5-dithiobis-2-nitrobenzoic acid (DTNB), glutathione, and linoleic acid were purchased from FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan. OneTouch^® was purchased from LifeScan Japan, Tokyo, Japan. Salivette^® was obtained from SARSTEDT, Nümbrecht, Germany. All water in assays was used deionized and distilled one. The ultraviolet plate was purchased from zell-kontakt GmbH, Nörten-Hardenberg, Germany. Hexadecyltrimethylammonium bromide (CTAB) was obtained from Tokyo Chemical Industry, Tokyo, Japan. The salivary amylase monitor and its chips were gifted by Nipro, Osaka, Japan.

We used human hepatoma cell lines established from hepatocellular carcinoma (Huh7) and a highly differentiated immortalized human hepatocyte (OUMS-29) [44 (link)]. Huh7 cells were obtained from RIKEN Bioresource center (Tsukuba, Japan). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and antibiotics. Cells were maintained in a 37 °C incubator with 5% CO₂.
The following materials were used: ALLN and epoxomicin as PIs (Calbiochem, La Jolla, CA, USA); copper sulfate (NACALAI TESQUE, Inc., Kyoto, Japan); PBA (Sigma-Aldrich, St. Louis, MO, USA); tunicamycin (Sigma-Aldrich); and zinc acetate (Wako Pure Chemical Industries, Ltd., Osaka, Japan).
Antibodies to the following antigens were used: LC3 (Medical and Biological Laboratories); Beclin 1 (Novus Biologicals, Littleton, CO, USA); Atg7, p-eIF2α (Cell Signaling Technology, Danvers, MA, USA); ubiquitin, XBP1, p62 (Santa Cruz Biotechnology, CA, USA); K8, actin (Sigma-Aldrich); Rubicon (Abcam, Cambridge, MA, USA); and ubiquitin (Dako, Glostrup, Denmark). Lamp1 antibody was a kind gift from J.T. August (Johns Hopkin University, Baltimore, MD, USA).