PMC:4925210 / 36666-45874 JSONTXT

{"project":"2_test","denotations":[{"id":"27214281-12771126-73835281","span":{"begin":5254,"end":5256},"obj":"12771126"},{"id":"27214281-12771126-73835281","span":{"begin":5254,"end":5256},"obj":"12771126"},{"id":"27214281-7556934-73835282","span":{"begin":6217,"end":6219},"obj":"7556934"},{"id":"27214281-7556934-73835282","span":{"begin":6217,"end":6219},"obj":"7556934"}],"text":"Functional expression of PKD1 and TRPP2 in Drosophila S2 cells\n\n-Molecular cloning and expression\nThe Bgl2-Age1 fragment encompassing ~13 kb of human PKD1 cDNA was moved from HA-PKD1 (HBF307N-hPKD1) to the Drosophila pMT/V5-HisA vector (Invitrogen). The remaining 5’ part of the cDNA was amplified by PCR incorporating the Drosophila optimal translational initiation start site (CAAA) immediately upstream of the first methionine and was cloned into pMT/V5-HisA upstream of the Bgl2-Age1 fragment of human PKD1 using EcoR1 (5’) and Bgl2 (3’). The EcoR1-Bgl2 fragment of PKD1 in pMT/V5-HisA was verified by complete sequencing. Mouse TRPP2 bearing the CAAA sequence upstream of its first methionine was cloned into the pMT/V5-HisA vector as EcoR1-Xho1 fragment. The human CD8α cDNA excluding the nucleotide sequence corresponding to the signal peptide was cloned into the pMT/Bip/V5-HisA vector as a Kpn1-EcoR1 fragment. CD8α or PKD1+TRPP2 in pMT/BIP or pMT vectors were transiently transfected into Drosophila S2 cells using Nucleofection (Program G-30, transfection kit V, Amaxa). Transfection was done in 12-well plates containing 2×106 (for functional expression) or 107 cells (for biochemical expression) using 2 μg of total DNA (1.5 μg PKD1, 0.3 μg TRPP2, and 0.2 μg CD8α). Transfection efficiency using pMT/GFP was routinely at 80-90%. Cells were treated with 700 μM CuSO4 24 h following transfection and for 48 h to induce the expression of CD8α, PKD1, and TRPP2.\n\n-Electrophysiology\nTwenty four hours following induction by CuSO4 (700 μM), cells were seeded onto poly-L-lysine coated coverslips and processed for electrophysiology after another 24 h. Whole cell currents were obtained as described above for MEFs and CHO-KI cells with the following exceptions: Pipette resistance was 6 MΩ when back-filled with intracellular solution where the Ca2+ concentration was adjusted to 100 nM (physiological). Typical capacitance was 9-11 pF. Extracellular solution was Tyrode solution. Cell capacitance was not compensated.\n\n-WNT9B cell surface binding\nCells were seeded onto poly-L-lysine-coated glass disks the day before the experiment. At the day of the experiment, cells were washed twice with PBS and incubated with 1 μg/ml purified WNT9B in 0.5% BSA at 4°C for 3 h. Cells were washed three times (10 min each wash) with cold PBS and fixed in 2% paraformaldehyde in PBS for 15 min at room temperature. Cells were washed three times with cold PBS and incubated overnight with 1:500 dilution (or 0.4 μg/ml) of goat α-WNT9B in 5% donkey serum in PBS. Next day, cells were washed there times in cold PBS and incubated for 1 h with donkey α-goat conjugated to Alexa-488. After three final washes with cold PBS, coverslips were mounted with ProLong (Molecular Probes), and cells were imaged using an Olympus IX81 inverted confocal microscope (60x). Images were analyzed by the Olympus Fluoview software.\n\nTOP/FOP Flash reporter assays\nOne million of MEFs or CHO-K1 cells were transfected with a Super TOP-Flash or FOP-Flash plasmid and Renilla luciferase pRL-TK vector together with pcDNA3 or ZNRF3 expression vector, using the Ingenio Electroporation Kit (Mirus-Bio) and the Nucleofector IIb Device (Lonza). Renilla luciferase was used as the internal control reporter to normalize transfection efficiency. All transfections were performed with 0.5 μg of Super TOP-Flash or FOP-Flash, 0.05 μg of pRL-TK, and 2 μg of pCDNA3 or ZNRF3. After 18 h following transfection, cells were split into 18 wells of a 24 well plate. After 24 hr, cells were treated with PBS, WNT3A (500 ng/ml), WNT9B (1 μg/ml), or a mixture of WNT3A and WNT9B for another 24 hr and lysed in a reporter lysis buffer (Promega, Madison, WI, USA). Luciferase assay was performed using the Dual Luciferase Assay System kit according to the manufacturer's protocols (Promega, Madison, WI, USA). Relative luciferase activity was reported as fold induction after normalization for transfection efficiency. Three independent experiments were performed in sextuplicates.\n\nRNA-isolation and Quantitative RT PCR\nTotal RNA from Pkd2+/+ and Pkd2−/− MEFs was extracted using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. 