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minutes with 2.5% trypsin (Sigma-Aldrich, St. Louis, MO, USA). Tissue was washed with incubation medium containing Dulbecco’s modified Eagle’s medium (DMEM)/HAM F10 (1:1) medium (Gibco, Life Technologies, Grand Island, NY, USA), supplemented with 50 units/mL penicillin, 50 μg/ml streptomycin and 10% fetal calf serum (FCS; Gibco, Life Technologies, Grand Island, New York, USA) and triturated by passing through a 70 μm mesh filter. Cell suspension was incubated at 37˚C, 5% CO2 for 48 h to let glial cells adhere the culture flask before it was washed with PBS to remove excess of myelin and cell debris. Cultures were subsequently refreshed twice a week. Cultures reached confluence after 2-3 weeks.
Primary SEGA cell cultures were derived from surgical brain specimens obtained from patients (age at surgery: 1 year; gender: 1 m/1 f) undergoing epilepsy surgery or surgery for tumor growth and/or related obstructive hydrocephalus at the Department of Pediatric Neurosurgery of the Anna Meyer Children’s Hospital (Florence, Italy). SEGA cultures were established in the same manner as described above for fetal cultures. One established SEGA cell line was generously provided by the Department of Pediatrics/ Institute of Neurology of the Medical University of Vienna (Vienna, Austria). The culture was prepared from tissue obtained at surgery from a TSC patient (age at surgery: 14 yrs; gender: m; mutation: TSC2). Further established SEGA cell lines, generously provided by the Laboratory of Molecular and Cellular Neurobiology of the International Institute of Molecular and Cell Biology (Warsaw, Poland; 42), were prepared from tissue obtained at surgery from two TSC patients (age at surgery 4 and 11 yrs; gender: m/f ; TSC2 mutation).
The astrocytoma cell line U373 was obtained from the American Type Culture Collection (Rockville, MD, USA); cells were cultured under the same conditions as pri- mary cell cultures.
Secondary astrocyte cultures for experimental manipulation were established by trypsinizing confluent cultures and sub-plating onto poly-L-lysine (PLL; 15 μg/mL, Sigma-Aldrich)-precoated 6 and 12-well plates (Costar; 1 × 105 cells/well in a 6-well plate for Western blot analysis or 5 ×104 cells/well in a 12-well plate for RNA isolation and PCR). In the present study astrocytes were used for analyses at passage 1-6.
Transfection and stimulation of cell cultures
Cells plated in PLL-coated plates were transfected with either mimic pre-miRNA (Applied Biosystems, Carlsbad, CA, USA; see supplementary Table 1) or antisense LNA oligonu- cleotides for miR146a, miR21 and miR155 (Ribotask ApS, Odense, Denmark). Fluorescent labeled scrambled mimic (FAMTM dye-labeled Pre-miRTM Negative Control #1, Applied Biosystems, Carlsbad, CA, USA) or antisense (FAMTM dye-labeled Anti-miRTM Negative Control #1) LNA oligonucleotides were used to control for non-specific effects of the oligonucleotides. Oligonucleotides were delivered to the cells using Lipofectamine® 2000 transfection reagent (Life Technologies, Grand Island, New York, USA) in a final concentration of 50 nM for a total of 24 h. Cell cultures were stimulated with human recombinant (r)IL-1β (Peprotech, NJ, USA; 10 ng/ml) for 24 hours. Viability of human cell cultures was not influenced by the treatment with IL-1β (not shown; 41). Cells were harvested after 24 hours of stimulation and/or transfection. Medium was collected for the detection of miRNA, IL-6 and HMGB1 release and cells were washed twice with PBS before harvesting the cell samples.



























































































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