Poly dl ornithine

Cells were grown on 8-well chamber slides (Labtek, Thermo Fisher Scientific), precoated for 1 h with fibronectin (10 μg/ml) for U87-MG or with poly-DL-ornithine (Sigma-Aldrich) (10 µg/ml) for GBM6, to be treated for 6 h with ProA, digoxin, bufalin or digitoxin. As previously described^{16 (link)}, cells were incubated with the anti-EB1 (clone 5; BD Biosciences, San Jose, CA) and α-tubulin (clone DM1A; Sigma-Aldrich) primary antibodies, and then with Alexa488 or 568-conjugated secondary antibodies (Invitrogen). Staining was observed using either a Leica DM-IRBE microscope or a Leica TCS SP5 confocal laser-scanning microscope (Leica, Heidelberg, Germany). Images were acquired using Metamoph software or the Leica Confocal software, and were processed using Image J software. For each experimental condition, at least 100 EB1 comets (in at least 10 cells) were examined to measure their length.

Primary culture of neurons from the cerebral cortex of 17-day-old embryonic rats was performed according to a previously reported protocol [24 (link),25 (link)]. Briefly, cortical tissues were minced into pieces in sterile phosphate-buffered saline (PBS), pH 7.4, and dissociated with 0.25% trypsin (Invitrogen, Carlsbad, CA, USA) and 0.01% DNase I (Sigma-Aldrich, St. Louis, MO, USA) in PBS for 12 min at 37 °C. The reaction was terminated by the addition of 0.25% soybean trypsin inhibitor (Sigma-Aldrich), and the cell suspension was centrifuged at 1000× g for 5 min. The pellet was resuspended in serum-supplemented D/F medium (1:1 Dulbecco’s modified Eagle’s medium/Ham’s F-12 medium (Invitrogen) containing 5% horse serum (HS), 5% fetal bovine serum (FBS), and 1% 2-mercaptoethanol (Invitrogen, San Diego, CA, USA)). Dissociated neurons were seeded at a density of 5 × 10⁵ cells/cm² onto poly-DL-ornithine (Sigma-Aldrich)-coated 24-well plates for extracellular ProTα measurement, and subsequently cultured at 37 °C in a 5% CO₂ atmosphere. Cytosine β-d-arabinofranoside (Ara-C; Sigma-Aldrich) at 0.3 μM was added to the culture at 24 h after seeding, followed by another 48 h of culture.