Human dermal fibroblasts were originally cultured from skin biopsies acquired under an IRB-approved protocol, as previously described [54] (link). Cells were grown at 37°C, 5% CO2 in a 50∶50 mix of RPMI-1640, supplemented with 1× Amphotericin B (JR Scientific, Woodland, CA), 1× Penicillin-streptomycin-Glutamine (Life Technologies, Carlsbad, CA), 1× MEM Non-Essential Amino Acids Solution (Life Technologies), and 10% Fetal Bovine Serum (JR Scientific) and AmnioMAX C100 media (Life Technologies). Fibroblasts were harvested at 80% confluency to avoid decreased transcription associated with contact inhibition.
SK-N-MC-rtTA cell lines harboring expanded CGG-repeat episomes were created, as previously described [36] (link). These cells were grown in DMEM (Life Technologies) +10% Tet-system-approved fetal bovine serum (Clontech, Mountain View, CA) and 1× Penicillin-streptomycin (Life Technologies) at 37°C, 5% CO2. DOX media was prepared from 1 mg/mL stock doxycycline hyclate (Sigma-Aldrich, St. Louis, MO) dissolved in sterile water. For the DOX washout, DOX media was aspirated and cells were washed once with DPBS (Life Technologies) before adding DOX-free media.
SK-N-MC-rtTA cell lines harboring expanded CGG-repeat episomes were created, as previously described [36] (link). These cells were grown in DMEM (Life Technologies) +10% Tet-system-approved fetal bovine serum (Clontech, Mountain View, CA) and 1× Penicillin-streptomycin (Life Technologies) at 37°C, 5% CO2. DOX media was prepared from 1 mg/mL stock doxycycline hyclate (Sigma-Aldrich, St. Louis, MO) dissolved in sterile water. For the DOX washout, DOX media was aspirated and cells were washed once with DPBS (Life Technologies) before adding DOX-free media.