Lncap

Human tumor cell lines were cultured in DMEM/10% FCS under a humidified 5% CO₂ atmosphere at 37 °C. The following cell lines were used: A-172, A549, A64-CLS, Hela, HL-60, LNCaP, MCF-7, MG-63, PC-3, SaOs, and SW-480 from Cell Lines Service (Eppelheim, Germany); B-CPAP, BHY, CAL-62, COLO320HSR, H1184, H146, HT29, LoVo, SKN-MC, SW-403, and THP-1 from DSMZ (Braunschweig, Germany); FTC-133, U251-MG, and U373-MG from Sigma-Aldrich (Taufkirchen, Germany); and SW948 and U87-MG from American Type Culture Collection (Manassas, VA, USA), as previously described [18 (link)].

The prostate cancer cell line LNCaP was obtained from Cell-Lines Service in Germany and was cultivated at 37 °C in a 5% CO₂ environment in media suggested by the provider, the cell line was not tested for mycoplasma and not authenticated. Cells were harvested during log-phase growth at 70–90% confluency. Fresh frozen tissues were embedded in OCT (4583, Tissue-Tek) and sections were collected during cryo-sectioning at −20 °C (CryoStar NX50 Cryostat), mixed with β-mercaptoethanol (444203, Calbiochem) in RLT lysis buffer (79216, Qiagen) at a 1:100 ratio, placed in Lysing Matrix D (116913050, MP Biomedicals), then lysed using FastPrep (MP Biomedicals, USA). For the post mortem brain specimen, GM and WM regions were separated using shallow indentations by a scalpel between regions prior to sectioning. Total RNA was extracted using RNeasy Plus Mini Kit (74134, Qiagen) according to manufacturer’s instructions. Quality of the total RNA was determined using RNA 6000 Nano or Pico chip on the 2100 Bioanalyzer automated electrophoresis system (Agilent Technologies Inc.), by calculating RIN. Concentration of the total RNA was determined using the Qubit RNA BR assay (Q10210, Life Technologies).

Human epithelial PC cell lines LNCaP and PC-3 (Cell Lines Service, Eppelheim, Germany) were propagated in RPMI 1640 medium supplemented with 10% fetal calf serum and 100 units/ml penicillin/streptomycin (PAN Biotech) at 37°C and 5% CO₂ in a humidified atmosphere. For cell transfer onto a cell culture plate adherent cells were detached by 0.1% trypsin/0.04% ethylenediaminetetraacetic acid (EDTA) and resuspended in RPMI medium. For further experiments RPMI medium was supplemented with 5 mM N-acetylcysteine (NAC; Carl Roth, Karlsruhe, Germany), 100 μM vitamin C (Carl Roth) or 5 μg/ml cycloheximide (Merck, Darmstadt, Germany), respectively.