However, the potential role of JWA in chemically induced skin carcinogenesis has not been elucidated

ring the transfection. Briefly, a mixture of 2 mg HIV-based lentiviral expression vector, 2 mg pCMV-dR8.2 dvpr, and 2 mg m 168 vector or pHIT-G, complexed with 18 mL Fugene 6 in 1 mL Dulbecco’s modified Eagle medium was added to HEK 293T cells in a 10-cm plate. Supernatants were collected 48 hr post-transfection, filtered through a 0.45 mm pore-size filter and stored at 280uC. Multiplicity of infection was calculated based on the titer of m 168 pseudotyped particles in the presence of the HLA-1 Ab on 293T cells. H9 cells, iPS cells, HEK 293T, and human foreskin fibroblast cells were infected by m 168-pseudotyped lentiviral particles at a MOI of 5 and conjugated with different antibodies for 24 hr. In CSP-1103 parallel, cell lines were infected with VSVG-pseudotyped lentiviral particles as a positive control. The transduction “7851485 efficiency was detected by the eGFP expression in the target cells using flow cytometry 9 days after infection. Flow cytometry was performed at Rutgers/UMDNJ FACS Core Facility with Beckman Coulter Cytomics FC500 and analyzed using CXP Software. Monoclonal antibodies Ab-mediated targeting transduction experiments and iPS immunofluorescence staining were performed using the following primary antibodies: Mouse anti-human HLA ABC, anti-SSEA4, antiCD9, anti-CD24, “7851485 TRA-160, and TRA-1-81, rat anti-human FZD7, anti-SSEA3. Secondary antibodies used were goat anti-mouse IgG-R-phycoerythrin antibody from Sigma, Goat anti-Rat IgG FITC conjugate, and Alexa Fluor 488 goat anti-rat IgM antibody from Invitrogen. The DyLightTM 488 mouse anti-Human TRA-1-60 antibody was used for live cell staining of the reprogramming cells by incubating for 30 minutes at 37uC and 5% CO2 in the iPS reprogramming medium. Cells were then washed 2 times with iPS reprogramming media and images were obtained on a Nikon Eclipse Ti microscope. Trophoblast differentiation and TK negative selection Before differentiation, 26104 cell hES H9 cells were treated with Accutase and plated in one Matrigel-coated six-well plate with mTeSRTM containing 5 mM ROCK-inhibitor Y27632. After 24 hr, media was replaced with MEF conditioned medium plus 20 ng/mL BMP4 and changed daily. Five days post BMP4 addition, cells were infected with either the SSEA4 or CD24 antibody conjugated m 168 pseudotyped virus bearing the pSin-EF2-TK-Puro lentiviral vector for 24 hours. The infected cells were cultured in MEF conditioned media medium with BMP4 plus 2 mM ganciclovir from day 7 to day 10. After 4 days selection in ganciclovir, cells were treated with trypsin/EDTA solution, fixed in 2% paraformaldehyde, and permeabilized by suspension in PBS containing 0.1% Triton X100. 56105 cells were mixed with 1 ml of mouse anti-human Cytokeratin 7 antibody . Secondary antibodies used were goat antimouse IgG-R-phycoerythrin antibody from Sigma. The samples were analyzed on a FACS Calibur flow cytometer. Isolation of African-American primary fibroblast cells The human primary fibroblast cells were prepared as previous reports. The skin punch biopsies were obtained from Cooperative Human Tissue Network, Univ. of Pennsylvania Medical Center. The skin biopsies were washed in DMEM/F12 and minced into approximately 0.51-mm size pieces before being seeded onto gelatin-coated 6-well cell culture plates containing mouse embryonic fibroblast media consisting of Dulbecco’s modified Eagle medium: Nutrient Mixture F-12 containing 10% fetal bovine serum and antibiotics & antimycotics. The culture medium was partially c