Pi3k inhibitor ly294002

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.

Human colonic myofibroblast cells CCD18COCo (ATCC CRL1459), colon adenocarcinoma cells HT-29 (ATCC HTB-38), lung cancer cells A549 (ATCC CCL185) and breast cancer cells MCF7 (ATCC HTB22) were obtained from ATCC (LGC Standards, Middlesex, UK) and bladder cancer cells EJ138 were obtained from ECACC (Public Health England, Salisbury, UK). All cells were used within 6 months of resuscitation and were authenticated by the ATCC or ECACC by using standard protocols prior to purchase. Cells were cultured in minimum essential medium from Sigma-Aldrich (Dorset, UK) supplemented with 10 % fetal bovine serum (Labtech International, East Sussex, UK). Human colonic myofibroblast cells were used up to passage 10. The human recombinant HMGB1, anti-HMGB1 primary antibody, anti-RAGE primary antibody and anti-TLR4 primary antibody were purchased from R&D Systems (Oxford, UK). MEK1/2 inhibitor (U0126) and PI3K inhibitor (LY294002) were purchased from Cell Signaling Technology (Massachusetts, USA). Protease and phosphatase cocktail set inhibitors were purchased from Calbiochem (Nottingham, UK). All other reagents were obtained from Fisher Scientific (Leicestershire, UK), BioRad Laboratories (Hertfordshire, UK) or Sigma-Aldrich.

A 3D collagen gel culture environment was established by embedding cells in 1.5 mg/ml collagen type 1 matrix (First Link Ltd., Birmingham, UK), as previously described^{20 (link),21 (link)}, using 0.74 × 10⁵ cells/ml orbital fibroblasts alone or with macrophages at 1:2 (1.5 × 10⁵ cells/ml) or 1:5 (3.7 × 10⁵ cells/ml) ratios. For gel contraction assays and immunofluorescence experiments, 150 μl gels were cast in the microwells of glass bottom MatTek dishes (MatTek Corporation, MA, USA), and then detached as free-floating gels^{20 (link)}. For all other experiments, gels were cast in 24-well plates with 210 μl gel solution and left attached. Where appropriate, inhibitors (IGF-1R blocking antibody 1H7, 5 μg/ml, BioLegend, CA, USA; TGF-β inhibitor SB431542, 10 μM, Cayman Chemical, MI, USA; PI3K inhibitor LY294002, 10 μM, Cell Signaling Technology, MA, USA) or control diluent at matching concentration were added to the culture medium immediately after gel polymerization and maintained throughout the experiment (IGF-1R blocking antibody 1H7, 10 μg/ml was also added to the gel mix).