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, RNA was extracted with the miRNeasy mini kit (Qiagen) following manufacturer's instructions. mRNA-seq was performed from 2 ?g of RNA that was first subjected to mRNA selection with Dynabeads® mRNA Purification Kit (Invitrogen). mRNA was fragmented 10 min at 95°C in RNAseIII buffer (Invitrogen) then adapter-ligated, reverse transcribed and amplified (6 cycles) with the reagents from the NEBNext Small RNA Library Prep Set for SOLiD. Small RNA-seq was performed from 500 ng RNA with the NEBNext Small RNA Library Prep Set for SOLiD (12 PCR cycles) according to manufacturer's instructions. Both types of amplified libraries were purified on Purelink PCR micro kit (Invitrogen), then subjected to additional PCR rounds (8 cycles for RNA-seq and 4 cycles for small RNA-seq) with primers from the 5500 W Conversion Primers Kit (Life Technologies), RNA sequencing of HAECs. For Supplementary Fig. 2B, three independent HAEC cultures (HAEC1, HAEC2, HAEC3) were triggered to differentiate in air-liquid interface (ALI) cultures for 2 days (ALI day, vol.2
, SOLiD data were analyzed with lifescope v2.5.1, using the small RNA pipeline for miRNA libraries and whole transcriptome pipeline for RNA-seq libraries with default parameters. Annotation files used for production of raw count tables correspond to Refseq Gene model v20130707 for mRNAs and miRBase v18 for small RNAs. Data generated from RNA sequencing were then analyzed with Bioconductor, Caliper Lifesciences), and finally quantified with the Bioanalyzer High Sensitivity DNA Kit (Agilent)
, Reads from ChIP-seq were mapped to the X. laevis genome release 7.1 using Bowtie2 50 . Peaks were called and annotated according to their positions on known exons with HOMER 51 . Peak enrichments of E2F4 binding site in the promoters of centriole genes and cell cycle genes 9 were estimated in presence or absence of Multicilin and a ratio of E2F4 binding (Multicilin vs no Multicilin) was calculated. Promoter reporter studies. The human CDC20B promoter was cloned into the pGL3 Firefly Luciferase reporter vector (Promega) with SacI and NheI cloning sites. The promoter sequenced ranged from ?1073 to +104 relative to the transcription start site. 37.5 ng of pGL3 plasmid were applied per well. pCMV6-Neg, pCMV6-E2F1 (NM_005225) and pCMV6-E2F4 (NM_001950) constructs were from Origene. 37.5 ng of each plasmid was applied per well. 25 ng per well of pRL-CMV (Promega) was applied in the transfection mix for transfection normalization (Renilla luciferase). HEK 293T cells were seeded at 20,000 cells per well on 96-well plates. The following day, cells were transfected with the indicated plasmids (100 ng of total DNA) with lipofectamine 3000 (Invitrogen), RNA-seq (samples GSM1434783 to GSM1434788) and ChIP-seq (samples GSM1434789 to GSM1434792) data were downloaded from GSE59309. Reads from RNA-seq were aligned to the Xenopus laevis genome release 7.1 using TopHat2 47 with default parameters. Quantification of genes was then performed using HTSeq-count 48 release 0.6.1 with "-m intersection-nonempty" option. Normalization and statistical analysis were performed using Bioconductor package DESeq2 49
, on the protection of animals used for scientific purposes. Experiments on X. laevis and mouse were approved by the 'Direction départementale de la Protection des Populations, Pôle Alimentation, issue.14, pp.62-12112012, 2010.
