PMC:3475485 / 4429-10348 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/3475485","sourcedb":"PMC","sourceid":"3475485","source_url":"https://www.ncbi.nlm.nih.gov/pmc/3475485","text":"Methods\n\nEstablishment of MEF derived from wild-type and PKD2 mutation embryos\nMEF were acquired from PKD2 TG embryos or Pkd2 KO embryos during development (13.5 days) [11]. Heads and limes were removed from embryos. The remaining embryonic tissues were minced and dispersed in 0.05% trypsin prior to incubation at 37℃ for 15 min. Cells were plated in Dulbecco's Modified Eagle's Medium of Defined Minimal Essential medium (Welgene Biotech, Taipei, Taiwan) supplemented with 10% fetal bovine serum (Welgene Biotech) and were cultured at 37℃ in an atmosphere of 5% CO2 until confluent growth was achieved. MEF cells were frozen as stocks at the second passage and were used for the subsequent studies at the third passage.\n\nMicrorray hybridization and data analysis\nTotal RNA was prepared using a commercial kit (Qiagen, Valancia, CA, USA) according to the manufacturer's instructions. Gene expression profiles were obtained using the CodeLink Uniset Mouse Expression Bioarray (Amersham, Buckinghamshire, UK). The 30,000 gene probes contained in the bioarray allows the detection of differences in gene expression as low as 1.3-fold with 95% confidence. Ten micrograms of total RNA was amplified, and labeled cDNA was produced. Biotin-labeled cDNA was hybridized to the array overnight in a shaking incubator at 37℃, and excess target sequences were washed away using a series of saline sodium citrate washes. The array was stained by treatment with streptavidin-Alexaflour 647 (Molecular Probes, Eugene, OR, USA), the excess was washed away, and the array was scanned at an excitation wavelength of 632 nm using a GenePix scanner (Molecular Devices, Sunnyvale, CA, USA). The resulting image was quantified, and the intensity of each spot was divided by the median spot intensity to provide a scaled and comparable number across multiple arrays.\n\nDNA microarray scanning and analysis\nMicroarrays were scanned using an Arraywox scanner (Applied Precision, Seattle, WA, USA), analyzed using ImaGene version 5.1 software (Biodiscovery, Segundo, CA, USA), and normalized using Genesight version 3.2 software (Biodiscovery). Normalization was performed by subtracting the means of all genes. The data normalized by Genesight were compared using an M/A plot. Genes differentially expressed were identified by intensity differences, after subtracting the background intensity. Genes showing expression changes of at least 2-fold were selected, and these selected genes were clustered by the hierarchical method.\n\nClustering algorithm\nThe normalized log ratio corresponding to each time point was exported to the software for clustering algorithm. Acuity version 3.1 (Molecular Devices) was used for the 'gene shaving' algorithm. To estimate the number of clusters in a dataset, the 'Gap Statistics' attribute of the Acuity software package was used. The cluster number, estimated by gap statistics, was used for an input parameter in gene shaving. After gene shaving, a single linkage was used for hierarchical clustering.