Dimethyl sulfoxide (dmso)

For brain metastasis treatment, we started therapy on day 14 once the mice had recovered from surgery and after checking the success of cell inoculation.
Lenalidomide (LND), a thalidomide derivative, was obtained from the Celgene Corporation (Summit, NJ) and from Sellek Chemicals LLC (Houston, TX). LND was injected intraperitoneally in DMSO (Sigma-Aldrich) at 50 mg/Kg/day, every day until the end of the experiment. Docetaxel (TXT) and NVP-AUY922 (NVP), both from LC Laboratories, were injected intraperitoneally in DMSO at a dosage of 15 mg/Kg/day and 30 mg/Kg/day respectively. TXT was administered every 4 days for 2 weeks and NVP every 2 days for 2 weeks.

Dinaciclib (CAS 779353-01-4) was purchased from Selleckchem and diluted in DMSO (Sigma-Aldrich) to a 100 μM concentration. Used dilutions were made in complete medium. Pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (z-VAD-fmk, CAS 187389-52-2) was purchased from ApexBio and diluted in DMSO to form a 50 mM stock solution. Used dilutions (40 μM) were made in complete medium. Generic BH3-mimetic ABT-737 (CAS 852808-04-9) was obtained from ApexBio and diluted in DMSO to an initial 10 mM stock. The Bcl-x_L specific inhibitor A-1331852 (CAS 1430844-80-6) was purchased from ChemieTek and a 3 mM initial stock solution in DMSO was employed. The nucleoside analog Gemcitabine (CAS 122111-03-9) was obtained as hydrochloride salt from LC Laboratories and diluted in DMSO to form a 1 mM stock. In every case, further dilutions were made taking into account not to surpass 0.1% (v/v) content of DMSO in media.

Schwann cell (SC)-dorsal root ganglia (DRG) co-cultures were prepared from either E15.5 rat or E13.5 mouse embryos according to standard procedure (Taveggia and Bolino, 2018 (link)). After DRGs were digested with 0.25% Trypsin (Invitrogen) at 37 °C for 45 min, the reaction was stopped by adding deactivated fetal calf serum (FCS; HyClone) and basic medium (1% Penicillin/ Streptomycin (Lonza), 10% FCS, 50 ng/ml nerve growth factor (NGF; Alomone Labs) in minimum essential medium (MEM; Gibco)) and cells were plated on collagen-coated coverslips. On day 7, myelination was induced by culturing the cells in basic medium with 50 ng/ml ascorbic acid (AA; Sigma). For PTEN inhibition in Pmp22^tg cultures, cells were treated with 1% DMSO (Sigma) as a control or with the PTEN inhibitor VO-OHpic (Rosivatz et al, 2006 (link)) (Sigma) at 50 nM, 500 nM and 5 μM, respectively. In Pmp22^+/- cultures, cells were treated with either 1% DMSO as control, 20 nM mTOR inhibitor Rapamycin (LC Laboratories) or 10 µM PI3K inhibitor LY294002 (Cell Signaling, #9901). Medium was changed every 2–3 days for 2 weeks. HEK293T cells (Sigma) were maintained in DMEM and regularly tested for mycoplasma contamination. Cells were transiently transfected with ALFA-tagged (Gotzke et al, 2019 (link)) PMP22 and Flag-tagged or untagged PTEN expression plasmids (Vectorbuilder) via polyethylenimine.