, Pericentrin was revealed using Alexa 514 (detection 535-564 nm, depletion 660 nm), ?-tubulin was revealed using Alexa 568 (detection 582-667 nm, depletion 775), and FOP was revealed using Alexa 488 (detection 498-531 nm, depletion 592 nm). Pictures were deconvoluted using Huygens software. Maximum intensity projection of 3 deconvoluted pictures is presented in Fig. 4g. Primary antibodies: rabbit anti-CDC20B (1:500; Proteintech, 133376-1-AP), mouse IgG anti-PLK1 (1:500; Ther-moFisher, 33-1700), rabbit anti-Pericentrin (1:500, Abcam, ab4448), mouse IgG1 anti-FoxJ1 (1:1000; eBioscience, Dissected brains were subjected to 12 min fixation in 4% paraformaldehyde, 0.1% Triton X-100, blocked 1 h in PBS, 3% BSA, incubated overnight with primary antibodies diluted in PBS, 3% BSA, and incubated 1 h with secondary antibodies at room temperature, p.250
, Ab 11316), rabbit anti-ZO1 (1:600; ThermoFisher Scientific, 61-7300), rabbit anti-Spag5 (1:500; Proteintech, 14726-1-AP), mouse IgG1 anti-ZO1 (1:600; Invitrogen, mouse IgG2b anti-FGFR1OP (FOP), pp.33-9100, 2000.
, A-11034), Alexa Fluor 647 goat antirabbit (1:800; ThermoFisher Scientific, A-21244), Alexa Fluor 514 goat anti-rabbit (1:800; ThermoFisher Scientific, A-31558), Alexa Fluor 488 goat anti-mouse IgG2b (1:800; ThermoFisher Scientific, A-21141), Alexa Fluor 568 goat anti-mouse IgG2b (1:800; ThermoFisher Scientific, A-21144), Alexa Fluor 488 goat anti-mouse IgG2a (1:800, mouse IgG1 anti-?tubulin (1:500; Sigma-Aldrich, T9026). Secondary antibodies: Alexa Fluor 488 goat anti-rabbit, vol.647, p.800
, Expression constructs containing shRNA targeting specific sequences in the CDC20B coding sequence under the control of the U6 promoter were obtained from Sigma-Aldrich (ref. TRCN0000088273 (sh273), TRCN0000088274 (sh274), TRCN0000088277 (sh277)). PCX-mcs2-GFP vector (Control GFP) kindly provided by Xavier Morin
, Electroporated animals were reanimated in a 37°C incubator before returning to the mother. Statistical analyses of mouse experiments. Analysis of CDC20B signal intensity in deuterosomes (dot plot in Fig. 3b). For each category, >25 cells from two different animals were analyzed. Deuterosome regions were delineated based on FOP staining and the intensity of CDC20B fluorescent immunostaining was recorded using ImageJ software, and expressed as arbitrary units, P1 pups were anesthetized by hypothermia
, Analysis of the number of centrioles per cell at 15dpe (Fig. 4j): > 100 cells were analyzed per condition. n = 3, 3, 3, and 3 animals for sh control, sh273, sh274, and sh277, respectively, from two independent experiments. Unpaired t test vs sh control: p < 0.0001 (sh273, sh274, sh277 ****). Analysis of ependymal cell categories at 15dpe (Fig. 4k): Data are mean ± sem from three independent experiments. More than 500 cells were analyzed for each condition, Analysis of Cdc20b shRNAs efficiency (Fig. 4c): For each cell at the deuterosomal stage, the intensity of CDC20B fluorescent immunostaining was recorded using ImageJ software and expressed as arbitrary units
, After dissection, tracheas were placed in cold DMEM:F-12 medium (1:1) supplemented with 15 mM HEPES, 100 U/mL penicillin, 100 ?g/mL streptomycin, 50 ?g/mL gentamicin sulfate, and 2.5 ?g/mL amphotericin B. Each trachea was processed under a binocular microscope to remove as much conjunctive tissue as possible with small forceps and was opened longitudinally with small dissecting scissors. Tracheas were then placed in supplemented DMEM:F-12 containing 0.15% protease XIV from S. griseus. After overnight incubation at 4°C, FCS was added to a final concentration of 10%, and tracheal epithelial cells were detached by gentle agitation, Mouse tracheal epithelial cells (MTECs)