\n\nSemi-quantitative and real-time RT-PCR\nTotal RNA was prepared using a commercial kit (Qiagen) according to the manufacturer's instructions. Single-strand cDNA was synthesized by incubating 5 µg total RNA with 200 units AMV, 100 nM oligo(dT)12-18, 1 mM dNTP mixture, and 40 units RNase inhibitor at 42℃ for 1 h in a final volume of 25 µL. The reaction was terminated by incubation at 70℃ for 15 min. The initial amount of mRNA and reaction conditions were optimized to obtain linearity for mouse 18s rRNA. For semi-quantitative reverse transcription (RT)-PCR, the used primers were: human PKD2, forward 5'-CGTGCCCCAGCCCAGTC-3' and reverse 5'-TTCCAGTACAGCCCATCCAATAAG-3'; mouse Pkd2, forward 5'-TGCGAGGGCTGCGAGGTC-3' and reverse 5'-TGTCAGCTTGCGTGTGGTTGC-3'; mouse 18s rRNA, forward 5'-GTAACCCGTTGCACCCCATT-3' and reverse 5'-CCATCCAATCGGTAGTAGCG-3'. RT-PCR cycling conditions were as follows: 10 min at 95℃, 25 cycles of 50 s at 94℃, 50 s at 57℃, 50 s at 72℃, and 5 min at 72℃. The amplified products were separated on a 1% agarose gel. For real-time PCR, the used primers were: mouse β-actin, (forward 5'-GACGATGCTCCCCGGGCTGTATTC-3' and reverse 5'-TCTCTTGCTCTGGGCCTCGTCACC-3') used as a positive control; mouse peroxisome proliferator-activated receptor γ (PPAR-γ), forward 5'-TCTTAACTGCCGGATCCACAAAAA-3' and reverse 5'-ATCTCCGCCAACAGCTTCTCCTTC-3'; mouse protein kinase α (PKCα), forward 5'-GGGCAGCCTCCGTTTGATGGT-3' and reverse 5'-CGCTTGGCAGGGTGTTTGGTC-3'; mouse Wnt4, forward 5'-GCCATCGAGGAGTGCCAATACC-3' and reverse 5'-GGCCACACCTGCTGAAGAGATG-3'; mouse integrin α4 (ITGα4), forward 5'-GTAGCCCCAGTGGAGAGCCTTGTG-3' and reverse 5'-ATGCCAGTGGGGAGTTTGTTATCG-3'; mouse IL6ST, forward 5'-TGAATCGGACCCACTTGAGAGG-3' and reverse 5'-CAGGAGCGGCTTGTTTGAGGTA-3'; mouse aquaporin 1 (AQP1), forward 5'-GGAGGCGCCGAGACTTAGGT-3' and reverse 5'-GCGGGTGAGCACAGCAGAGC-3'; mouse transforming growth factor-β2 (TGF-β2), forward 5'-TCATCCCGAATAAAAGCGAAGAGC-3' and reverse 5'-AGGGCAACAACATTAGCAGGAGAT-3'. Real-time RT-PCR was performed using the real-time SensiMixPlus SYBR kit as described by the manufacturer's instructions (Quantance, London, UK).\n\nWestern blot analysis\nProteins were extracted using radio-immunoprecipitation assay buffer in MEF cells. Proteins were separated by 12% sodium dodecyl sulfate-polyacryamide gel electrophoresis and were transferred to a polyvinylidene fluoride membrane (Millipore, Bellerica, MA, USA). Membranes were blocked with 5% non-fat dry milk and were incubated with various antibodies. Antibodies to PC2 (anti-human and anti-mouse) and AQP1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies to β-actin were obtained from Sigma-Aldrich (St. Louis, MO, USA). Anti-Wnt4 was obtained from R\u0026D Systems (Minneapolis, MN, USA). Each membrane was washed with phosphate-buffered saline-Tween, and immunocomplexes were detected by enhanced chemiluminescence (Amersham).","divisions":[{"label":"Title","span":{"begin":0,"end":7}},{"label":"Section","span":{"begin":9,"end":721}},{"label":"Title","span":{"begin":9,"end":78}},{"label":"Section","span":{"begin":723,"end":1844}},{"label":"Title","span":{"begin":723,"end":764}},{"label":"Section","span":{"begin":1846,"end":2503}},{"label":"Title","span":{"begin":1846,"end":1882}},{"label":"Section","span":{"begin":2505,"end":3014}},{"label":"Title","span":{"begin":2505,"end":2525}},{"label":"Section","span":{"begin":3016,"end":5139}},{"label":"Title","span":{"begin":3016,"end":3054}},{"label":"Title","span":{"begin":5141,"end":5162}}],"tracks":[{"project":"2_test","denotations":[{"id":"23105924-19098310-44845729","span":{"begin":169,"end":171},"obj":"19098310"}],"attributes":[{"subj":"23105924-19098310-44845729","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#93c3ec","default":true}]}]}}