in vitro tubulin polymerization assay kit  (Millipore)

Journal: Pharmaceutics

Article Title: Anti-Breast Cancer Properties and In Vivo Safety Profile of a Bis-Carbazole Derivative

doi: 10.3390/pharmaceutics17040415

Figure Lengend Snippet: Docking simulations suggested that 1 binds a region proximal to the Paclitaxel-binding site of tubulin. The most important amino acid residues involved in the interactions are evidenced. Cyan ribbons: Tubulin, α-subunit, Salmon ribbon Tubulin β-subunit. Yellow sticks, 1 binding mode. Oxygen atoms belonging to evidenced aminoacids are colored in red, while nitrogen in blue. Bromide moieties are evidenced in amaranth.

Article Snippet: The tubulin polymerization inhibition was detected using the in vitro Tubulin Polymerization Assay Kit (EMD Millipore Corporation, Burlington, MA, USA), following the procedure previously published [ ].

Techniques: Binding Assay

Journal: Pharmaceutics

Article Title: Anti-Breast Cancer Properties and In Vivo Safety Profile of a Bis-Carbazole Derivative

doi: 10.3390/pharmaceutics17040415

Figure Lengend Snippet: In vitro tubulin polymerization assay. The assembly of tubulin into microtubules was followed by measuring the turbidity (absorbance, A) at 350 nm for 3600 s at 37 °C. The polymerization curves, in the presence of 1 (1 μM) or not (CTRL, DMSO), are shown. Moreover, the graphic shows the curves obtained using two reference molecules, Vinblastine and Paclitaxel, (both at 10 μM concentration), used as tubulin-destabilizing and tubulin-stabilizing agents, respectively.

Article Snippet: The tubulin polymerization inhibition was detected using the in vitro Tubulin Polymerization Assay Kit (EMD Millipore Corporation, Burlington, MA, USA), following the procedure previously published [ ].

Techniques: In Vitro, Polymerization Assay, Concentration Assay

Journal: Pharmaceutics

Article Title: Anti-Breast Cancer Properties and In Vivo Safety Profile of a Bis-Carbazole Derivative

doi: 10.3390/pharmaceutics17040415

Figure Lengend Snippet: Immunostaining studies on MDA-MB-231 cells treated with compound 1 (0.3 µM), Vinblastine, Paclitaxel (both at 1 µM), or vehicle (CTRL, DMSO) for 24 h. In the control experiment (CTRL), cells exhibited a regular arrangement of cytoskeleton. Vinblastine and Paclitaxel, instead, dramatically impacted the tubulin network. Vinblastine produced tubulin crystal formation (panel ( B ), V), whereas Paclitaxel induced tubulin bundles and thickened fibers (panel ( B ), P). Compound 1 exposure produced a morphology similar to Paclitaxel treatment (white arrows). Panels ( A ): DAPI, excitation/emission wavelength 350 nm/460 nm. Panels ( B ): β-tubulin (Alexa Fluor ® 488) excitation/emission wavelength 490 nm/515 nm. Panels ( C ): overlay. Images were taken at 20×, and representative fields were shown.

Article Snippet: The tubulin polymerization inhibition was detected using the in vitro Tubulin Polymerization Assay Kit (EMD Millipore Corporation, Burlington, MA, USA), following the procedure previously published [ ].

Techniques: Immunostaining, Control, Produced

Journal: Molecular Oncology

Article Title: Repositioning VU ‐0365114 as a novel microtubule‐destabilizing agent for treating cancer and overcoming drug resistance

doi: 10.1002/1878-0261.13536

Figure Lengend Snippet: Prediction of novel microtubule‐targeting agents by the CMap analysis. (A) Connections of drug‐gene signatures were analyzed using the “Touchstone” tool in the CMap database ( https://clue.io/ ). Connections were viewed as a heat map ranked by the summary connectivity score. Drugs highlighted in red were potential microtubule‐targeting agents. Some drug names were duplicated because they have multiple gene signatures in the CMap database. An enlarged image was provided in Fig. . (B) L1000FWD visualization of drug‐gene signatures. Drugs sharing similar mechanisms of action (MOAs) were clustered together. The red box indicates the cluster of tubulin polymerization inhibitors. The lower panel shows the representative L1000FWD figures of microtubule‐targeting agents (vinblastine and paclitaxel). An enlarged image is provided in Fig. . (C) L1000FWD visualization of candidate drugs that were identified as microtubule‐targeting agents in other studies. (D) L1000FWD visualization of candidate drugs that had not been previously identified as microtubule‐targeting agents. In panels B–D, each clustered point represents the gene signature of a drug in different cell lines, treated with varying doses and time intervals. The yellow circles highlight the queried drugs' gene signature.

Article Snippet: In vitro tubulin polymerization was measured with a commercial kit (#BK006P; Cytoskeleton) according to the manufacturer's instructions.

