Vinblastine

Drugs, suppliers, and concentrations used were Barasertib (Aurora B inhibitor; alternative name AZD1152‐HQPS; SelleckChem S1147; 1.11 nM); CHR‐6494 (Haspin inhibitor; MedChem Express HY‐15217; 500 nM); CW069 (HSET inhibitor; SelleckChem S7336; 25.0 μM); Etoposide (Topoisomerase II inhibitor; SelleckChem S1225; 333 nM); GSK461364 (PLK1 inhibitor; SelleckChem S2193; 2.20 nM); GSK923295 (CENP‐E inhibitor; SelleckChem S7090; 3.20 nM); Ispinesib (KIF11 inhibitor; alternative name SB‐715992; SelleckChem S1452; 1.70 nM); MK‐5108 (Aurora A inhibitor; alternative name VX‐689; SelleckChem S2770; 0.576 nM); MK‐8776 (CHK1 inhibitor; alternative name SCH 900776; SelleckChem S2735; 9.00 nM); Paclitaxel (microtubule inhibitor; SelleckChem S1150; 2.67 nM); Vinblastine (microtubule inhibitor; Sigma V1377; 2.40 nM); and YM155 (BIRC5 inhibitor; SelleckChem S1130; 0.540 nM).

Mouse keratinocytes were isolated from the backskins of newborn or embryonic mice. Mouse keratinocytes were grown in E low calcium medium and maintained in a 37°C incubator with 7.5% CO₂. Primary cells were isolated from mouse backskin by incubating the backskin overnight in a mixture of 1X phosphate-buffered saline (PBS) and Dispase II (Hoffman-La Roche, Basel, Switzerland) at a 1:1 ratio. The epidermis was then removed from the dermis and placed in a mixture of trypsin and versene at a 1:1 ratio for 3 min. Cells were resuspended in fresh media, filtered, pelleted, and plated onto glass coverslips coated with 100 μM laminin (Invitrogen, Waltham, MA), or onto fibroblast feeders for long-term passage. For drug treatments, 10 μM nocodazole (Sigma-Aldrich, St. Louis, MO) and the corresponding concentration of DMSO was incubated with cells for 30 min before fixation. In mitotic cells, in which MTs are much more dynamic, nocodazole treatment was with 0.5 μM for 5 min. Vinblastine (2 nM) and taxol (10 μM) were both from Sigma, and treatments were for 5–10 min. For spindle orientation experiments on cultured keratinocytes, cells were plated on glass coverslips coated with 100 μM laminin (Invitrogen). HeLa cells were grown at 37°C with 7.5% CO₂ in DMEM media containing 10% Fetal Bovine Serum with antibiotics.

HT1080 and HCA2T cells were split to a density of 1 × 10⁵ cells per well of a six well plate 24 hours prior to treatment with the indicated concentration of DMSO (Sigma), Doxorubicin (Sigma), Vinblastine (Sigma) and LS1. All drugs were concurrently applied to the cells. Cell survival was measured 48 hours after treatment by counting the adherent cells in each group using a Z2 particle counter (Beckman Coulter). The ratio of adherent drug-treated cells to adherent cells treated with DMSO represents the raw survival. Experiments using Doxorubicin were repeated six times; experiments using Vinblastine were performed in triplicate.

Porcine tubulin (Cytoskeleton; 10 μM; T238P-A) was incubated in the absence or presence of colchicine (TCI; C0380), vinblastine (Sigma; V1377), and Taxol (TCI; P1632). A photoaffinity-based target ID probe of 1 (5 μM) was added and incubated for 75 min at room temperature. UV light (365 nm) was irradiated for 5 min to generate a covalent bond between the probe and tubulins. A click reaction between the acetylene group of target ID probe 1 and Cy5-azide was conducted at room temperature for 1.5 h. For the click reaction, 5% t-BuOH (TCI; B0706), 1 mM CuSO₄ (Sigma; #209198), 100 μM TBTA {tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, Sigma; #678937}, 2 mM TCEP {tris(2-carboxyethyl) phosphine hydrochloride, Alfa; J60316}, and 40 μM Cy5-azide (Lumiprobe; #33030) were added. Tubulin protein was analyzed by SDS‒PAGE, and Cy5-labeled tubulin was measured by a fluorescent gel scanner (Azure Biosystems; Sapphire). The whole loading level of tubulins was visualized by silver staining and imaged by ChemiDoc (Bio-Rad).

Nocodazole, paclitaxel (Taxol), vinblastine, and colchicine were purchased from Sigma-Aldrich (St. Louis, MO). SRF was purchased from ChemDiv while SB203580, PD98059 and SP600125 were from Calbiochem (San Diego, CA). Antibodies were obtained from the following companies: vimentin, α- and β-tubulin, JNK-1, Mdr-1, Bcl-2, pBcl-2 (T56), pBcl-2 (S70), pBcl-2 (S87) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); pp38, pERK1/2, pJNK (Cell Signaling Technology, Denver, MA); monoclonal anti P-glycoprotein (MDR) (Sigma, USA) and monoclonal actin antibodies were from BD Pharmingen (San Diego, CA). [³H]vinblastine (specific activity, 11.6 Ci/mmol) and streptavidin-coated yttrium silicate scintillation proximity assay (SPA) beads were purchased from GE Healthcare (Buckinghamshire, UK). [³H]colchicine (specific activity, 80.4 Ci/mmol) was obtained from PerkinElmer (Boston, MA, USA).