Bk004p

The effect of the studied compounds on the polymerization of purified porcine tubulin was monitored using a commercial assay kit (cat. number BK004P), purchased from Cytoskeleton, USA. Stock solutions of porcine brain tubulin solutions (4 mg.mL⁻¹) were prepared immediately prior to use in general tubulin buffer (80 mM piperazine-N,N′-bis(2-ethanesulfonic acid, PIPES) pH 6.9, 2 mM MgCl₂ and 0.5 mM (ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, EGTA) containing 1 mM guanosine triphosphate (GTP) (G-PEM buffer). Stock solutions of Paclitaxel (100 µM), Noconazole (100 µM) or tested samples (100 µM) in G-PEM buffer containing 5% DMSO were prepared.
The standard reaction mixture contains 95 μL of 4 mg.mL⁻¹ porcine brain tubulin and 5 μL of standards, test samples or G-PEM buffer only (control). Then, the microplate with loaded reaction mixtures was incubated for 90 min. at 37 °C in a thermostated spectrophotometer chamber. The turbidity of the mixtures was measured at 340 nm every 30 s. The experiment was performed on SpectroStar Nano (BMG Labtech, Aylesbury, UK) equipped with software for kinetic measurements.

Avermectin B1a was purchased from BioAustralis (BIA-A1010, BioAustralis, New South Wales, Australia), paclitaxel was obtained from Cytoskeleton (TXD01, Cytoskeleton, Denver, CO, USA), and colchicine was provided by Sigma Aldrich (C9754, Sigma Aldrich, St. Louis, MO, USA). Dulbecco’s Modified Eagle Medium (DMEM), the buffer Dulbecco’s PBS, and L-Glutamine (200 mM) were purchased from Capricorn (Capricorn, Dusseldorf, Germany), while fetal bovine serum (FBS), penicillin/streptomycin, and trypsin/EDTA were obtained from Biological Industries (Biological Industries, Jezreel Valley, Israel). The cell-counting dye trypan blue (0.4%) was purchased from Thermo Fisher Scientific (15250-061, Thermo Fisher Scientific, Waltham, MA, USA). Dimethyl sulfoxide (DMSO) was obtained from Carlo Erba (445106, Carlo Erba, Val de Reuil, Normandie, France).
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was used for cell viability assay and was purchased from Thermo Fisher Scientific (M6494, Thermo Fisher Scientific, USA). A tubulin polymerization assay was performed by using a tubulin polymerization assay kit from Cytoskeleton (BK004P, Cytoskeleton, Denver, CO, USA), whereas the apoptosis assay was performed by using annexin V-FITC/PI apoptosis kit purchased from Elabscience (E-CK-A211, Elabscience, Huston, TX, USA).

The interaction of mdivi-1 with tubulin was monitored in terms of intrinsic tryptophan fluorescence of tubulin^{50 (link)}. When excited at 295 nm, tubulin displays a typical tryptophan emission spectrum with a maximum at 355 nm. Association of the drug to tubulin reduces the fluorescence emission. The affinity of mdivi-1 to tubulin was thus measured by detecting the emission spectrum of the intrinsic tryptophan fluorescence of tubulin, according to procedures described previously^{45 (link)}. The reductions of fluorescence intensity at 355 nm at varying mdivi-1 concentrations were calculated, and the values were plotted on a double reciprocal plot. All the experiments were repeated at least three times.
A tubulin polymerization assay kit BK004P (Cytoskeleton Inc., Denver, CO, USA) was used according to the manufacturer’s instructions to examine the effects of mdivi-1 on in vitro tubulin polymerization. Briefly, 1 μl aliquots of mdivi-1 solubilized in DMSO at varying concentrations were added into 99 μl of the reaction mixture (3 mg/ml tubulin in 80 mM PIPES pH 6.9, 2 mM MgCl₂, 0.5 mM EGTA, 1 mM GTP, 10.2% glycerol) in the wells of a 96-well plate. Then, the plate was immediately placed onto a pre-warmed chamber of a spectrophotometer (EnSpire™ Multilabel Plate Reader, PerkinElmer, Waltham, MA, USA), and the absorption at 340 nm was measured every 60 s for 1 h at 37 °C.