Sh sy5y

The SH-SY5Y cell line was acquired from American Type Culture Collection (ATCC) (Manassas, VA, USA). We grew the human neuroblastoma cell line SH-SY5Y in a monolayer at 37 °C in a 5% CO₂ humidified atmosphere using Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham (DMEM/F12) medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich), 1% glutamine and 1% penicillin-streptomycin (100 U-100 µg/mL). To induce neuronal differentiation of SH-SY5Y cells, we incubated them for 5 days with 10 µM of all-trans RA (Sigma-Aldrich) [7 (link),65 (link)]. We dissolved Δ⁸-THC in DMSO and then diluted in PBS. We added it in the medium at the final concentration (the final DMSO concentration was <0.1%). We treated the differentiated SH-SY5Y cells with 20 µM Δ⁸-THC for 24 h.

The SH-SY5Y human neuroblastoma cell line was purchased from CLS (Cell Lines Service GmbH, Eppelheim, Germany). Cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS, Sigma-Aldrich, St. Louis, MO, USA), 100 IU/mL penicillin G, 100 μg/mL streptomycin, 1% L-glutamine, 1% non-essential amino acids, without sodium pyruvate at 37 °C in 5% CO₂-humidified atmosphere. At the time of 85% confluence the cells were subcultured, and the medium was changed every 3–4 days. For cell differentiation, SH-SY5Y cells were switched to a DMEM/F12 medium supplemented with 1% FBS in presence of 80 nM phorbol 12-myristate, 13 acetate (PMA, Sigma-Aldrich) for at least 6 days and the actual differentiation was evaluated by the observation of neurites outgrow by microscopy and the evaluation of βIII-tubulin mRNA levels by real-time PCR. Oxidative stress was induced by treating undifferentiated or differentiated SH-SY5Y cells with 10 μM catechol (Sigma-Aldrich). All the synthesized compounds as well as a reference antioxidant, Trolox (TRX, Sigma-Aldrich), were used at a concentration of 10 μM.

Human neuroblastoma cell‐line SH‐SY5Y (Sigma‐Aldrich, St. Louis, MO, USA) was thawed and seeded out in Poly‐l‐lysine (Sigma‐Aldrich, St. Louis, MO, USA) coated 12‐well plates (Nunc, Thermo Fisher Scientific, Waltham, MA) and Lab‐Tek 8‐well chamber slides (Thermo Fisher Scientific, Waltham, MA) coated with either Poly‐l‐Lysine or collagen I rat tail (Thermo Fisher Scientific, Waltham, MA). The SH‐SY5Y cells were grown in medium containing 41.5% Minimum Essential Medium Eagle (Sigma‐Aldrich, St. Louis, MO, USA), 41.5% Nutrient mixture F‐12 Ham (Sigma ‐Aldrich, St. Louis, MO, USA), 15% Fetal bovine serum (Thermo Fisher Scientific, Waltham, MA), 1% l‐glutamine (Thermo Fisher Scientific, Waltham, MA), 1% MEM non‐essential Amino Acids (Sigma‐Aldrich, St. Louis, MO, USA) at 37℃, 5% CO₂ and 5% H₂O until 60%–80% confluency.

The human HeLa (ATCC CCL-2) and SH-SY5Y (ATCC CRL-2266) cell lines were maintained in Dulbecco’s Modified Eagle Medium (DMEM) or minimum essential medium α (MEM-α) medium, respectively. Media were supplemented with 10% (v/v) Fetal Bovine Serum (FBS). Both cell lines were grown under a highly humidified atmosphere of 95% air with 5% CO₂ at 37 °C. The HeLa hnRNPDL KO cell line was generated as described elsewhere^{3 (link)}. HeLa cells have been authenticated within the last 3 years by STR analysis. The HeLa and SH-SY5Y cell lines were regularly tested for the presence of mycoplasma using qPCR Detection Kit (SIGMA), and both cell lines are mycoplasma negative.
For recombinant protein expression, the genes encoding the three isoforms of hnRNPDL (hnRNPDL-1, hnRNPDL-2 and hnRNPDL-3) were inserted into pETite (Lucigen corporation) vector with a His-SUMO N-terminal tag. For subcellular localization experiments, the same genes were cloned into pEGFP-C3 (Clontech). In all the cases, the correctness of the DNA sequence was verified by sequencing.

HTLA-230 (kindly donated by Dr E. Bogenman, Los Angeles, USA) and SH-SY5Y (purchased from Banca Biologica and Cell Factory, Genoa, Italy) NB cell lines were cultured in RPMI 1640 medium supplemented with 10% heat inactivated FCS, 50 mg/ml streptomycin, 50 mg/ml penicillin and 2 mM glutamine (Sigma-Aldrich, Milan, Italy). The HTLA-230 cells present MYCN amplification and the SH-SY5Y cells have normal MYCN status. Both cell lines were checked for morphology, proliferation rate, mycoplasma contamination and MYCN amplification after thawing and within four passages in culture.
To evaluate the effect of miR-659-3p over-expression and suppression on gene expression, 1×10⁶ HTLA-230 and SH-SY5Y cells were transfected with specific miR-659-3p mirVana™ mimic and inhibitor, respectively (Ambion, Life Technologies, Carlsbad, CA, USA, catalog# MC and MH 11582). Samples transfected with mirVana™ miRNA Mimic Negative Control #1 and mirVana™ miRNA Inhibitor Negative Control #1 were used as reference conditions. Transfection was performed at 20 nM miRNA concentration in OptiMEM^© medium (Sigma-Aldrich), using Lipofectamin RNAiMAX^© (Life Technologies), according to manufacturer's protocol. Cells were then cultured for 48 hours, checked for viability and processed for miRNA and total mRNA extraction, as described below. Transfection experiments were performed twice.

Human neuroblastoma cell line SH-SY5Y (ATCC, Manassas, VA, USA) was maintained in DMEM (Invitrogen, Grand Island, NY, USA) containing 10% heat-inactivated fetal bovine serum (FBS; Invitrogen) and 1% penicillin-streptomycin at 37°C in a humid incubator with 5% CO₂. To produce an experimental PD model in vitro, SH-SY5Y cells were exposed to 0.25, 0.5, 1, and 2 mM MPP⁺ (Sigma, St. Louis, MO, USA) for 24 h or treated with 1 mM MPP⁺ for 6 h, 12 h, 24 h, and 48 h.

The murine microglia BV2 cells were a generous gift from Dr. Alba Minelli (University of Perugia, Perugia, Italy). The SH-SY5Y human neuroblastoma cells were purchased from American Type Culture Collection (ATCC CRL-2266; VA, USA). The BV2 and SH-SY5Y cells were cultured in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (HyClone, UT, USA), 2 mM l-glutamine, 50 U/mL penicillin, and 50 μg/mL streptomycin (Sigma-Aldrich). The cells were maintained at 37 °C in a humidified atmosphere of 95% air and 5% CO₂ and grown to 80% confluence. These cells were subcultured two or three times per week using 0.25% trypsin (Sigma-Aldrich).