Sh sy5y human neuroblastoma cell line

SH-SY5Y human neuroblastoma cell line (Sigma 94030304) was cultured in DMEM/F12 (Gibco 11330032) supplemented with 1× GlutaMAX (Gibco 35050-061), 1× Penicillin/Streptomycin (Sigma P4333-100ML) and 15 % heat-inactivated FBS (Gibco 10270-106).
Cells (200,000 per well of a 6-well plate) were plated. Cells were transduced the day after and each lentivirus was mixed with spent medium (500 ng (GV) ml⁻¹, 1 ml total). Three wells were transduced individually for each shRNA-coding lentivirus (2–4 shRNA sequences per target or 1–2 per nontargeting shRNA were tested; see cloning for details). Medium was changed the day after transduction and cells were then collected 4 days post-transduction in RIPA buffer (500 µl per well) containing 2 tablets of cOmplete EDTA free protease inhibitor cocktail (Roche 11873580001) and 1 tablet of PhosSTOP (Roche 0490684500) per 20 ml of buffer.

To test the previously described neurotoxic effects of the examined aggregates in vitro, we used a differentiated SH-SY5Y human neuroblastoma cell line (Sigma-Aldrich), with the related culturing and differentiating methods based completely on our previous works [7 (link), 26 (link)]. Five-day-old animals (as determined by BSI) were chosen for the experiments, an age that precedes the beginning of the reproductive stage (i.e., egg production) by 2 days. Before treatment (using micropipette) of individual rotifers with aggregated molecules (0.1, 10 and 100 μg/mL), the stock solutions (1 mg/mL) were ultrasonicated (Emmi-40 HC, EMAG AG, Mörfelden-Walldorf, Germany) for 10 min at 45 kHz to achieve sterilization and homogenization. During the in vivo experiments, the viability assay, the assessments of descriptive characteristics (such as normalized mean lifespan /NML/, body size index /BSI/, bright light disturbance /BLD/, mastax contraction frequency /MCF/, and cellular reduction capacity /CRC/), and the assembly of the experimental setup (with the oil-covered microdrop) were carried out as presented in detail previously [34 (link)].

The SH-SY5Y human neuroblastoma cell line was provided by Sigma-Aldrich. Cells were grown in Dulbecco’s modified Eagle´s medium (DMEM) supplemented with 2 mM L-glutamine, 100 mU/mL penicillin, 0.1 mg/mL streptomycin, and 10% heat-inactivated fetal bovine serum (FBS). Cells were maintained at 37 °C and incubated in humidified atmospheric air containing 5% CO₂. After 1 day, the cells were exposed to 10 nM tau and/or 10 nM MB for 24 h. Afterward, The Ca²⁺-ATPase activity was also assayed in membranes prepared from SH-SY5Y cells, as described above.

The SH-SY5Y human neuroblastoma cell line was obtained from Sigma-Aldrich (cat. n° 94030304) (St. Louis, MO, USA). Cells were expanded in a growth culture medium composed of high glucose Dulbecco’s Modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 50 U/mL of penicillin, and 50 μg/mL of streptomycin, and cultured at 37 °C with 5% CO₂ as previously reported [31 (link)]. Cell differentiation was induced by reducing serum levels of the medium to 1% with 10 µM retinoic acid (RA) for seven days prior to treatments [32 (link)]. SF (LKT Laboratories, Minneapolis, MN, USA), EGCG and PB (Sigma-Aldrich, St. Louis, MO, USA) were dissolved in DMSO, and 10 mM stocks were kept at −20 °C until use. Differentiated SH-SY5Y cells were treated with 1 μM SF, 2.5 μM EGCG, and 0.5 μM PB and SEP (1 μM SF + 2.5 μM EGCG + 0.5 μM PB,) for 24 h or 6 h according to the different experiments. Oxidative stress was induced, as previously reported [33 (link)], by exposing cells to 700 µM H₂O₂ in 1% FBS DMEM.

The SH-SY5Y human neuroblastoma cell line was obtained from Sigma-Aldrich (Milan, Italy). Cells were grown in DMEM supplemented with 10% (v/v) FBS, 2 mM glutamine, 50 U/mL of penicillin and 50 μg/mL of streptomycin and maintained at 37 °C in a humidified incubator with 5% CO₂ as reported in [40 (link)]. Cell differentiation was induced reducing serum levels of the medium to 1% with 10 µM of RA for seven days prior to treatments. Differentiated SH-SY5Y were treated with plain and hill extracts for 24 h. Extracts were dissolved in DMSO. The control group was treated with an equivalent volume of the vehicle alone. Oxidative stress was induced exposing cells to 700 µM H₂O₂ for different times depending on the assay.