Inhibitor cocktails

Serum neuronal exosomes were isolated with the method reported by previous studies (26 (link), 28 (link), 33 (link)). Briefly, 500 μl of Dulbecco's phosphate buffered saline (DPBS, Thermo Fisher Scientific, cat# 14190144) containing the inhibitor cocktails (Roche, cat# 11873580001) was added into 1 ml serum. The mixture was centrifuged (relative centrifugal force, 4,500 g) for 20 min at 4°C. The supernatant was transferred into a new tube on ice. 200 μl of total exosome isolation reagent (Invitrogen, Thermo Scientific, Vilnius Lithuania) was added into the supernatant. After mixing, the sample was incubated 1 h at 4°C. The mixture was centrifuged (relative centrifugal force, 10,000 g) for 10 min at 4°C. The supernatant was discarded. The pellet was completely resuspended in 200 μl DPBS. Each sample received 100 μl DPBS containing 3% bovine serum albumin (BSA, KeyGen Biotech, cat# KGY00810) and was incubated for 1 h at 4°C each with 2 μg of mouse anti-human NCAM antibody (Santa Cruz Biotechnology, Santa Cruz, CA, cat# SC-106), which had been biotinylated with the EZ-Link sulfo-NHS-biotin system (Thermo Scientific, cat# A39256). The mixture was put on the rotating mixer 2 h at 4°C. Then 25 μl of streptavidin-agarose resin (Thermo Scientific, cat# 20347) was added in the mixture and put on the rotating mixer 1 h at 4°C. After centrifugation at 500 g for 10 min at 4°C and removal of the supernatant, each pellet was suspended in 50 μl of 0.05 M glycine-HCl (pH 3.0) with vortex for 10 s. Then the supernatant pH was adjusted to 7.0 with 1 M Tris-HCl (pH 8.6) and was added 150 μl DPBS. The serum neuronal exosomes were stored at −80°C.

5 × 10⁵ HepG2 cells were cultured in 2 mL of DMEM medium supplemented with 10% FBS and 1% P/S. Once the cell reached 80% confluence, they were treated with varied concentration of CuSO₄ (Sigma Aldrich, 209198) from 200 µM to 800 µM for 24 h. For the starvation induction, cells were additionally treated with Earle’s Balanced Salt Solution (EBSS) media (Sigma Aldrich, E3024) for 2 h with or without 50 µM chloroquine (CLQ) prior to lysis with RIPA buffer containing inhibitor cocktails (Roche Life Science, 11697498001). Protein concentration was determined to be sure that the protein of each sample was equally loaded on the Criterion TGX precast gel for western blot detection. ATP7B, GAPDH and LC3B-II/LC3B-I was determined using the above-mentioned antibodies. Western Blot analysis was carried out as described above.