Taxol

HEK293 cells and HEK293T cells were cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco) containing 10 % fetal bovine serum (FBS, Gibco) and 1 % glutamine (Gibco). THP-1 cells and Jurkat cells were cultured in Roswell Park Memorial Institute medium (RPMI, Gibco) supplemented with 10 % FBS and 1 % glutamine. The cells were cultured at 37 °C with 5 % CO₂.
The antibodies and reagents used in this work were as follows: anti-HA (Cell signaling technology,CST), anti-p24 (Santa Cruz Biotechnology), anti-TRIM11 (Sigma-Aldrich), anti-Myc (Roche); puromycin and MG132 (Sigma-Aldrich), chloroquine (CST), nocodazole (Beoytime) and taxol (Tocris Bioscience).

Embryos for immunostaining were collected every three hours at different temperatures, as indicated and dechorionated. Embryos were transferred in a 1:1 solution of heptane and 4% formaldehyde in PBS for 20 min. Alternatively, embryos were incubated in a 1:1 solution of PEM Buffer containing 1 μM Taxol (Tocris Bioscience, Bristol, UK) and heptane to which 1 mL of 20% formaldehyde was added for 10 min. Taxol was added to stabilise microtubules and improve the staining of this cytoskeletal component [76 (link)]. After fixation, formaldehyde was replaced with methanol and vigorously shaken. After two washes in methanol, embryos were washed in PBS-0.1% Tween-20 (3 times, 15 min each) and incubated in blocking solution for 1 h at room temperature. Primary antibodies were incubated overnight at 4 °C, rotating. Primary antibodies were used at the following dilutions: rabbit anti-Centrosomin 1:250 (kindly provided by Professor J. Raff, Oxford, UK), and mouse anti-β-tubulin 1:50 (DSHB, E7; Iowa City, IW, USA). Fluorophore-conjugated secondary antibodies (Invitrogen by Thermo Fisher Scientific, Paisley, UK) were used at 1:250 and DAPI at 1 µg/mL. Specimens were mounted in MOWIOL/DABCO and imaged on Olympus BX-61, TRF fluorescence microscope. Images were processed using Volocity (Improvision, Perkin Elmer, Seer Green, UK) and Adobe Photoshop.

The CIPN model was induced as described previously (Polomano et al., 2001 (link)). Briefly, paclitaxel (Taxol^®) (2.0 mg Kg^–1 – cumulative dose of 8.0 mg Kg^–1) (Tocris, United Kingdom) was dissolved in a solution of 4% Dimethyl Sulfoxide (DMSO). Rats weighing 190-200 g received an intraperitoneal (i.p.) injection of the paclitaxel solution in 4 alternate days (day 1, 3, 5, and 7). The injections were performed between 9 a.m. and 11 a.m. Control animals were injected with 4% DMSO. The DMSO concentration was elected based on previous studies showing that it is the minimal concentration of DMSO required to resuspend paclitaxel and it does not induce any toxic effects (Worthley and Schott, 1969 (link); Chen et al., 2011 (link); Braz et al., 2015 (link)).

Paclitaxel (Taxol, Tocris Bioscience #RDS109750, Bristol, United Kingdom) was made fresh daily by dissolving in absolute ethanol, cremophor EL (Sigma-Aldrich) and 0.9% saline at a ratio of 1:1:18, respectively. Experimental procedures were as previously described (Deng et al., 2015 (link); Paton et al., 2017 (link); Atigari et al., 2020 (link)). Mice were administered paclitaxel 4 mg/kg i.p. injections on four alternate days to give a cumulative dose of 16 mg/kg. Mechanical and cold allodynia were assessed every second day to measure the progression of paclitaxel-induced effects. Mice were placed in transparent plastic chambers upon a metal mesh stand. After a 20 min habituation to the apparatus, each hind paw was measured in duplicate for each type of stimulation, always beginning with mechanical testing. On days with behavioural measurements and a paclitaxel dose, measurements were always taken before the administration of paclitaxel.

2-Deoxy-d-glucose (2-DG, Sigma D6134-1G); NMDA (Tocris, 0114); D-AP5 (Tocris, 0106); MK801 (Tocris, 0924); CGP (Tocris, 1493); DIDS (Sigma, D3514); Veratridine (Tocris, 2918); Bumetamide (Sigma, B3023); GlyH-101 (Sigma, 219671); NPPB (Tocris, 0593); Mannitol (Spectrum Chemical, MA165); Ionomycin (Tocris, 1704); SMIFH2 (Sigma, S4826-5MG); CK-666 (Sigma, SML_0006_5MG); Latrunculin A (Lat-A, Sigma, L5163); Thapsigargin (Tocris, 1138); Jasplakinolide (JASP, Tocris, 2792); Bovine Serum Albumin (BSA, Sigma, A4919-5G); DAPI (Biotium, 40043); SAHA (Tocris, 4652); Santacruzamate A (Tocris, 7191); CI994 (Tocris, 2952); FK228 (Tocris, 3515); Tubastatin (Tocris, 6270); Nicotinamide (Tocris, 4106); EX527 (Tocris, 2780); anti-sirtuin 5 inhibitor peptides (Dr.C.A. Olsen, NRE139; NRD167); Suramin (Fisher, AC328540500); C646 (Sigma, SML0002); Cycloheximide (Sigma, C4859); Blebbistatin (Sigma, B0560); Taxol (Tocris, 1097); Nocodazole, (Tocris, 1228); MyoVin-1, (Calbiochem, 475984); Mibefradil, (Tocris, 2198); ω-Conotoxin GVIA, (Tocris, 1085); NBQX (Tocris, 0373); Nifedipine, (Tocris, 1075). Each chemical was dissolved according to the manufacturer’s guidelines, either in water or DMSO, or ethanol.

Small molecule inhibitors used in experiments were the microtubule ligands taxol and nocodazole (Tocris, Bristol, UK), the focal kinase (FAK) inhibitor, PF-573228 (Cayman Chemical, Ann Arbor, MI, USA), and the Syk inhibition GS-9973 (Sigma, St. Louis, MO, USA). All small molecule inhibitors were dissolved in DMSO and used at the concentrations indicated in the figure legends. 0.1% DMSO was used for mock treatment. Cells were pretreated 20 min prior to performing degranulation assays or immunofluorescence microscopy. Commercial antibodies were obtained for CD63 (clone AD1, BioRad, Hercules, CA, USA), GEF-H1 (GenTex, Zeeland, MI, USA), phospho(Ser886)-GEF-H1 (clone E1L6D, Cell Signaling Technology, Danvers, MA, USA), Rac1 (clone 23A8, Sigma, St. Louis, MO, USA), RhoA (clone 26C4, Santa Cruz), β-tubulin (clone EPR16774, Abcam, Cambridge, UK), HA (clone HA-7, Abcam, Cambridge, UK), and vinculin (Proteintech, Rosemont, IL, USA).