Paclitaxel taxol

XBP1 Biochip assay kit was obtained from Randox Laboratories (XBP1 Array (EV4357), Randox Laboratories Ltd., Crumlin, UK). Thapsigargin (Tg) (Sigma-Aldrich, St. Louis, USA), Tunicamycin (Tm) (Sigma-Aldrich, St. Louis, USA), Brefeldin A (BFA) (Sigma-Aldrich, St. Louis, USA), Dithiothreitol (DTT) (Sigma-Aldrich, St. Louis, USA), 4μ8C (Sigma-Aldrich, St. Louis, USA), MKC-8866 (Probechem, St Pete Beach, USA), Paclitaxel (Taxol) (Sigma-Aldrich, St. Louis, USA), Lipopolysaccharide (LPS) (Sigma-Aldrich, St. Louis, USA) and Nigericin (Nig) (InvivoGen, San Diego, USA) were diluted in DMSO (Sigma-Aldrich, St. Louis, USA).

Microtubules were polymerised from porcine tubulin (Cytoskeleton Inc., Denver, CO) and labelled with fluorophores and biotin by stochastic incorporation of labelled dimers into the microtubule lattice. Mixes of 1.66 μM unlabelled tubulin, 0.15 μM Hilyte488-tubulin, and 0.4 μM biotin-tubulin were incubated in BRB80 (80 mM PIPES pH 6.85, 2 mM MgCl₂, 0.5 mM EGTA, 1 mM DTT) with 0.5 mM GMPCPP (Jena Bioscience, Jena, Germany) for 2–4 hr at 37°C. Polymerised microtubules were pelleted in a room temperature table top centrifuge at 18,400 x g for 8.5 min, and washed once with pre-warmed (37°C) BRB80. After pelleting once more, the microtubules were gently resuspended in pre-warmed (37°C) BRB80 containing 40 μM paclitaxel (taxol; Sigma-Aldrich) and used on the same day.

Lipopolysaccharide (LPS) and Paclitaxel (Taxol) were obtained from Sigma Chemicals Inc. and stored as 1 mg/ml and 10 mM, respectively. The Griess reagents were obtained from Promega Corporation, Madison, WI.

Human B-cell [RPMI8866 and Tat-GFP expressing RPMI8866 (RPMI8866-Tat-GFP)] and T-cell (Jurkat) lines were used in the study. Cells were maintained in EX-CELL Medium (SAFC Biosciences, United States) supplemented with L-glutamine and 10% FBS (Paneco, Russia). The effects of three MT inhibitors on cell cycle and cell death were evaluated in the study: paclitaxel (taxol; Sigma, United States), nocodazole (Biochem, United States) and vinorelbine (Sigma, United States). MT inhibitors were added to the medium 24 h prior to analysis at final concentrations of 3; 10; 30; 100; 300; and 1000 nM.

Paclitaxel (Taxol, CAS: 33069‐62‐4), doxorubicin (CAS: D1515) and cisplatin (CAS: D15663‐27‐1) were purchased from Sigma‐Aldrich. FITC‐labelled paclitaxel, which was used as an indicator of cytoplasmic drug accumulation, was donated by Dr. Han Zou of Nanjing University. The synthetic mature miR‐495 mimic (CAS: hsa‐miR‐495) and the nonsense RNA were purchased from Cell Biolabs Inc. (San Diego, CA, USA). The antibodies against MDR1 (CAS: sc‐13131) and GAPDH (CAS: sc‐32233) were obtained from Santa Cruz Biotech (Santa Cruz, CA, USA). The plasmids pSi‐ABCB1siRNA, which targets ABCB1, and pSi‐miR‐495 sensor, along with their respective negative control pSi‐negatives, were provided by Genepharm (Pallini, Greece). The p‐MIR‐reporter plasmid and β‐galactosidase (β‐gal) expression plasmid were bought from Ambion (Grand Island, NY, USA). Luciferase Reporter Assay Kits were purchased from BioVision Inc. (Cat: K801‐200; Milpitas, CA, USA) and Promega (Cat: E1483; Madison, WI, USA). In addition, five primary ovarian and six primary gastric cancer samples were obtained from the excised tissue tumour tissues donated by cured patients in Taizhou municipal hospital, and recurrent ovarian and gastric tumour tissues were independently obtained from five patients with ovarian cancer and six patients with gastric cancer.

Paclitaxel (Taxol) and vinblastine sulphate salt were obtained from Sigma-Aldrich, Burlington, MA, USA. All other materials used in this study were of analytical grade and commercially available.

Pelleting of Taxol-stabilized microtubules from cytosolic fractions was performed essentially as described [47 (link)]. Asynchronously growing HEK293 cells expressing eGFP or SIRT4-eGFP were lysed in PHEM buffer [60 mM PIPES, 25 mM HEPES, 1 mM EGTA, 1 mM magnesium acetate, pH 6.8; 1 × cOmplete^™ protease inhibitor cocktail (Sigma-Aldrich, München, Germany)] using a Dounce homogenizer. Following centrifugation (14,000× g for 30 min) of the total cell lysate, the supernatant (cytosolic fraction) was supplemented with GTP (1 mM) and Paclitaxel/Taxol (20 µM) (both from Sigma-Aldrich, München, Germany). Samples were incubated at room temperature for 30 min and subjected to centrifugation (14,000× g for 15 min) through a sucrose layer (15% sucrose in PHEM buffer) to obtain supernatant and the microtubules containing pellet fraction. The latter was washed one time in Taxol containing PHEM buffer, centrifuged, and sample fractions were analyzed by SDS-PAGE.