Sodium tetrahydroborate

PVP was carboxylated by partially hydrolyzing the pyrrolidone ring following a previously published method that allows an amount of ring opening of 15% [30 (link)] (Figure 1a). Briefly, 0.2 g of PVP (MW = 29 kDa, Sigma Aldrich, St. Louis, MO, USA) was dissolved in 10 mL of 0.1 N NaOH and heated at 140 °C for 48 h in a beam calorimeter (Parr Instrument Company, Moline, IL, USA). In order to prevent the closing of the opened pyrrolidone ring, its γ-amino butyric was methylated by adding 600 μL of 35% formaldehyde solution (Sigma Aldrich, St. Louis, MO, USA) followed by adjusting the solution to pH 9 and then cooling to 0 °C. Next, 1.5% of sodium tetrahydroborate (Sigma Aldrich, St. Louis, MO, USA) was added; the solution was stirred for 45 min at room temperature (RT) and then vacuum-dried at 60 °C overnight. Prior to use, the carboxylation of PVP was assessed by Fourier transform-infrared spectroscopy (FT-IR) to monitor the ring opening and the presence of the carboxyl groups. FT-IR spectra were acquired with an FT-IR Nicolet 6700 (ThermoFisher Scientific Inc., Waltham, MA, USA) equipped with an attenuated total reflectance (ATR) stage, with 64 scans per sample with a resolution of 4 cm⁻¹.

Cells were fixed and permeabilised with 3% paraformaldehyde (Sigma) containing 0.25% Triton X-100 (Sigma) and 0.05% glutaraldehyde (Sigma) for 15 min, before being washed in PBS (Lonza). Autofluorescence was quenched using 0.01% sodium tetrahydroborate (Sigma) in PBS. Cells were incubated with anti-tubulin (DM1A, Sigma) antibody, at 1∶500 dilution, followed by secondary antibodies conjugated to DyLight 488, 594 or 649 (Jackson ImmunoResearch Laboratories, Suffolk, UK). For actin, Texas-Red-, FITC- or Alexa-Fluor-633-labelled phalloidin (Invitrogen) was added together with the secondary antibody. For the EB1 antibody (1A11/4, Santa Cruz Biotechnology), cells were fixed in −20°C methanol for 5 min, before being rehydrated in PBS and stained using anti-tubulin antibodies (as above). For the experiments involving nocodazole (Sigma) and Cell-Tak, cells were spread on glass-bottomed dishes in the presence of 10 or 4 µM nocodazole or DMSO (control) and Cell-Tak, respectively, for 1 h prior to fixation. Cells were then imaged using an oil-immersed 100× objective, with 1.35 numerical aperture, on an inverted microscope (IX71; Olympus) controlled by a Deltavision system (Applied Precision, Washington, USA). Images were captured using a Coolsnap HQ CCD camera (Princeton Instruments, Lurgan, UK).