Ham s f 12k nutrient medium

Mouse motor neuron-like hybrid NSC-34 cell line [51 (link)] was stably transfected with the pTet-ON plasmid (Clontech, Palo Alto, CA, USA), coding for the reverse tetracycline-controlled transactivator, used to construct inducible cell lines expressing the cDNAs encoding human wild-type-SOD1 (WT) or human SOD1 mutant G93A (G93A). Cells were transfected with human wild type SOD1 (WT) or mSOD1 G93A expressing vector, as previously described [39 (link)]. Cells were grown in a mixture of 1:1 Dulbecco’s modified Eagle’s medium (DMEM) and Ham’s F-12K Nutrient Medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 15 mM HEPES (Sigma-Aldrich, St. Louis, MO, USA), 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA), 100 U/mL penicillin and 100-μg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA), and incubated at 37 °C in 5% CO₂. The expression of hSOD1 was induced by adding 2 µg/mL doxycycline (doxy; Sigma-Aldrich, St. Louis, MO, USA) to the culture medium for the last 24 h of culture.

Mouse MN-like hybrid NSC-34 cell line (provided by Dr. Cinzia Volontè from the National Research Council, Institute for Systems Analysis and Computer Science “Antonio Ruberti”, Cellular Neurobiology Unit, IRCCS Fondazione Santa Lucia, Rome, Italy) [29 (link)] was stably transfected with the pTet-ON plasmid (Clontech, Palo Alto, CA, USA), carrying the reverse tetracycline controlled transactivator used to induce the expression of human wild-type (WT) or mutant G93A SOD1 (hSOD1-G93A) cDNAs, as previously described [30 (link),31 (link)]. Cells were grown in a medium composed by 1:1 DMEM and Ham’s F-12K Nutrient Medium (Sigma-Aldrich, Munich, Germany), 15 mM HEPES (Sigma-Aldrich, Munich, Germany), 10% of heat-inactivated FBS (Invitrogen, New York, NY, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin (Sigma-Aldrich, Munich, Germany). Cell cultures were incubated at 37 °C in 5% CO₂. The addition of 2 µg/mL doxycycline (Sigma-Aldrich, Munich, Germany) for the last 24 h of culture was used to induce the expression of human WT or mutant (G93A) SOD1.