5 μg of total RNA was used for cDNA synthesis with 1:1 mixture of 50 ng/μl random hexamers (Invitrogen) and 0.5 μg/μl Oligo dT (Invitrogen). Quantitative real-time PCR were performed on the resultant cDNA using the PerfeCTa CYBR Green SuperMix for iQ (Quanta BioSciences, Gaithersburg, MD) following manufacturer's instructions. Primer sequence information is presented in Suppl. Table 2. Real-time PCR was carried out using a Bio-Rad iCycler Thermal Cycler (Bio-Rad, Hercules, CA). Reactions were run in triplicate in three independent experiments. The mRNA levels of each target protein were normalized relative to housekeeping gene GAPDH mRNA levels in each sample.\n\nXenopus Embryo Manipulations\nXenopus embryos obtained by in vitro fertilization were maintained in 0.1x modified Barth medium 62 and staged according to Nieuwkoop and Faber 63. The sequences of the antisense morpholino oligomers (GeneTools, LLC) used in this study are listed in Suppl. Table 2. The efficacy of Dvl2-MO and Pkd1-sMO1 was demonstrated previously64, 65. The efficacy of Pkd1-sMO2 was verified by RT-PCR of RNA extracted from stage 35 embryos (Supplementary Fig. 7e) using primers spanning exons 17/18 of the predicted Xenopus laevis Polycystin1 (Xenbase Gene #483413, 5’-TGT CCT TGA AGT GCG TGT CA-3’ and 5’-CCC GTA GAT GTG GTG CTT GA-3’).In the case of Wnt9A-MO, it was verified using a Wnt9A-GFP fusion construct injected into Xenopus embryos in the presence or absence of the Wnt9A-MO. For morphological scoring of edema formation embryos were injected at the 2- to 4-cell stage with the indicated amounts of MO and cultured until sibling embryos reaches stage 43. For whole the imaging of the individual pronephric kidneys embryos were injected unilaterally at the 2-cell stage embryo with the indicated amounts of MOs, cultured until stage 40 and fixed in Dent's fixative (4:1 Methanol:DMSO). Whole mount immunofluorescence was performed by incubating the embryos sequentially with the 3G8 and 4A6 antibodies66 and developing them using AlexaFluor-448 and AlexaFluor-555-labeled α-mouse secondary antibodies (Life Technologies). Embryos were analyzed individually by confocal microscopy comparing the injected to the contralateral control side. Pronephric kidneys with dilated and shortened nephron segments were classified as dysplastic. In individual cases 3-dimensional (3D) reconstruction of the kidneys was used to verify differences in tubule diameter. Analysis was performed in a blinded fashion to avoid bias.\nRescue experiments for the Dvl2-MO were performed by injecting 0.5 μg synthetic mRNA encoding wild type human DVL2 or mutant DVL2-E499G into a single blastomere at the 2-cell stage followed by injection of 3.2 pMol Dvl2-MO into all four blastomeres at the 4-cell stage. Embryos were cultured until stage 40 and processed for whole kidney imaging as outlined above. Scoring was based on comparing the kidneys of the Dvl2-MO-injected side to the one of the Dvl2-MO/mRNA-injected side\n\nCell surface biotinylation\nWild type, Pkd1−/− MEFs, or HEK293T cells co-transfected with TRPP2 and wild type PKD1 (PKD1WT) or PKD1S99I were washed 3 times in PBS (pH 8.0), scraped, and re-suspended in 1 ml PBS (pH 8.0). Cell surface proteins were labelled with 1 mM EZ-Link NHS-PEG4-biotin (Thermo Scientific, 21329) for 30 min at room temperature. Biotinylation was terminated by 3 washes with PBS containing 100 mM Glycine, pH 3.0, and lysed in 1% Triton X-100, 150 mM NaCl, 10 mM Tris-HCl at pH 7.5, 1 mM EDTA, 1 mM EGTA, 0.5% NP-40, and 10% sucrose with protease inhibitor cocktail (Roche Applied Science) at 4°C for 30 min. Lysates were collected by centrifugation (18,000 × g, 20 min) and biotin-labelled surface proteins were captured on streptavidin–agarose beads at 4°C by an overnight incubation. Proteins bound to beads were collected by a brief (1 min) centrifugation, and pellets were washed 3 times with lysis buffer for 20 min at 4°C. Biotin-labeled surface proteins were eluted with SDS gel-loading buffer (50 mM Tris-HCl at pH 6.8, 100 mM DTT, 2% SDS, 0.1% bromophenol blue, and 10% glycerol) and analyzed by western blotting.\n\nEndogenous co-immunoprecipitations\nFive 10-cm dishes containing confluent cultures of wild type MEFs were pooled and lysed in 1 ml of non-denaturing lysis buffer containing 1% Triton X-100, 150 mM NaCl, 10 mM Tris-HCl at pH 7.5, 1 mM EDTA, 1 mM EGTA, 0.5% NP-40, and 10% sucrose with protease inhibitor cocktail (Roche Applied Science) at 4°C for 30 min. Lysates were cleared by centrifugation (18,000 × g, 20 min) and incubated with either mouse α-PKD1 (E4, 5 μg) or mouse IgG (Santa Cruz biotechnology) at 4°C overnight. Mouse antibodies were captured by Protein G coupled to agarose beads and immunocomplexes were immunoblotted with rabbit α-Dvl1 (1:200, Thermo Fisher, Rockford, IL, USA), rabbit α-Dvl2 (1:1000, Thermo Fisher) or mouse α-PKD1 (7e12, 1:5,000). Proteins were visualized with an enhanced chemiluminescence detection kit (ECL, Thermo).\n"}