, Cells were plated on regular cell culture plates and maintained in a humidified atmosphere of 5% CO 2 at 37°C for 4 h to allow attachment of putative contaminating fibroblast. Medium containing cells in suspension was further centrifuged at 400×g for 5 min and cells were resuspended in supplemented DMEM:F-12 containing BEGM Singlequots kit supplements and 5% FCS. Cells were plated on rat tail collagen I, FCS
, Air-liquid interface culture was conducted once transepithelial electrical resistance had reached a minimum of 1000 ohm/cm 2 (measured with EVOM2, World Precision Instruments)
, Air-liquid interface culture was obtained by removing medium at the apical side of the Transwell®, and by replacing medium at the basal side with supplemented DMEM:F-12 containing 2% Ultroser-G TM, p.10
, 5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester) (Sigma) was added one day after setting-up the air-liquid interface, vol.3
, Immunostaining on HAECs and MTECs. Three days after setting-up the air-liquid interface, MTECs on Transwell membranes were pre-extracted with
, A-11034), Alexa Fluor 647 goat antimouse (1:1000; ThermoFisher Scientific, A-21235). For co-staining of CDC20B and DEUP1, CDC20B primary antibody was directly coupled to CF TM 633 with the Mix-n-Stain TM kit (Sigma-Aldrich) according to the manufacturer's instruction. Coupled primary antibody was applied after secondary antibodies had been extensively washed and after a 30 min blocking stage in 3% normal rabbit serum in PBS. Western blot and immunofluorescence on transfected cells. Cos-1 or Hela cells cells were grown in DMEM supplemented with 10% heat inactivated FCS and transfected with Fugene HD (Roche Applied Science) according to manufacturer's protocol. Transfected or control cells were washed in PBS and lysed in 50 mM Tris HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, containing 1% NP-40 and 0.25% sodium deoxycholate (modified RIPA) plus a Complete Protease Inhibitor Cocktail (Roche Applied Science) on ice. Cell extracts separated on polyacrylamide gels were transfered onto Optitran membrane (Whatman) followed by incubation with rabbit anti-mouse CDC20B (1:500, Proteintech, 24579-1-AP) or homemade rabbit anti-Xenopus Cdc20b (1:300) antibody and horseradish peroxidase conjugated secondary antibody, 5% Triton X-100 in PBS for 3 min, and then fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. HAECs were treated 21 days after setting-up the air-liquid interface. They were fixed directly on Transwells® with 100% cold methanol for 10 min at ?20°C (for CDC20B and Centrin2 co-staining, Supplementary Figure 4a, b) or with 4% paraformaldehyde in PBS for 15 min at room temperature (for CDC20B single staining, p.1000
, using lipofectamine 3000 according to manufacturer's instructions, were rinsed on ice with chilled Ca2+ and Mg2+ free Dulbecco's PBS (DPBS, Invitrogen), harvested using a cell scraper and lysed on ice for 5 min, Asynchronous HEK cells transfected with the plasmids described below
, Incubation was performed overnight at 4°C. Columns were washed and eluted with 50 ?L elution buffer. Samples were denatured at 70°C for 10 min with Bolt? LDS Sample Buffer and Bolt reducing agent, then separated on 4-12% gradient Bolt precast gels (ThermoFisher), transferred onto nitrocellulose (Millipore), and subjected to immunoblot analysis using either anti-CDC20B (Pro-teinTech, 133376-1-AP, 1/500) or anti-c-myc antibody (clone 9E10, 1/1000). In Fig. 6, note that the high level of expression of myc-PLK1 (Fig. 6a) and myc-SPAG5 (Fig. 6b) drained out locally the ECL reagent at the peak of the protein. The resulting double bands correspond in fact to unique ones. Human SPAG5, subcloned into pCMV6-MT, was from OriGene. Human DEUP1 and PLK1 were cloned into pCS2-MT vector (Addgene)
