Tetracycline free fbs

The inducible MEF line used in this study was generated as described previously51 (link). Briefly, the gene encoding oncogenic-Dbl was sub-cloned into vector pTRE-HA (Clontech). Parental MEFs containing the transcriptional transactivator tTA (Clontech) were then co-transfected with the resulting pTRE-HA-onco-Dbl vector along with vector pMET-puro, in a 20:1 ratio. Following puromycin selection (∼3 weeks), colonies were screened for inducible expression of oncogenic-Dbl. Cells were maintained at 37 °C, 5% CO₂ atmosphere, in DMEM medium containing 4 mM glutamine (Gibco) and supplemented with 10% (v/v) tetracycline-free FBS (Gibco) and 0.6 μg ml⁻¹ doxycycline. Expression of oncogenic-Dbl was induced by re-plating cells in doxycycline-free medium (10% FBS). Residual doxycycline was removed by replacing the medium (with the appropriate concentration of FBS, and small-molecule inhibitor where relevant) after 5 h. Uninduced control samples were treated in the same way: re-plating followed by media change at 5 h, but with 0.6 μg ml⁻¹ doxycycline present at all times. For glutamine-withdrawal experiments, glutamine-free DMEM medium (Gibco) was supplemented with dialysed FBS (Gibco). Growth medium containing other concentrations of glutamine was prepared by mixing appropriate volumes of DMEM (4 mM glutamine) and glutamine-free DMEM.

Human hTERT‐RPE‐1 (human retinal epithelial; RRID: CVCL_4388; RPE‐1) cells were grown in Dulbecco's modified Eagle's medium/nutrient mixture F‐12 Ham (DMEM‐F12; Sigma) supplemented with 10% Fetal Bovine Serum (FBS; Gibco) and 100 U/ml Penicillin/Streptomycin (P/S; Gibco) and maintained at 37°C with 5% CO₂ atmosphere. Tetracycline‐free FBS (Gibco) was used to grow cells expressing the PLK4 tet‐inducible construct. The FBS was heat inactivated at 56°C water bath for 30 min. RPE‐1 cells were routinely tested for mycoplasma.

The NSC34 motor neuronal-like cell lines were used in their neural-precursor form in continuation with our previous work. Cells stably transfected with pTet-ON plasmid (Clontech, Mountain View, CA, USA) harbouring sequences encoding for human SOD1 WT (NSC34-SOD1WT) or G93A mutant (NSC34-SOD1G93A) were a kind gift of prof. Maria Teresa Carrì (University of Tor Vergata, Rome, Italy) [18 (link)]. Cells were cultured in 5% CO₂ in DMEM/F12 (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% tetracycline-free FBS (GIBCO, Waltham, MA, USA), penicillin/streptomycin antibiotic and 200 µg/mL G418 (Carlo Erba, Milan, Italy) for selection maintenance. The maximal expression of SOD1 proteins was achieved by the addition of 2 µg/mL doxycycline (Sigma-Aldrich) to the medium after 48 h. The parental NSC34 cells (CELLutions Biosystem Inc., Duluth, GA, USA) were used as control and cultured according to the manufacturer’s instructions. NSC34-SOD1WT and NSC34-SOD1G93A cells were plated in 96-well plates (10⁴ cells/well) and kept in a controlled environment (37 °C and 5% CO₂). After 24 h from doxycycline induction, 1, 5, 10, or 50 μg/mL of unlabeled NHK1, previously dissolved in DMSO, were diluted in the culture medium and cells were incubated for additional 24 h. Cell viability was assessed by MTT assay [39 (link)]. Parental NSC34 were used as control.