Penicillin streptomycin glutamine (psg)

Cells were maintained in Dulbecco's modified Eagle medium (DMEM; D6429; Sigma‐Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum and 1× penicillin–streptomycin–glutamine (161‐23201; Wako Pure Chemical Industries, Osaka, Japan) in the presence of 5% CO₂ at 37 °C. One day before incorporating the beads, cells were seeded onto 35‐mm glass‐bottom culture dishes (P35G‐1.5‐10‐C; MatTek, Ashland, MA, USA) at a density of 1.5 × 10⁵ cells per dish in the absence of antibiotics.

Prominin-1⁺ cells were selected from dissociated IC using a magnetic-activated cell sorting (MACS) system with anti-mouse prominin-1 magnetic beads (Miltenyi Biotec) according to the manufacturer’s protocol. To generate primary neurospheres, MACS-selected cells were cultured on uncoated plates at clonal density (1–2 cell/mm²) in Neurobasal medium (Life Technologies, CA, USA) containing MACS NeuroBrew-21 without vitamin A (Miltenyi Biotec) supplemented with epidermal growth factor (EGF) (20 ng/ml; Miltenyi Biotec), fibroblast growth factor-2 (FGF2) (20 ng/ml; Miltenyi Biotec), and penicillin-streptomycin-glutamine (Wako, Tokyo, Japan). The number of neurospheres was counted by microscopy following 6 days of culture at 37 °C under a 5% CO₂ atmosphere. To generate secondary neurospheres, primary neurospheres were dissociated with Accutase (Life Technologies, Carlsbad CA, USA) and then seeded on new plates. For clonal cell analysis, prominin-1⁺ cells were seeded at one per well by the limiting dilution method or into 96-well plates (Sumitomo Bakelite, Tokyo, Japan) by a fluorescence-activated cell sorter (FACSAria I, BD). Clusters of cells > 50 μm in diameter were counted as neurospheres.

HeLa S3 (ATCC CCL-2.2), HEK293 (ATCC-CRL-1573) and U2OS cells (ATCC HTB-96) were obtained from the American Type Culture Collection. MEF cells (wild-type) and p62 KO MEF cells (p62^−/− cells) were kindly provided by Dr. Tetsuro Ishii56 (link). The cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich) and supplemented with 10% foetal bovine serum and 1× penicillin-streptomycin-glutamine (Wako). The cells were cultured in an atmosphere of 5% CO₂ at 37 °C. During nutrient starvation experiments, MEF cells or U2OS cells were washed twice with phosphate-buffered saline (PBS) and incubated in Hank’s balanced salt solution (HBSS; Invitrogen) containing 1 mM MgCl₂ and 1 mM CaCl₂ (starvation medium).