, After concentration on a ?-Precolumn Cartridge Acclaim PepMap 100 C 18 (i.d. 5 mm, 5 ?m, 100 Å, ThermoFisher Scientific) at a flow rate of 10 ?L/min, using a solution of H 2 O/ACN/FA 98%/2%/0.1%, a second peptide separation was performed on a 75 ?m i.d. × 250 mm (3 ?m, 100 Å) Acclaim PepMap 100 C 18 column (ThermoFisher Scientific) at a flow rate of 300 nL/min. Solvent systems were: (A) 100% water, 0.1% FA, (B) 100% acetonitrile, 0.08% FA. The following gradient was used t = 0 min 6% B; t = 3 min 6% B; t = 119 min, 45% B; t = 120 min, 90% B; t = 130 min 90% B (temperature at 35°C). NanoHPLC was coupled via a nanoelectrospray ionization source to the Hybrid Quadrupole-Orbitrap High Resolution Mass Spectrometer (ThermoFisher Scientific). MS spectra were acquired at a resolution of 70,000 (200 m/z) in a mass range of 300-2000 m/z with an AGC target 3e6 value of and a maximum injection time of 100 ms. The 10 most intense precursor ions were selected and isolated with a window of 2 m/z and fragmented by HCD (Higher energy C-Trap Dissociation) with normalized collision energy (NCE) of 27. MS/MS spectra were acquired in the ion trap with an AGC target 2e5 value, the resolution was set at 17 500 at 200 m/z combined with an injection time of 100 ms. Data were reprocessed using Proteome Discoverer 2.1 equipped with Sequest HT. Files were searched against the Swissprot Homo sapiens FASTA database, -gel digestion, NanoHPLC, and Q-exactive plus analysis. For mass spectrometry analysis, protein spots were manually excised from the gel and destained with 100 ?L of H2O/ACN (1/1), 2016.
, For sense probe, it was linearized by SpeI and transcribed by T7. For antisense probe it was linearized by ApaI and transcribed by Sp6 RNA polymerase. Synthetic capped mRNAs were produced with the Ambion mMESSAGE mMACHINE Kit. pCS105/GFP-CAAX was linearized with AseI and mRNA was synthesized with Sp6 polymerase. pCS2-mRFP and pCS2-GFP-gpi were linearized with NotI and mRNA was synthesized with Sp6 polymerase. pCS-Centrin4-YFP (a gift from Reinhard Köster, Technische Universität Braunschweig, Germany) was linearized with Notl and mRNA was synthesized with Sp6 polymerase. pCS2-GFP-Deup1 and pCS2-Multicilin(MCI)-hGR were kindly provided by Chris Kintner; both plasmids were linearized with ApaI, and mRNAs were synthesized with Sp6 polymerase. Embryos injected with MCI-hGR mRNA were cultured in Dexamethasone 20 ?M in MBS 0,1× from st11 until fixation, Xenopus embryo injections, plasmids, RNAs, and morpholinos. Eggs obtained from NASCO females were fertilized in vitro, dejellied and cultured using standard protocols 54
, Quantities of mRNA injected: 500 pg for GFP-CAAX, RFP, GFP-gpi, Separase and Separase(PD); 25 to 500 pg for GFP-Deup1; 40 to 500 pg for MCI-hGR; 1 ng for Venus-cdc20b, cdc20b-Venus, cdc20b, and cdc20b-RFP; 500 pg to 1 ng for RFP-cdc20b, cdc20b-Venus, and cdc20b were generated by GATEWAY? Cloning Technology
, Spl Mo 5?-acacatggcacaacgtacccacatc-3?. 20 ng of MOs was injected per blastomere or 10 ng of each Mo for co-injection
