Ly294002

Total cell lysates and biotinylated proteins were subjected to SDS-PAGE electrophoresis. The primary antibodies used include mouse anti-HA (901501, 1:5000, BioLegend), mouse anti-GAPDH (FD0063, 1:10,000, FuDeBio), mouse anti-Akt (sc-5298, 1:2000, Santa Cruz Biotechnology), rabbit anti-p-Akt (4060, 1:2000, Cell Signaling Technology). The secondary antibodies used were IRDye® 800CW Goat anti-Mouse IgG Secondary Antibody (926–32210, LI-COR Biosciences), IRDye® 680RD Goat anti-Rabbit IgG Secondary Antibody (926–68071, LI-COR Biosciences). LY294002 (L832989, 10 μM, Macklin) was applied for 24 h to inhibit PI3K signaling.

To promote differentiation into osteoclasts, RAW264.7 cells were treated with
RANKL (Sigma-Aldrich, USA) for six days. After treatment with 50 ng/mL RANKL,
CoQ10 (10^-3 M, Aladdin Reagent Co., Ltd., China) with or without
10^-4 M H₂O₂ was added to the cell line
(11 (link)). These cells were preconditioned
with the inhibitors PI3K (LY294002, 30 μM; #L832989; Macklin, China) for 1 h
(19 (link)), ERK (PD98059, 10 μM; MedChem
Express, USA) for 1 h (20 (link)), p38MAPK
(SB203580, 10 μM; Yuanye Bio-Technology Co., Ltd., China) for 1 h (21 (link)), or mitophagy agonist Torin1 (4 nM;
#T861013; Macklin) for half an hour.

RWPE-1, LNCAP, C4-2, PC3, 22RV1, and DU145 cell lines were obtained from the Cell Bank of Typical Culture Preservation Committee of the Chinese Academy of Sciences (Shanghai, China). The RWPE-1 cells were cultured in primary keratinocyte culture medium (iCell Bioscience Inc., cat. PriMed-iCell-010, China), and the other cell lines were cultured in RPMI 1640 (Gibco, cat. 11875-093, United States) supplemented with 10% fetal bovine serum (FBS; Gibco, cat. 10270-106, United States) and 1% penicillin–streptomycin solution (Gibco, 15140-122, United States) at 37°C under 5% CO₂. As per experimental requirements, the cells were treated with 20 ng/ml LY294002 (Macklin, cat. HY-10108, China) in dimethyl sulfoxide for 24 h (Bao et al., 2015 (link)).