Recombinant human β ngf

Parental PC12 cells were provided by RIKEN BRC (Tsukuba, Japan). Recombinant human BMP4 (PeproTech, Rocky Hill, NJ, USA) and recombinant human β-NGF (PeproTech) were dissolved in LF6 buffer solution (5 mM Glutamic acid, 5 mM NaCl, 2.5% glycine, 0.5% sucrose, 0.01% Tween 80, pH 4.5). LDN-193189, a selective inhibitor of BMP type I receptors [17 (link),27 (link)], was obtained from Cayman Chemical (Ann Arbor, MI, USA) and dissolved in dimethyl sulfoxide (Fujifilm Wako Pure Chemical, Tokyo, Japan). Penicillin–streptomycin solution was from Sigma-Aldrich (St. Louis, MO, USA). Glutamic acid, NaCl, glycine and sucrose were from Fujifilm Wako Pure Chemical. Tween 80 was obtained from MP biomedicals (Solon, OH, USA).

DRG excised from adult BALB/c mice (5–10-week-old) were collected in a dish containing cold F12 (Nutrient Mixture F12 Ham) medium (Sigma–Aldrich Inc., Italy). After accurate de-sheathing, DRG were transferred into a sterile 35 mm dish containing collagenase from Clostridium hystoliticum 0.125% (Sigma–Aldrich Inc., Italy) and DNase (Sigma–Aldrich Inc., Italy) in F12 (Nutrient Mixture F12 Ham) medium and incubated at 37 °C. After 1 h incubation, DRG were triturated using a 1000 µL tip. Myelin and nerve debris were eliminated by centrifugation through bovine serum albumin (BSA) cushion. Cell pellets were re-suspended in Bottenstein and Sato medium (BS): 30% F12 (Nutrient Mixture F12 Ham medium), 40% DMEM (Dulbecco’s Modified Eagle’s medium, Sigma–Aldrich Inc., Italy), 30% Neurobasal A medium (Life Technologies, Italy), 100X N2 supplement (Life Technologies, Italy), penicillin 10 U/mL and streptomycin 100 mg/mL (Sigma–Aldrich Inc., Italy), supplemented with Recombinant Human β-NGF, Recombinant Murine GDNF and Recombinant Human NT3 (Peprotech, Rocky Hill, NJ, USA) and plated onto 24 mm glass coverslips pre-coated with laminin (Sigma–Aldrich Inc., Italy). The administration of OHP and amiloride was done 48 h after the isolation of DRG neurons, and all the experiments were performed from 54 to 96 h of culture.

DRG from adult BALB/c mice were excised and cultured as previously described^{7 (link)}. Briefly, DRG from cervical to sacral (up to S2) level were bilaterally excised, collected and accurately de-sheathing in a dish containing cold F12 (Nutrient Mixture F12 Ham) medium (Sigma–Aldrich Inc., Italy). After incubation at 37 °C (1 h) with collagenase from Clostridium hystoliticum 0.125% (Sigma–Aldrich Inc., Italy), DRG were dissociated. Cells were plated on laminin (Sigma–Aldrich Inc., Italy) coated glass coverslips (24 mm) and cultured at 37 °C with 5% CO₂ for 48 h in Bottenstein and Sato medium (BS)^{7 (link)} supplemented with Recombinant Human β-NGF, Recombinant Murine GDNF and Recombinant Human NT3 (Peprotech, Rocky Hill, NJ, USA). The administration of OHP (0.1 μg/ml) and 5-FU (500 nM) was performed 48 h after the isolation of DRG neurons and all the experiments were performed from 48 to 54 h of culture.

Cell culture reagents were purchased from Life Technologies (Carlsbad, CA, USA). RNA extraction and Ultra ECL chemiluminescence system reagents were purchased from BioTeke (Beijing, China). PrimeScript™RT reagent Kit with gDNA Eraser was purchased from Takara Biotechnology (Takara Bio Inc, Shiga, Japan). SsoFast^™EvaGreen^® Supermix was purchased from Bio-Rad Laboratories (California, USA). Recombinant Human β-NGF was purchased from Peprotech (Rocky Hill, NJ, USA). Anti-β-NGF, anti-TrkA, anti-P75 and anti-GAPDH antibodies were purchased from Epitomics (Burlingame, CA, USA). Ro 08-2750 was purchased from R&D Systems (Minneapolis, MN, USA). K252a was purchased from Biovision (Milpitas, CA, USA). LM11A-31 was purchased from Sigma-Aldrich (Saint Louis, MO, USA). Anti-β-catenin and anti-C-myc were purchased from Arigo Biolaboratories (Taiwan, China). Anti-CD44, anti-MMP2, anti-MMP7 and anti-TIMP2 were purchased from OriGene Technologies, Inc (Rockville MD, USA). p-CTNNB1-S552 and p-TrkA-Y490 antibodies were purchased from ABclonal Technology (Woburn, MA, USA). PVDF membranes were purchased from Millipore (Bedford, MA, USA).

DRG obtained from adult BALB/c mice (5/10-wk-old) were excised and collected in a dish containing cold F12 (Nutrient Mixture F12 Ham) medium (Sigma Aldrich Inc.). Working under a dissecting microscope and using fine forceps, the surrounding membranes were gently teased away from each DRG; nerves and sheath were cut. All de-sheathed DRG were then transferred into a sterile 35 mm dish containing collagenase from Clostridium hystoliticum 0.125% (Sigma Aldrich Inc.) and DNase (Sigma) in F12 (Nutrient Mixture F12 Ham) medium and incubated at 37 °C for 1 h. After incubation, DRG were triturated using a 1000 µl tip. Myelin and nerve debris were eliminated by centrifugation through a bovine serum albumin (BSA) cushion. Cell pellets were re-suspended in Bottenstein and Sato medium (BS) (30% F12 (Nutrient Mixture F12 Ham medium), 40% DMEM (Dulbecco’s Modified Eagle’s medium (Sigma Aldrich Inc., Italy), 30% Neurobasal A medium (Life Technologies, Italy), 100 X N2 supplement (Life Technologies, Italy), penicillin 10 U/mL and streptomycin 100 mg/mL (Sigma Aldrich Inc., Italy), supplemented with Recombinant Human β-NGF, Recombinant Murine GDNF and Recombinant Human NT3 (Peprotech, USA) and plated onto 24 mm glass coverslips pre-coated with laminin (Sigma Aldrich Inc., Italy).