Mt 1 2

The cells were lysed in ice-cold RIPA buffer (Millipore, Temecula, CA, USA) containing a protease inhibitor cocktail (Roche, IN, USA). Cell lysates were centrifuged at 15,000 rpm for 20 min at 4 °C, and total protein concentrations were determined using a Bio-Rad protein assay kit (Bio-Rad Laboratories). Equal amounts of total protein from each sample were run through a gradient SDS-PAGE gel, followed by immunoblotting onto PVDF membranes. The membranes were blocked and probed with primary antibodies against actin (Sigma-Aldrich), tubulin (Sigma-Aldrich), and MT-I/II (Invitrogen). The membranes were immersed in 0.1% PBST containing horseradish peroxidase-conjugated secondary antibodies, and the protein levels were determined using enhanced chemiluminescence reagents.

For immunohistochemical analysis, kidney tissues from the sacrificed mice were harvested and fixed in 10% formaldehyde. Paraffin cross-sections with a thickness of 5 μm were stained with antibodies against MT-I/II (Invitrogen), followed by N-Histofine^® MOUSESTAIN KIT (414321F; Nichirei Biosciences Inc.; Tokyo, Japan). To measure renal fibrosis, the paraffin-embedded sections were stained using Masson’s Trichrome stain kit (ScyTek Laboratories Inc., Logan, USA). Quantification of kidney immunohistochemical assay and fibrosis on Masson’s trichrome-stained sections were performed using ImageJ analysis. The raw pixel values of the selected areas were estimated using the ImageJ software. These values indicated the distribution area times the stain intensity of the images.