Vinorelbine tartrate

Eribulin mesylate (hereafter, eribulin) was supplied by Eisai Inc. (Cambridge, MA, USA) as laboratory-grade dry powder active pharmaceutical ingredient (API) obtained from the same manufacturing stream used for Eisai’s branded clinical product, Halaven^®. Paclitaxel and vinorelbine tartrate (hereafter, vinorelbine) were obtained as dry powders from Selleckchem (Shanghai, China). Eribulin, Paclitaxel and vinorelbine were prepared as 10 mM stock solutions in 100% (v/v) DMSO, aliquoted into small volumes and stored at −20 °C until day of use. Although not a designated test agent for this study, cisplatin was included in all assays as an internal reference control for assay performance; cisplatin was obtained as a laboratory grade liquid formulation from Hospira Australia Pty Ltd. (Melbourne, Australia), with storage per manufacturer’s instructions and dilutions for cell culture studies on day of use.

CR0 and PM cells were seeded in 96-well plates at 2000–2500 cells per well in LHC-9 medium and incubated for at least 8 h. The medium was then changed, and the cells were treated with various concentrations of cisplatin (Hansoh Pharmaceutical, Jiangsu, China), gemcitabine (Selleck, Shanghai, China), vinorelbine tartrate (Selleck, Shanghai, China), or H₂O₂ (Sinopharm Chemical Reagent Co., Shanghai, China). Cells were incubated for a designated period of time, and at the end of the experiments, cell proliferation or cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay according to manufacturer’s protocol (Promega, Madison, WI, USA).

Monoclonal anti-Alix (ab117600) and anti-Calnexin (ab22595) antibodies were purchased from Abcam, whilst polyclonal anti-β-tubulin (#2146) was purchased from Cell Signalling Technologies. Secondary antibodies (rabbit- and mouse- anti-goat IgG HRP conjugate #170–6515 and #170–6516) were purchased from Bio-Rad. Rhodamine-conjugated phalloidin (PHDR1) was purchased from Cytoskeleton and DAPI (D9542) was purchased from Sigma-Aldrich. DMEM, OptiMEM and fetal bovine serum (FBS) were purchased from Thermofisher Scientific. Vinorelbine tartrate (#S4269) was purchased from Selleckchem.