1 mg/ml streptomycin and 0.5 μg/ml amphotericin B under a humidified atmosphere of 95% air and 5% CO2. Establishment of Stable
TGF-β1 Transfectants A cDNA clone encoding full-length mouse TGF-β1 mRNA (GenBank accession no. BC013738) in the pCMV-SPORT6 vector was purchased from OpenBiosystems (Huntsville, AL) and subcloned into pIRES2-PF-04929113 AcGFP1 vector (Clontech, Inc. Palo Alto, CA). The IRES2-AcGFP1 vector harboring TGF-β1 was then transfected into SCCVII cells using Lipofectamine 2000 reagent (Life Technologies, Inc. Grand Island, NY). TGF-β1 transfectants were selected by culture for 2 weeks in medium containing 400 μg/ml G418 (Life Technologies, Inc.); the resistant clones were then obtained using the method of limiting dilution. As a negative control, SCCVII cells were transfected with pIRES2-AcGFP1 vector without
GSK3326595 cell line the inserted TGF-β1 cDNA. The levels of TGF-β1 expression in the stable transfectants were then determined using RT-PCR and an ELISA (R&D Systems Inc., Minneapolis, MN). For RT-PCR, total RNA was isolated from the samples using a Fast RNA Kit Green (Qbiogene, Carlsbad, CA) according to the manufacturer’s instructions. After quantifying the isolated RNA using a spectrophotometer, 1-μg aliquots were reverse transcribed using Superscript II reverse transcriptase (Gibco BRL, Gaithersburg, Md.,). The following primer sets were used: for TGF-β1, 5′-ATCTCGAGCTCCGCCATGCCGCCCTCGGGG-3′ (forward) and 5′-TCGACTGCAGAATTCTCAGCTGCACTTGCA-3′ (reverse); for AcGFP1, 5′-GAGCTGTTCACCGGCATCGT-3′ (forward) and 5′-GATGGGGGTATTCTGCTGGT-3′ (reverse). Hedgehog antagonist Cultured bone marrow-derived DCs Bone marrow-derived DCs (bmDCs) were generated using the method previously described by Labeur et al., with some modification . Briefly, bone marrow was collected from the tibias and femurs of male C3H/He N mice, passed through a 100-μm nylon mesh to remove small pieces of bone and debris, resuspended in CM, and plated in tissue culture dishes for 2 h. Nonadherent cells were collected and plated at a density of 2 × 106 cells/well in 6-well
plates containing 1 ml of CM. Then on Endonuclease days 0, 3 and 5, two-thirds of the medium were replaced with CM containing 20 ng/ml recombinant murine GM-CSF (Peprotech, Rocky Hill, NJ). By day 8 of culture, most of the nonadherent cells had acquired typical DC morphology, and those cells were used as the source of bmDCs. For in vitro experiments, 1 μg of lipopolysaccharide (LPS; Sigma-Aldrich Flanders NJ) was added to the CM on day 7; then after an additional 48 h the mature bmDCs were used. At the end of the procedure, DC purity was assessed based on CD11c expression using single color flow cytometry and was found to be 90% or greater. TDLN cell preparation To prepare TDLNs, tumor cells (1 × 106 cells/mouse) were inoculated unilaterally into the ears of C3H/He N mice.