The results are shown in 2 Effects of HBs on Sperm Functions pretreated with N-Acetylcysteine also markedly declined

plates by Piclidenoson transfecting MLE 12 cells with control siRNA or C/EBPb/c siRNA using 5 ml LipofectamineTM 2000 in 500 ml of Opti-MEM I medium. 12 h after siRNA transfection, the cells were treated with or without 20 ng/ml IL-1b for different time points. Supernatants were collected for ELISA. ELISA Both MLE12 cells and primary cultured alveolar type II epithelial cells were stimulated by IL-1b for the indicated time. The supernatants were centrifuged at 3000 rpm for 5 min, and the cell-free supernatants were harvested for IL-6 measurements by using a commercially available ELISA kit according to the manufacturer’s protocol. Invitrogen, Carlsbad, CA). Cells were maintained in a humidified incubator at 37uC with 5% CO2. Recombinant mouse IL-1b was purchased from R&D Systems, Minneapolis, MN. RNA Isolation and Detection of mRNA by Quantitative Real-Time PCR Total RNAs were extracted from cells with Trizol according to the manufacturer’s procedure. After isolation, total cellular RNA was incubated with RQ1 RNase-free DNase to remove contaminating DNA. 2 mg of total RNA was submitted to reverse transcription by using the Superscript II RNase H2 Reverse Transcriptase. The levels of mRNA of C/EBPc and GAPDH were determined by qPCR. PCR was performed with primers for C/EBPc: 59 20688974 primer, 59-GCA AGC AAA GCA AAA AGA GC-39 and 39 primer, 59-GCT TCC AAC CGT TCA TTC TC-39; GAPDH: 59 primer, 59-GCC TCG TCT CAT AGA CAA GAT G-39 and 39 primer, 59-CAG TAG ACT CCA CGA CAT AC-39. Following reverse transcription, the cDNA was amplified and quantified using a Sequence Detection System and a PCR universal protocol as follows: AmpliTaq Gold activation at 95uC for 15 s and, annealing/extension at 60uC for 1 min. The fluorescence of the double-stranded products accumulated was monitored in real time. The relative mRNA levels were normalized to levels of GAPDH mRNA in the same sample. The expression of IL-6 by RT-PCR was determined as previously described. Expression Vectors and Promoter Reporters Full-length mouse C/EBPc cDNA was amplified from total RNA of the mouse lung using reverse transcription-PCR, sequenced, and inserted into pcDNA3.1. Recombinant adenovirus containing mouse C/EBPc was constructed by using BD Adeno-XTM Expression System 1. To generate the virus, Adeno-C/EBPc was digested with PacI and transfected to HEK-293 cells according to the manufacturer’s instructions. Recombinant adenoviruses were purified by BD Adeno-X virus purification kit, and stored in aliquots at 280uC. The viral stocks were tittered using Adeno-X Rapid Titer Kit. The mouse IL-6 promoter-reporter, IL-6 promoter-reporter containing a mutated NF-kB binding site, as well as the C/EBPb and NF-kB p65 expression plasmids were kindly provided by Richard C. Schwartz. Mouse IL-6 promoter-reporter containing a mutated C/EBP binding site was kindly provided by Gail A. Bishop. NF-kB promoter reporter was obtained from Promega. C/EBP promoter reporter, 26C/EBPLuc containing two copies of a C/EBP binding site, was kindly provided by Peter F. Johnson. Western Blot Analysis Both MLE12 and primary cultured AEC II were lysed in cold RIPA buffer. Samples containing 80 mg protein were electrophoresed in a 12% polyacrylamide gel and “2436504 transferred to a PVDF membrane. Membranes were incubated with rabbit anti-C/EBPc antibody and rabbit anti-GAPDH antibody, respectively. After 3 washes in TBST, the membranes were incubated with a 1:5,000 dilution of horseradish peroxidase-conjugated donkey anti-rabbit Adenov