Quinacrine

The karyotype study was repeated on three different populations and karyotypic stability was assessed on three different generations. Cytogenetic analysis was carried out by setting up two independent cultures, each obtained by seeding 10⁵ cells on a slide (amnio-dish) placed on the bottom of Petri dishes (Corning, NY, USA). Cells were seeded at a density of 1.2 × 10³ cell/cm² in proliferative medium. In the first culture, cells were blocked with colchicine after 24–96 h from seeding, at a confluence of 20%, while the second culture was blocked 24 h after. Subsequently, the metaphases were blocked with colcemid (Merck) and cells were maintained in culture for 12–16 h. The osmotic treatment was then carried out by incubation in hypotonic solution (KCl 0.075 M, Merck) and fixation with a solution of methanol, ethanol and acetic acid (2:1:1, v/v) (Merck). The slides were then extracted from the Petri dishes and dried under controlled temperature (26 °C) and humidity (36% RH) conditions. For the Q-banding method using quinacrine (QFQ), the slides were immersed in a quinacrine (Merck) solution in McIlvaine buffer, kept in the dark for 10 min and, after rinsing in the same buffer, mounted. The metaphases were visualized by fluorescence optical microscopy and acquired through an image analysis system.

Quinacrine [4-N-(6-chloro-2-methoxyacridin-9-yl)-1-N,1-N-diethylpentane-1,4-diamine] was used to visualize vesicular ATP, a fluorescent dye widely used to detect ATP-enriched vesicles (Gutierrez-Martin et al., 2011 (link)). Quinacrine staining was carried out by incubating granule cells for 15 min at 37°C in Locke’s buffer (140 mM NaCl, 4.5 mM KCl, 2.5 mM CaCl₂, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 5.5 mM glucose and 10 mM HEPES [pH 7.4]) containing 4 μM Quinacrine (Sigma–Aldrich). Time-lapse studies were performed with an Olympus IX81 inverted microscope equipped with a 100X, 1.45 NA, oil-immersion objective. Images were acquired every 200 ms with a Hamamatsu C9100 EM-CCD digital camera (Hamamatsu) controlled by CellR software (Olympus). Vesicular fusion and release into the extracellular space was observed as a rapid loss of the Quinacrine signal. During the assays, cells were continuously perfused with Locke’s buffer at a rate of 1.5 ml/min at 37°C. Perfusion was gravity-driven and solution exchange was performed by manually operating electronic valves of a VC-6 drug application system (Warner Instruments). Images were processed using the ImageJ free software v.1.50i (National Institutes of Health, Bethesda, MD, United States).

AA, AdOx, quinacrine, monensin, MG132, and AZA were obtained from Merck (Madrid, Spain). Antibodies used in this study are listed in Supplementary information Methods.