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Al Care and Use Committee at the Oklahoma State University (VM-
Al Care and Use Committee at the Oklahoma PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27919709 State University (VM-15?8). Bleomycin or saline was delivered to the lungs of C57BL/6 male mice (6? weeks) via nasal instillation at a dose of 3 U/kg body weight. On day 14 mice were sacrificed, and lung tissues were collected. Fibroblasts and alveolar epithelial type II cells (AEC II) were isolated from the lungs of saline or bleomycin-treated mice according to the previously described protocols [16, 17]. Alveolar epithelial type I cells (AEC I) were obtained by culturing AEC II in Dulbecco's Modified Eagle Medium (DMEM) for 5 days .RNA isolationLL29 lung fibroblasts were purchased from American Type Culture Collection (ATCC, Manassa, VA, CCL134) and were maintained in F12K medium supplemented with 10 fetal bovine serum (FBS) and 1 penicillin/streptomycin (P/S). The fibroblasts were seeded on 6-well plates at a density of 2? ?105/well. After 24 h, cells were infected with a lentiviral miR-27b or its control at a multiplicity of infection (MOI) of 50. 48 h post infection, cells were stimulated with TGF (5 ng/ml). After another 48 or 72 h, (S)-Hydroxychloroquine cells were collected for RNA and protein analyses.Real-time PCRTotal RNAs were isolated from lung tissues or cells by using Tri Reagents (Molecular Research Center,Total RNA was reverse-transcribed into cDNA using Moloney Murine Leukemia Virus reverse transcriptase.Zeng et al. BMC Cell Biology (2017) 18:Page 3 ofFor miRNA quantitation, total RNA was poly (A)-tailed using an A-Plus Poly (A) Polymerase Tailing Kit (Epicentre, Madison, WI) before reverse transcription. The following primers were used: COL1A1, Forward: CGAA GACATCCCACCAATCAC, and reverse: CAGATCAC GTCATCGCACAAC; COL3A1, Forward: TGGCTA CTTCTCGCTCTGCTT, and reverse: TTCCAGACA TCTCTATCCGCATAG; COL4A1 forward: CTCTGG CTGTGGCAAATGTG, and reverse: CCTCAGGTCC TTGCATTCCA; -SMA, forward: GTGTTGCCCC TGAAGAGCAT, and reverse: CGCCTGGATAGCCA CATACAT; microRNA-universe reverse primer: GC GAGCACAGAATTAATACGAC; RNU6 forward primer: AGAGAAGATTAGCATGGCCCCT; miR-27b forward primer: TTCACAGTGGCTAAGTTCTGC. Real-time PCR was performed using SYBR Green master mix on an ABI 7500 fast system (Applied Biosystems, Foster City, CA). The thermal temperature were: 95 for 10 min, followed by 40 cycles of 95 for 15 s, 60 for 30 s, and 65 for 30 s. The endogenous reference genes were glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or RNU6. The comparative Ct method was used to calculate the relative mRNA and miRNA expression levels.Luciferase reporter assayFibroblast contraction assayLL29 fibroblasts were seeded in 6-well plates at a density of 1? ?105 cells per well overnight and infected with miR-27b or its control lentivirus at a MOI of 50 for 48 h. The cells were treated with TGF1 (5 ng/ml) for another 48 h. The cells were trypsinized and mixed with rat tail collagen I (BD Bioscience, Cat# 354236) to a final concentration of 1 ?105cells/ml and 1 mg/ml of collagen 1, followed by the addition of 15 l 0.5 M NaOH to 1 ml of the cells. The cells were then added to 24-well BSAcoated plates (500 l/well). After a 30-min incubation, medium with or without TGF (5 ng/ml) were added (500 l/well). Cells were incubated for 48 h and images were taken. The gel areas were quantified PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28526414 using Image J software.StatisticsAll experiments were performed with at least three independent replicates. For statistical analysis, student t-test was used for two group comparisons and ANOVA, followed by Turkey's test for multiple group comparisons.
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