Gm01651

NIH3T3 mouse fibroblasts (CRL-1658), CaCo2 cells human colorectal adenocarcinoma (HTB-3), and the N2a neuroblastoma cell line (CCL-131) were obtained from the American Type Culture Collection and were validated by genomic PCR. Primary human fibroblasts (GM01651) were from Coriell Repository. Cells were cultured to full confluence in a 37 °C incubator with 5% CO2 in complete Dulbecco’s modified Eagle’s medium (DMEM, Sigma #D5030) supplemented with 10% heat-inactivated newborn calf serum (HyClone #SH30401.02HI), 1% penicillin/streptomycin/fungizone (Invitrogen, #10072322). All the cell lines were tested for mycoplasma contamination using DNA staining protocol with Hoechst 33258 dye and none of them was found positive. Knock-down of Ncor1 was done using lentiviral mediated shRNA (SHCLNG-NM_011308) from the Sigma Mission library following standard procedures. The efficiency of knock-down was tested by immunoblot. Cell viability was measured using the CellTiter-Blue kit (Promega, #G9241) 24 h after the addition of the different stressors according to the manufacturer’s instructions. All cell lines were validated by genomic PCR.

Primary human skin fibroblasts of control fibroblasts (HF6, HF19) and of DYT1 patient's fibroblast (HF48) were generated in Dr. Breakefield ‘s laboratory as described previously [42] (link), under IRB approved guidelines. Fibroblast control cell lines GM00023, GM00024, GM01651, GM02912, and DYT1 patient fibroblast cell lines GM02551, GM03208, GM032011, GM02306 were purchased from Coriell Institute for Medical Research (Camden, NJ). The Coriell Cell Repository maintains the consent and privacy of the donor fibroblast samples. DYT1 patient fibroblast cells, FFF13111983 [43] (link), were provided from Dr. Mirella Filocamo (Biobank from Patients affected by Genetic Diseases, L'Istituto Giannina Gaslini, Genoa, Italy), a member of the Telethon Network of Genetic Biobanks (project no. GTB12001). The primary fibroblast cells were grown in culture, as described [24] (link). For more information about the human fibroblast cell lines, see Table S1. Human fibroblast cells were seeded, and incubated overnight before the cAMP assay.

Seven primary human skin fibroblast lines—GM09503 (passage frozen (P), P3), GM01652 (P11), GM01651 (P14), GM00288 (P9), GM01681 (P12), GM01680 (P12), and GM03525 (P7) —were purchased from the Coriell Institute (Camden, NJ, USA). The fibroblasts were cultured in MEM (Gibco) supplemented with 15% (v/v) heat inactivated FBS (Gibco), 1% (v/v) MEM non-essential amino acids (Gibco), and 1% (v/v) Pen-Strep (Gibco) in the incubator (37 °C, 5% CO₂). The cells within 7 passages from the initial P were used for RT-qPCR assays.