Mouse Tracheal Epithelial Cell Culture MTEC were prepared and propagated as previously described

proteinase K for 10 min and rinsed in cool running water for 5 min. Endogenous peroxide activity 23902761 was quenched with 1.5% hydrogen peroxide in methanol for 10 minutes, and incubation with the primary antibodies antiMAP17 and anti-SGLT1 was performed for 40 min. After incubation, immunodetection was performed with the BIRB-796 supplier EnVision visualization system using diaminobenzidine chromogen as the substrate, according to the manufacturer’s instructions. Immunostaining was performed in a TechMate 500 automatic immunostaining device and measured through a double-blind visual assessment using microscopic observation according to the anatomopathological experience of pathologists. A monoclonal MAP17 antibody was generated from bacterialpurified GST-MAP17 protein. Several clonal antibodies were tested for specificity and validated through antigen competition. Immunodetection of MAP17 The cells were trypsinized and cytospin onto glass coverslips. The following day, the cells were fixed with acetone for 10 min and then incubated with MAP17 monoclonal antibody for 30 min. The cells were washed three times with PBS and incubated for additional 30 min with a secondary goat anti-mouse antibody diluted 1:50 in fetal bovine serum. After incubation, immunodetection was performed with the EnVision visualization system using diaminobenzidine chromogen as the substrate. After washing, the slides were mounted with Aquatex. The images were acquired using an Olympus Provis Microscope AX70. Cell Culture Hela malignant cervical tumor cells were obtained from the ECACC human cell line repository and maintained in Dulbeccos modified Eagles medium containing 10% fetal bovine serum, penicillin, streptomycin and fungizone. MAP17 full-length cDNA was cloned into pBabe puro and mass culture generated by stable gene transfer in Hela cells. After selection with 2 mg/ML puromycin, mass cultures were used for the study. As a control, Hela cells were transfected with pBabe puro alone and selected. Materials and Methods Patient Selection This research followed the tenets of the Declaration of Helsinki and was approved by the Hospital Val d’Hebron institutional ethical review board. All samples were obtained after informed consent was provided by the patients. The participants provided their written informed consent to participate in the study. The patients included in this retrospective study were selected 26243621 from a clinical database at Hospital Val d’Hebron. Cytotoxicity Studies Cytotoxicity studies were performed as described previously. Briefly, 4,000 cells/well were seeded in 96-well plates and left to attach and grow for 24 hours before treatment. The drugs were weighed and then diluted with DMSO to generate a 10-mM solution. From here, a “mother plate”with serial dilutions was prepared at 200X the final concentration in culture. Eleven serial dilutions of the drug from an initial 30-mM dose were assayed per compound in a minimum of three independent experiments. The medium was removed from the cells and replaced with 0.2 ml of medium dosed with drug. Each concentration was assayed in triplicate. Two sets of control wells were included on each plate consisting of either medium only or medium with the same concentration of DMSO. A third control set was established consisting of untreated cells just before addition of the drugs. The cells Tissue Acquirement and Preparation Human cervical carcinoma tissues were obtained from surgical procedures and sent to the pathology laboratory for