Lipofectamine plus

For high-content imaging acquisition and cell culturing, we chose a black glass-bottom 96-well PS SensoPlate™ (Greiner Bio-One, Kremsmünster, Austria). Initially, the control proteasome or marine natural products were diluted and dispensed into culture plates at the concentrations stated in the text.
HEK293T cells transiently expressing EGFP-UL76 were used in this HCS assay. HEK293T cells were seeded at 1 × 10⁶ cells onto 6-cm culture dishes one day before transfection. Then, 3 µg of plasmid DNA pEGFP-UL76 was transfected into HEK293T cells mediated by Lipofectamine Plus and Lipofectamine (Thermo Fisher Scientific, Waltham, MA, USA). After 3 h of transfection, the transfected cells were trypsinized and dispensed into black glass-bottom 96-well plates at 1 × 10⁴ cells per well in a volume of 200 µL per well, including the indicated compound at each concentration with three repeats. The culture plates containing the cells and tested compounds were incubated at 5% CO₂ and 37 °C for 48 h. Subsequently, the cells were fixed in 1% paraformaldehyde for 10 min and simultaneously permeabilized with 0.1% IGEPAL^® CA-630, then stained with 1.5 µg/mL DAPI on ice for 30 min. After extensive washing with PBS, the cells were submerged in PBS, sealed in the dark, and stored at 4 °C.

Murine mammary epithelial cells (Eph4 cells) and HEK293 cells were cultured in DMEM supplemented with 10% FCS. Transfection was performed using Lipofectamine Plus or Lipofectamine 2000 (Thermo Fisher Scientific) following the manufacturer’s instructions. To establish stable transfectants, the transfected cells were selected by incubation in medium containing 500 μg/ml G418 (Nacalai Tesque) and clones derived from single cells were picked up.

We seeded LoVo CRC cells in 24-well plates and transfected them with pcDNA3-ING1, pCMV-p53WT, both, or control empty vector plasmid (pcDNA3) with firefly luciferase and Renilla luciferase reporter plasmids using LipofectAMINE-Plus (Catalog number 15338100, Thermo Fisher Scientific). Two days after transfection, we measured luciferase activities using a Dual Luciferase Assay System (Catalog number E1910, Promega) and a luminescence imaging instrument (Atto, Tokyo, Japan). We normalized the firefly luciferase activities to those of the Renilla luciferase control as described previously [20 (link)].

Plasmid pCMV_TOM20_GFP was transfected into HEK293 cells. For transfection, the cells were plated in 6-well plates at 5 × 10⁵ cells/well in DMEM without antibiotics one day prior to transfection. Transfections were performed using Lipofectamine Plus (ThermoFisher Scientific, MA, USA) according to the manufacturer’s protocol. Stable transfectants were screened using geneticin (G-418; ThermoFisher Scientific, MA, USA) treatment. Specifically, the transfected cells were treated with geneticin (400 μg/mL), and TOM20_GFP stable transfected cell lines were screened after 5–10 days as follows: single stable clones were cultured confluently in 24-well plates and washed twice with 1× phosphate-buffered saline (PBS), lysed with 100 μL of cell lysis buffer for 15 min on ice, and then centrifuged for 15 min at 12,000× g to prepare the TOM20_GFP solution. This protein solution was then transferred into a new tube and mixed with an equal volume of SDS lysis buffer. Finally, the mixture was heated in boiling water for 5 min and subjected to western blotting analysis.

Parental RKO and DLD-1 cells, plus RKO Flp-In T-Rex derivatives expressing Bim, Myc-tagged Bcl-xL and GFP-tagged Mcl-1, were as described [12 (link),49 (link),68 (link)]. All lines were cultured in DMEM plus 10% fetal calf serum (Life Technologies), 100 U ml⁻¹ penicillin, 100 U ml⁻¹ streptomycin and 2 mM glutamine (all from Sigma), then maintained at 37°C in a humidified 5% CO₂ atmosphere. A stable RKO line harbouring a tetracycline-inducible GFP-tagged Bcl-xL was generated as described [69 (link)]. In brief, a Bcl-xL cDNA [12 (link)] was cloned into a pcDNA5/FRT/TO-based vector (Invitrogen) then co-transfected with pOG44 into Flp-In T-Rex RKO cells using Lipofectamine Plus (Thermofisher). Stable integrants were selected in 400 µg ml⁻¹ hygromycin B (Roche) and 8 µg ml⁻¹ blasticidin (Melford), colonies pooled and expanded to create an isogenic population. To synchronize cells in S-phase, cells were treated with 2 mM thymidine for 16 h.