Opti minimal essential medium

The procedure used to produce lentivirus was based on the Lenti-Pac™ HIV Expression Packaging kit user manual as previously described (31 (link)). In addition, mCherry fluorescent protein (mCherryFP) was fused to the plasmid vector. Briefly, 293Tα lentiviral packaging cells (GeneCopeia Co., Guangzhou, China) were cultured in DMEM supplemented with 10% heat-inactivated FBS and incubated at 37°C in 5% CO₂. Following this, 1.25 µg lentiviral expression plasmid, 2.5 µl (0.5 µg/µl) Lenti-Pac HIV Expression Packaging mix and EndoFectin™ Lenti transfection reagent (all from GeneCopeia Co.) were diluted with Opti-Minimal Essential medium (Invitrogen; Thermo Fisher Scientific, Inc.) for 25 min at room temperature. When cell confluence reached 70–80%, the mixture was added to the culture medium of 293Tα cells. The culture medium was replaced 8 h after transfection with DMEM supplemented with 10% FBS. In addition, 10 µl TiterBoost reagent (GeneCopeia Co.) was added to improve the percentage of virus generation. A total of 72 h post-transfection, the culture medium was collected, centrifuged at 4°C, 3,000 × g for 30 min, and the supernatant was filtered. Lentiviral stocks were aliquoted and stored at −80°C for further use.

In order to evaluate the functional role of Parkin, small interfering (si)RNA was used to reduce its expression. The selective siRNA duplex (5′-GAGGAUGACAACGACAUAATT-3′, antisense, 5′-UUAUGUCGUUGUCAUCCUCTT-3′) and a nonspecific control duplex (5′-UUCUCCGAACGUGUCACGUTT-3′; antisense, 5′-ACGUGACACGUUCGGAGAATT-3′). These were obtained from YangZhou Ruibo Biotech Co., Ltd. (Yangzhou, China). Transfection of siRNA into cells was performed using Lipofectamine^® 2000 transfection reagent (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol (37 (link)). Cultured cells were washed with Opti-Minimal Essential Medium (Invitrogen; Thermo Fisher Scientific, Inc.) without serum or antibiotics and seeded in 6-well plates to 30–40% confluence. The transfection reagent and siRNA were diluted separately in serum-free medium, mixed and incubated for 10 min at room temperature to form the siRNA/lipid complex. This complex was added to each well at a final concentration of 70 nM/well siRNA. At 48 h post-transfection, cells were harvested to determine Parkin protein expression levels by western blot analysis according to the protocol described above.

The full-length coding sequence of the human cyclin B1 plasmid with a green fluorescent protein (GFP) tag (chloroxylenol/cyclin B1-GFP; lot no., 26061) was purchased from Addgene, Inc. (Cambridge, MA, USA). In total, 1.5×10⁵ ovarian cancer cells were plated per well in a 6-well plate on day 0. On day 1, the A2780 cells were transfected with 2 µg plasmid in Opti-minimal essential medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) using Lipofectamine 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.). On day 2, the cells were treated with taxane alone or combined TSA and PS341 for an additional 24 h. The small interfering RNA (siRNA) corresponding to positions 776–796 of cyclin B1 (5′-CUCGUACAGCCUUGGGAGACAtt-3′) and the control siRNA targeting GFP were ordered from Invitrogen (Thermo Fisher Scientific, Inc.). The A2780T cells were transfected with siRNA using Lipofectamine 2000 according to the protocol of the manufacturer (Invitrogen; Thermo Fisher Scientific, Inc.). A total of 24 h subsequent to transfection, the cells were treated with taxane or combined TSA and PS341 for an additional 24 h.