Glutamaxtm 1

The human prostate cancer cell lines PC-3, DU145, 22Rv1, VCaP, and LNCaP were obtained from ATCC (Manassas, VA, USA). In literature PC-3 cells are described to be docetaxel-resistant [29 (link)]. All cell lines except LNCaP cells are androgen-independent and abiraterone/enzalutamide-resistant due to the absence of AR (PC-3 and DU145) or the presence of AR-V7 (22Rv1 and VCaP). Cells were incubated at 37°C in a humidified atmosphere with 5% (v/v) CO₂. Cells were continuously kept in culture for a maximum of 3 months, and were routinely inspected microscopically for stable phenotype and regularly checked for contamination with mycoplasma. All cell lines were recently authenticated by a commercial service (Multiplexion, Heidelberg, Germany) using single nucleotide polymorphism (SNP)-profiling method.
PC-3, DU145 and 22Rv1 cells were cultured in 10% FBS/RPMI medium (RPMI medium supplemented with Glutamax^TM-I (Invitrogen, Paisley, UK) containing 10% fetal bovine serum (FBS, Invitrogen) and 1% penicillin/streptomycin (Invitrogen)). LNCaP cells were cultured in 10% FBS/RPMI medium (RPMI medium supplemented with Glutamax^TM-I containing 10% FBS, 1% penicillin/streptomycin, and 1 mM sodium pyruvate (Invitrogen)). VCaP cells were cultured in 10% FBS/DMEM medium (DMEM medium supplemented with Glutamax^TM-I (Invitrogen) containing 10% FBS and 1% penicillin/streptomycin (Invitrogen)).

Media and reagents for culture cells: L-15 Medium (1×) + GlutaMAX^TM-I, trypsin-EDTA, RPMI Medium 1640 (1×) + GlutaMAX^TM-I, DPBS, penicillin, streptomycin and fetal bovine serum Gold (FBS Gold) were provided by Gibco (San Diego, CA, USA). Kits for apoptosis detection: FITC Annexin V apoptosis detection Kit I was obtained from BD Pharmingen^TM (San Diego, CA, USA); FAM-FLICA Caspase-8 and FAM-FLICA Caspase-9 Assay were provided by ImmunoChemistry Technologies (Davis, CA, USA). Chemical treatment of cells: reduced graphene oxide (rGO) was obtained from Sigma-Aldrich (St. Louis, MO, USA), while the proteasome inhibitor (MG-132) was provided by Selleckchem (Cologne, Germany). Kit for cytotoxicity analysis: the LDH-Cytotoxicity Assay kit was provided by BioVision (St. Louis, CA, USA). The compounds for oxidative stress analysis: SDS, TCA, TBA, Folin–Ciocalteu reagent and 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) were provided by Sigma-Aldrich (St. Louis, MO, USA), and GSH/GSSG-Glo^TM Assay kit by Promega (Madison, WI, USA). The compounds for ultrastructure analysis: glutaraldehyde, paraformaldehyde, cacodylate buffer, osmium tetroxide, ethanol and glycid ether were provided by Sigma-Aldrich (St. Louis, MO, USA).

In the next stage, PA5 hOMCs were co-cultured with primary human DRG neurons (Fig. 5A). DRG neurons were expanded on T-75 flasks (Thermo-Scientific) freshly coated with poly-L-lysine (PLL, Sigma, 100 µg/mL) for two passages in DMEM/F12 (Gibco, Life Technologies) + GlutaMAX^TM-I (Gibco, Life Technologies) + 10% FBS (Sigma) medium. To start the co-cultures, PA5 and F7 cells were cultured, and re-plated at 6,000 cells/cm² onto 24-well plates (Thermo-Scientific) freshly coated with PLL (Sigma, 100 µg/mL). 4-OHT, which activates the c-MycER^TAM conditional immortalisation system, was removed from PA5 hOMC media during cell seeding. After 24 hours, DRG neurons were detached and plated at a seeding density of 500 cells/well onto 24-well plates with monolayers of PA5 or F7 cells, and onto negative control wells with DRG neurons only. Cells were fed with DMEM/F12 (Gibco, Life Technologies) + GlutaMAX^TM-I (Gibco, Life Technologies) + 10% FBS (Sigma) every 48 hours, and fixed after 3 and 5 days of co-culture. Immunocytochemistry was carried out as described previously. A total of 15 frames were taken per condition (5 per technical triplicate) at 100 total magnification in the EVOS^® FL Imaging system (Thermo-Scientific). Neurite quantification was performed manually in ImageJ using the NeuronJ plugin^{51 (link)}. Data were recorded in Microsoft Excel files and analysed by GraphPad Prism^® 7.