Nih3t3

B6 fibroblasts were generated as previously described.^{83 (link)} Qa-1b expressing L cells were generated as previously described.^{28 (link)} NIH 3T3 (ATC#CRL-1658) and RAW 264.7 mouse macrophages (ATCC TIB-71) were purchased from ATCC. QFL hybridomas (BEko8Z cells) were established in the Shastri lab as described.^{28 (link)} B6 fibroblasts and splenocytes were cultured in complete RPMI (cRPMI) with 10% FBS (Invitrogen, Carlsbad CA), 100 U/ml Penicillin/Streptomycin (Invitrogen), 2mM L-Glutamine, and 50 μM 2-ME. NIH 3T3 and RAW 264.7 cells were cultured in complete DMEM with 10% FBS (Invitrogen, Carlsbad CA) and 100 U/ml Penicillin/Streptomycin (Invitrogen). All cell lines were maintained at 37°C, 5% CO₂, and 95% humidity.

The mouse embryonic fibroblast cell lines NIH3T3 and the human lung fibroblasts MRC-5 were purchased from UNC Tissue Culture Facility. The human bladder transitional cell line UMUC3 was provided by Dr. William Kim. The human pancreatic cancer BXPC3-Luc2 was purchased from PerkinElmer (Waltham, MA). UMUC3 and NIH3T3 were maintained in Dulbecco’s Modified Eagle’s Media (Invitrogen, CA), supplemented with 10% fetal bovine serum (FBS) (Sigma, MO) or 10% bovine calf serum (Sigma, MO). BXPC3-Luc2 cells were cultured in full RPMI-1640 medium (Invitrogen, CA), while MRC-5 were cultured in full αMEM (Invitrogen, CA). Cell lines were authenticated by Dr. William Kim’s group and UNC Tissue Culture Facility using the Short Tandem Repeat (STR) profiling method. Female nude mice 6–8 weeks-old were obtained from and raised by the University of North Carolina animal facility. All animal handling procedures were approved by the University of North Carolina at Chapel Hill’s Institutional Animal Care and Use Committee. Primary and secondary antibodies used for Western blot (WB), flow cytometry (flow cyt), immunofluorescence staining (IF), and immunohistochemistry (IHC) staining are listed in Table S1.

HEK293T and NIH3T3 cells (NIH3T3 CRL-1658) cells were obtained from the American Type Culture Collection (ATCC)(Manassas, VA). These cells were authenticated and tested for mycoplasma contamination by ATCC at the time of purchase. L cells stably expressing Jagged1 (Jag1) or Delta-like 1 (Dll1) were a gift of Dr. Gerry Weinmater (UCLA). MS5 cells stably expressing Delta-like 4 (Dll4) were a gift from Dr. Stephen Blacklow. HEK293T, NIH3T3, L cells, and MS5 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM)(Invitrogen) supplemented with 10% bovine calf serum.

SHSY5Y and NIH3T3 cells were purchased from the American Type Culture Collection (Manassas, VA) and maintained in 5 % CO₂ at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM) containing 10 % fetal calf serum (FCS), 100 U/ml penicillin, and 100 μg/ml streptomycin in 10 cm plates. After 70 to 80 % confluence, cells were rinsed with 1× PBS. Following trypsinization and mechanical dissociation, cells were subcultured into 24-well plates and incubated at 37 °C for 2 days preceding transfection. Transfection was done after the cells reached 70 to 80 % confluence. pBDNF constructs, pGL3-Basic, or pGL3-SV40 vector was transiently transfected into SHSY5Y and NIH3T3 cells using Lipofectamine 2000 (Invitrogen). Briefly, for each transfection, 2.0 μg of DNA and 5 μl of Lipofectamine were first diluted in OPTI-MEM (GibcoBRL), mixed gently, and incubated at room temperature for 5 min. Cells were rinsed once with PBS and OPTI-MEM was added before incubation with the DNA/Lipofectamine. The pRL-SV40 Renilla luciferase vector (50 ng) was co-transfected in all experiments. Cells were allowed to incubate at 37 °C and the media was replaced after 12 h by fresh DMEM. Cells were harvested 48 h after the start of transfection.