H1152

Experiments were carried out using mAG-hGeminin–expressing L1210 FUCCI cell line, which was generated in a previous study (5 (link)) and originated from ATCC (catalog no. CCL-219). However, the RO-3306 treatment experiments were carried out using the parental L1210 cells, as the FUCCI cells displayed higher RO-3306 toxicity than the parental cells. Cells were grown in RPMI media (containing 11 mM glucose, 2 mM l-glutamine, 10% FBS, 1 mM sodium pyruvate, 20 mM Hepes, and antibiotic/antimycotic) at 37 °C in 5% CO₂ and 21% O₂ atmosphere. Cells tested negative for mycoplasma. Barasertib (also known as AZD1152-HQPA; Cayman Chemical; catalog no. 11602), H-1152 (Sigma-Aldrich; catalog no. 555550), and RO-3306 (Cayman Chemical; catalog no. 15149) were dissolved in DMSO. The chemical concentrations used were selected based on cell cycle phenotypes observed in control experiments.

HEK (human embryonic kidney) 293 cells were maintained in DMEM (Dulbecco’s modified Eagle’s medium) supplemented with 10% (v/v) fetal bovine serum and 100 units/ml penicillin/100 µg/ml streptomycin at 37°C in 5% CO₂ atmosphere. Transient expression in HEK293 cells was performed by transfecting the plasmids using FuGENE6 (Roche) or polyethylenimine (Sigma) according to the manufacturer’s instructions. LRRK2-IN-1 was provided by Professor Dario Alessi (University of Dundee). Sunitinib and H-1152 were purchased from Sigma and Calbiochem, respectively. LRRK2-IN-1 and Sunitinib were dissolved in dimethyl sulfoxide (DMSO). H-1152 was dissolved in sterilized distilled water (SDW). Thirty-six hours after transfection, cells were treated with inhibitors or an equivalent volume of solvents (DMSO or SDW). The final concentrations of solvents were 0.1% (v/v) for DMSO and 1% (v/v) for SDW.

Lymphoma cells were plated at 2×10⁵ cells per well of 12-well tissue culture plates, then on the next day, they were treated with 10 µM H1152 (R&D Systems) for 4 h, 10 µM ABT199 (Selleckchem) plus 10 ng/ml Chx (Sigma-Aldrich) for 1 h, or a combination of H1152 (4 h) plus ABT199 and Chx (1 h). Cell cultures were placed on ice and conditioned medium containing apoptotic bodies was collected in ice-cold centrifuge tubes, then ice-cold PBS was added, cells were scraped and transferred to the corresponding ice-cold centrifuge tubes. After centrifugation at 2000 g for 5 min at 4°C, supernatants were discarded and pellets were washed in 1 ml of PBS, followed by another centrifugation at 2000 g for 5 min at 4°C. Supernatants were discarded and pellets were resuspended in 200-250 µl of sodium dodecyl sulphate (SDS) lysis buffer [1% (w/v) SDS, 50 mM Tris-HCl pH 7.5], which was transferred to QIAshredder tubes (QIAGEN) and centrifuged for 1 min at 20,000 g. The flow-through was transferred to fresh Eppendorf tubes and frozen (either rapid freezing or overnight freezing at −80°C). The lysates were thawed on ice before being centrifuged at 20,000 g for 15 min at 4 C. The supernatants were transferred to new microfuge tubes, the protein concentration determined, and samples stored at −20°C.