, RT reactions were carried out using iScript? Reverse Transcription Supermix for RT-qPCR (BIO-RAD). qPCR reactions were carried out using SYBRGreen on a CFX Bio-rad qPCR cycler. To check cdc20b temporal expression by qPCR we directed primers to exons 9/10 junction (Forward: 5?-ggctatgaattggtgcccg-3?) and exons 10/11 junction (Reverse: 5?-gcagggagcagatctggg-3?) to avoid amplification from genomic DNA. The relative expression of cdc20b was normalized to the expression of the housekeeping gene ornithine decarboxylase (ODC) for which primers were as follows: forward: 5?-gccattgtgaagactctctccattc-3?: reverse: 5?-ttcgggtgattccttgccac-3?. To check the efficiency of Mo SPL, expected to cause retention of intron 1 in the mature mRNA of cdc20b we directed forward (5?-cctcccgagagttagagga-3?) and reverse (5?-gcatgttgtactttctgctcca-3?) primers in exon 1 and exon2, respectively. To check the expression of p53 in morphants by qPCR, Xenopus embryos were snap frozen at different stages and stored at ?80°C. Total RNAs were purified with a Qiagen RNeasy kit (Qiagen). Primers were designed using Primer-BLAST Software. PCR reactions were carried out using GoTaq® G2 Flexi DNA Polymerase (Promega)
, Embryos were fixed in 4% paraformaldehyde (PFA) overnight at 4°C and stored in 100% methanol at -20°C. Embryos were rehydrated in PBT and washed in MABX (Maleic Acid Buffer + Triton X100 0,1% v/v). Next, embryos were
, For immunofluorescence, embryos were fixed at RT in PFA 4% in PBS, and incubated in the CDC20B antibody diluted 1/150 in BSA 3% in PBS. For all experiments, secondary antibodies conjugated with Alexa were used. GFP-CAAX in Supplementary Figure 5g was revealed using a rabbit anti-GFP antibody together with a secondary antibody coupled to Alkaline Phosphatase (AP), which was revealed as follows: embryos incubated with the AP-conjugated antibody were washed twice in alkaline phosphatase buffer, BR + 15% Serum + MABX with respective primary and secondary antibodies
, REF:11681460001) until appropriate staining. Finally embryos were washed twice in MABX and fixed in MEMFA 30 min at RT. To mark cortical actin in MCCs, embryos were fixed in 4% paraformaldehyde (PFA) in PBT (PBS + 0.1% Tween v/v) for 1 h at room temperature (RT), washed 3 × 10 min in PBT at RT, then stained with phalloidin-Alexa Fluor 555 (Invitrogen, 1:40 in PBT) for 4 h at RT, and washed 3 × 10 min in PBT at RT. Primary antibodies: mouse anti-Acetylated??-Tubulin (Clone 6-11B-1, :1000), rabbit anti-?-Tubulin (Abcam, Ab 16504, 1:500), mouse anti-?-Tubulin (Clone GTU88, Ab 11316, Abcam, 1:500), Chicken anti-GFP (AVES, GFP-1020, 1:1000), rabbit anti-GFP (Torrey Pines Biolabs, TP401, 1:500), mouse anti-Centrin (Clone 20H5, EMD Millipore, vol.1, p.500
, For single staining, all RNA probes were labeled with digoxigenin. For FISH on section, embryos were fixed in 4% paraformaldehyde (PFA), stored in methanol for at least 4 h at ?20°C, then rehydrated in PBT (PBS + Tween 0.1% v/v), treated with triethanolamine and acetic anhydride, incubated in increasing sucrose concentrations and finally embedded with OCT (VWR Chemicals). 12 ?m-thick cryosections were made. Double FISH on sections was an adaptation of the whole-mount FISH method. 80 ng of cdc20b digoxigenin-labeled sense and antisense riboprobes and 40 ng of antisense ?-tubulin fluorescein-labeled riboprobe 56 were used for hybridization. All probes were generated from linearized plasmids using RNA-labeling mix (Roche). FISH was carried out using Tyramide Signal Amplification -TSA TM Plus Cyanine 3/Fluorescein System, situ hybridization on Xenopus embryos. Whole-mount chromogenic in situ hybridization and whole-mount fluorescent in situ hybridization (FISH) was performed as detailed by Marchal and colleagues 54 , and Castillo-Briceno and Kodjabachian 55 , respectively
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