Techniques:

Journal: Molecular Oncology

Article Title: Repositioning VU ‐0365114 as a novel microtubule‐destabilizing agent for treating cancer and overcoming drug resistance

doi: 10.1002/1878-0261.13536

Figure Lengend Snippet: Identification of VU‐0365114 as a potent microtubule‐targeting agent. (A) The chemical structures of VU‐0365114, HMN‐214, acitretin, and GW‐843682X. (B, C) Purified tubulins were incubated with 10 μ m of the drugs (B) or the indicated concentration of the drugs (C) for 60 min. Tubulin polymerization was detected by measuring the absorbance at 340 nm. Data represent three independent experiments. (D) HeLa cells were treated with VU‐0365114 (VU; 10 μ m ), colchicine (Col; 10 μ m ), or paclitaxel (Txl; 0.5 μ m ) for 3 h. Soluble and assembled (pellet) tubulins were isolated and analyzed by Western blotting. The corresponding nitrocellulose membrane stained with Ponceau S was used as a loading control. Data represent three independent experiments. (E) HeLa‐Kyoto‐tubulin‐EGFP/H2B‐mCherry cells were treated with VU‐0365114 (10 μ m ), colchicine (10 μ m ), or paclitaxel (0.5 μ m ) for 4 h. The EGFP and mCherry fluorescence was observed under a fluorescence microscope (scale bar: 20 μm). Data represent three independent experiments. (F) The binding site of VU‐0365114 on tubulin was examined by the competition of tubulin‐binding with colchicine or BODIPY FL‐vinblastine. The intrinsic fluorescence of the colchicine‐tubulin complex or the fluorescence of BODIPY FL‐vinblastine was analyzed on a plate reader. The error bars are the mean ± SD ( n = 3). Statistical significance, compared to untreated controls (* P < 0.05, ** P < 0.01, and *** P < 0.001), was determined using a one‐way ANOVA with Tukey's post hoc test. (G) Tubulins were treated with DMSO (Con), colchicine (Col; 100 μ m ), vinblastine (Vin; 100 μ m ), or VU‐0365114 (VU; 100 μ m ), and then digested by TPCK‐treated trypsin. Samples were analyzed by SDS/PAGE. M, protein molecular weight marker; −, tubulin without digestion. Data represent three independent experiments. (H) AsPC‐1 and PANC‐1 cells were treated with 10 μ m VU‐0365114 for 18 h, and then RNA sequencing analysis was performed. The differentially expressed genes were used to query the CMap database. The gene expression‐based similarity of VU‐0365114 to CMap drugs was visualized as a heat map. The original image was provided in Fig. . Some drug names were duplicated because they have multiple gene signatures in the CMap database. Data were obtained from one experiment conducted on two technical replicates.

Article Snippet: In vitro tubulin polymerization was measured with a commercial kit (#BK006P; Cytoskeleton) according to the manufacturer's instructions.

Techniques: Purification, Incubation, Concentration Assay, Isolation, Western Blot, Membrane, Staining, Fluorescence, Microscopy, Binding Assay, SDS Page, Molecular Weight, Marker, RNA Sequencing Assay, Expressing

Journal: bioRxiv

Article Title: Sodium Thiosulfate acts as an H 2 S mimetic to prevent intimal hyperplasia via inhibition of tubulin polymerization

doi: 10.1101/2021.09.09.459573

Figure Lengend Snippet: A) α -tubulin immunolabelling in carotids of native (D0) or CAS-operated mice treated or not (Ctrl) with STS 4g/L. L=Lumen; M= Media; IH= Intimal Hyperplasia. Images are representative of 5 to 8 mice per group. B) WB analysis of α-tubulin over total protein in aortas of mice treated or not (Ctrl) with STS 4g/L for 7 days. Data are scatter plots of 7 mice per groups with mean±SEM with **p<.01, as determined by one-way ANOVA with Tukey’s multiple comparisons tests. C) α-tubulin immunolabelling in human vein segments kept or not (D0) in culture in presence or not (Ctrl) of 15mM STS for 7 days. Scale bar 40µm. L=Lumen; M= Media. Images are representative of 5 different veins. D) WB analysis of tubulin over total protein in human vein segments kept or not (D0) in culture in presence or not (Ctrl) of 15mM STS or 100 µM NaHS for 7 days. *p<.05, **p<.01, ***p<.001, as determined by repeated measures one-way ANOVA from 7 different veins with Dunnett’s multiple comparisons tests. E ) α-tubulin immunofluorescent staining in VSMC exposed or not to 15mM STS or 100 µM NaHS for 8 hours. Images are representative of 5 independent experiments. Bar scale 10 µm. F) Quantitative assessment of microtubule filaments immunostaining per cell. Data are representative of 3 independent experiments, 3 to 4 images per experiment per condition. ***p<0.001 as determined by one-way ANOVA with Tukey’s multiple comparisons tests. G) In vitro tubulin polymerization assay in presence or not (Ctrl) of 15mM STS, 100µM NaHS or 10µM Nocodazole. Data are mean±SEM of 3 independent experiments. H) Flow cytometry analysis of cell cycle (DNA content) using DAPI-stained VSMC treated or not (Ctrl) for 48 hours with 15mM STS or 10nM Nocodazole. Upper panel : representative histograms; lower panel : table with mean±SD of 5 independent experiments. *p<0.05, **p<0.01 as determined by one-way ANOVA with Dunnett’s multiple comparisons tests.

Article Snippet: The assay was performed using the In Vitro Tubulin Polymerization Assay Kit (≥99% Pure Bovine Tubulin; 17-10194 Sigma-Aldrich), according to the manufacturer’s instruction.

Techniques: Staining, Immunostaining, In Vitro, Polymerization Assay, Flow Cytometry