Sh sy5y human neuroblastoma cells

SH-SY5Y human neuroblastoma cells (cat. 94030304; Sigma-Aldrich, Milan, Italy) were seeded (1 × 10⁴ cells/cm²) and cultured as monolayer in moist air, at 37 °C, 5% CO₂, in a cell incubator (model MCO-15AC; SANYO Electric Co., Ltd., Moriguchi, Japan), using 10% (v/v) foetal bovine serum (FBS)-supplemented RPMI-1640 medium (cat. ECS0180L and ECM0505L, respectively; Euroclone, Milan, Italy), containing 2 mM L-glutamine, 100 IU/ml penicillin and 100 mg/ml streptomycin (cat.10378-016; Life Technologies Italia, Monza, Italy), as described in previous papers^{10 (link), 12 (link)}.

SH-SY5Y human neuroblastoma cells (Sigma-Aldrich, St. Louis, MO, USA, from The European Collection of Authenticated Cell Cultures, ECACC, Public Health England) were used. This cell line is not listed as a commonly misidentified cell line by the International Cell Line Authentication Committee (ICLAC; http://iclac.org/databases/cross-contaminations/, on 16 January 2023) and was not further authenticated during the last five years. The SH-SY5Y cells were grown in Dulbecco’s Modified Eagle Medium plus F12 in a 1:1 ratio, containing 10% bovine serum, 1% L-glutamine, and 1% penicillin–streptomycin (all from Euroclone, Italy); they were maintained at 37 °C in a humidified 5% CO₂ atmosphere and used within passage 30. For the Western blot (WB) and Co-IP experiments, the cells were seeded in six-well plates at a density of 500,000/4 mL/well. Under these conditions, the cells are not differentiated into neurons. Twenty-four hours after the onset of the culture, the cells were treated for 5 min with CGS 21680 (300 nM) or DHPG (10 µM). ZM 241385 (500 nM) and MPEP (10 µM) were applied 20 min before and then along with CGS 21680 or DHPG. The SH-SY5Y cells were maintained at 37 °C under 5% CO₂ for the duration of the experiments.

SH‐SY5Y human neuroblastoma cells (Sigma‐Aldrich #94030304) at P11 were transduced in a 6‐well plate using 300 µl of Auto‐hLAG3 LV pre‐incubated with 30 µl of Lenti‐X™ Accelerator (Clontech #631256) for 30 min. The LV mixture was added onto the cells, evenly spread over the whole well and put into incubator onto a neodymium magnetic sheet (supermagnete #NMS‐A4‐STIC; adhesive force of 450 g/cm²) for 10 min. Cells were then removed from the incubator, LV mixture was completely removed, and fresh SH media added. Cells were kept until confluency and then sub‐cultured with the neodymium magnet sheet kept under the plate. To remove remaining magnetic beads, cell pellet was resuspended in 1.5 ml of SH media, pipetted into 1.5‐ml Eppendorf tube and inserted into DynaMag™‐2 Magnet (Invitrogen #12321D). Cell suspension was then removed from the tube leaving all magnetic beads in the tube. Quantification of P11+1 SH‐10 cells showed that 76% of cells were expressing hLAG3 upon induction by 1 µg/ml of Doxycycline (DOX; Clontech #631311). This decreased to 59% in P11+2 and remained around 50% in the following passages. P11+3 was used for all experiments except for the CO‐IP, where a later passage was used.