Human schwann cells

A human Schwann-like cell line (ATCC crl-2885) and human Schwann cells (ScienCell) were maintained as described in the distributor's manual. Human Schwann-like cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) with high glucose (WelGene) supplemented with 10% fetal calf serum (WelGene) and 1× penicillin/streptomycin (WelGene). human Schwann cells were maintained in the provided Schwann Cell medium (ScienCell) and cultured on 1× poly-_D-Lysine coated culture dishes. For differentiation, cells were cultured with DMEM with low glucose (WelGene) supplemented with 1% fetal calf serum (WelGene) and myelination signals, consisting of 100 ng/ml Nrg1 (Peprotech) and 100 μM dbcAMP (Sigma-Aldrich), for 7 days. For transfection of CRISPR/Cas9 components, ribonucleoprotein (RNP) complexes were formed by incubating 4 μg of Cas9-HA protein (ToolGen) with 1 μg of sgRNA at room temperature for 15 min. Then, 2 × 10⁵ cells were electroporated with the RNP complexes using a Neon electroporator (ThermoFisher) with 10 uL electroporation tips. For targeted deep sequencing, genomic DNA (gDNA) was collected from cells 72 h after transfection.

Human neuroblastoma CHLA-20 was a gift from Thomas Inge (Cincinnati Children’s Hospital Medical Center); the origin and culture conditions were previously described [26 (link)]. Athymic nude mice (nu/nu, NIH) (15 mice per group), were injected with 7.5 × 10⁶ cells subcutaneously to initiate tumor growth. When tumors reached a volume of 400 mm³, five doses of either SapC-DOPS (SapC 4 mg/kg body weight, DOPS 2 mg/kg body weight) or PBS (control) were intratumorally administered once every 3 days. Tumor growth was assessed periodically with a caliper, and after 16 days, tumors were excised, weighted, and processed for hematoxylin and eosin staining and apoptosis (TUNEL) assays.
The human neuroblastoma SK-N-SH and IMR-32 cell lines were obtained from American Type Culture Collection and grown in AMEM supplemented with 10% FBS. Human Schwann cells (ScienCell Research Laboratories, Carlsbad, CA) were grown as recommended by the supplier. After overnight attachment, cells were treated with either DOPS or SapC-DOPS for concentration- or time-dependence assays. Where indicated, cells were pretreated for 60 min with bongkrekic acid, cyclosporine A or N-acetyl cysteine.

Human cancer cell lines MDA-MB-231, H1299, Gli36 and U373 were obtained from ATCC (Manassas, VA, USA). MDA-MB-231-luc-D3H2LN cells were obtained from Caliper Life Sciences, Mountain View, CA. U87ΔEGFR-Luc cells were obtained from Dr. Balveen Kaur, Ohio State University, Columbus, Ohio. Human Schwann cells were obtained from ScienCell (Carlsbad, CA, USA). U87ΔEGFR-Luc, Gli36 and U373 were cultured in DMEM medium (Fisher Scientific). H1299 was cultured in RPMI medium (Fisher Scientific). MDA-MB-231 non metastatic and MDA-MB-231- Luc-D3H2LN metastatic cell lines were cultured in AMEM medium (Invitrogen). The above cell lines were cultured in their respective media supplemented with 10% FBS and 1% Penicillin / Streptomycin. Normal Human Schwann cells were cultured in Schwann cell medium (ScienCell, Carlsbad, CA, USA) supplemented with the provided growth factor supplement, FBS and antibiotics. All cells were cultured in a 5% CO₂ incubator at 37°C. Cells were routinely tested for mycoplasma contamination. No cross-contamination was observed in the used cell lines, as evidenced by the cellular morphology and growth parameters. No authentication of the cell lines was done by the authors.