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1:6000); Glial fibrillary acidic protein (GFAP, polyclonal rabbit, DAKO, 1:2000); SRY (sex determining region Y)-box 2 (SOX2, polyclonal rabbit, Millipore, Darmstadt, Germany, 1:1000); Nestin (monoclonal mouse, R&D, Minneapolis, MN, USA, 1:500) and the follow- ing secondary antibodies or counterstaining: Alexa Fluor® 488 donkey-anti-mouse anti- body (Invitrogen, Eugene, OR, USA, 1:200); Alexa Fluor® 568 goat-anti-rabbit antibody (Invitrogen, 1:200); Alexa Fluor® 594 Phalloidin (Life Technologies, 1:200, Eugene, OR, USA). Coverslips were mounted with Vectashield with DAPI (H-1200, Vector Laboratories Inc., Burlingame, CA, USA). Fluorescent microscopy was performed using Leica Confocal Microscope TCS SP-8X (Leica, Son, the Netherlands).
Proliferation assay
Proliferation of cell cultures was determined 72 hours after start of transfection by cell cycle flow cytometric analysis and Ki67 staining. Ki67 positive cells and the total number of DAPI-stained nuclei were manually counted with ImageJ (1.44p, National Institutes of Health, Bethesda, MD, USA). For each culture, conditions were plated in triplicate and fifteen fields per coverslip, with on average 1100-1300 cells per condition, were counted using a 20x magnification objective. Staining was evaluated with a Leica DM5000B flu- orescence microscope equipped with a Leica DFC 500 camera and Leica Application Suite X software (Leica Microsystems CMS GmbH, version 1.5.1, Wetzlar, Germany). The number of Ki67 positive cells was compared with the number of DAPI nuclei to deter- mine the percentage of proliferating cells.
For flow cytometric cell cycle analysis, cells were suspended in PBS/1% BSA and stained with Fixable Viability Dye eFluor® 780 (eBioscience, San Diego, CA, USA) on ice for 30 minutes. After fixation with 100% ethanol, cells were incubated with Propidium Iodide (1:100, Life Technologies) and RNAse A (1 g/ml used 1:1000, Sigma-Aldrich) in PBS for 10 minutes at 37˚C. Flow cytometric analysis of stained cells was performed using a FACSCanto Flow Cytometer equipped with FACSDiva software (BD Biosciences, San Jose, CA, USA) and data analysis was performed using FlowJo 7.6 (FlowJo LLC, Ashland, OR, USA). Viable cells showing a DNA content between G1 and G2 (S-phase) were selected as proliferative population.
Human material
The cases included in this study were obtained from the archives of the departments of neuropathology of the AMC, the UMCU, VU Medical Center Amsterdam (The Netherlands), Motol University Hospital (Prague, Czech Republic) and Medical University Vienna (Austria). We evaluated 22 TSC and 16 TLE-HS patients from whom we obtained anatomically well preserved epileptogenic brain tissue and sufficient clinical data (TSC: 19 surgical specimens and 3 autopsy specimens; mean age at resection = 16.7 years; stan- dard deviation (SD)= ± 14.46 years; range = 0.83-47 years; localization: 15 frontal, 6 tem- poral, 1 parietal; 12 males, 10 females; TSC1/TSC2 mutation status 5/17; mean duration of epilepsy: 13.7 ± 12.7 years; seizures >5/day. TLE-HS: 16 surgical specimens, mean age at resection = 39 years ± 12 years; range = 24-66 years; localization: hippocampal; 9 males, 7 females; mean duration of epilepsy: 20 ± 12 years; seizures: 13/month). The age- and localization-matched control group consisted of 31 autopsy cases of which 17 cortex (male/female: 7/10; years/range: 0.2-48; frontal:/temporal/parietal: 9/7/1) and 14 hippocam-