Abstract
Centrosomes, composed of two centrioles and pericentriolar material, organize mitotic spindles during cell division and template cilia during interphase. The first few divisions during mouse development occur without centrioles, which form around embryonic day (E) 3. However, disruption of centriole biogenesis in Sas-4 null mice leads to embryonic arrest around E9. Centriole loss in Sas-4−/− embryos causes prolonged mitosis and p53-dependent cell death. Studies in vitro discovered a similar USP28-, 53BP1-, and p53-dependent mitotic surveillance pathway that leads to cell cycle arrest. In this study, we show that an analogous pathway is conserved in vivo where 53BP1 and USP28 are upstream of p53 in Sas-4−/− embryos. The data indicates that the pathway is established around E7 of development, four days after the centrioles appear. Our data suggest that the newly formed centrioles gradually mature to participate in mitosis and cilia formation around the beginning of gastrulation, coinciding with the activation of mitotic surveillance pathway upon centriole loss.
Introduction
Centrosomes are major microtubule organizing centers (MTOCs) of animal cells and are composed of two centrioles, one mature mother centriole with distal and sub-distal appendages and one daughter centriole, surrounded by a proteinaceous pericentriolar material (PCM) (Conduit et al, 2015). During mitosis, centrosomes help assemble the mitotic spindle, and during interphase, the mother centriole forms the basal body template for cilia (Bornens, 2012). In proliferating cells, centrioles can form de novo without pre-existing centrioles or use the scaffold of existing centrioles to duplicate once per cell cycle in late G1 and S phases (Loncarek & Khodjakov, 2009). The centriole formation pathway has been defined in cell culture and in different organisms and relies on a set of core proteins that include spindle assembly defective protein 4 (SAS-4, also called CENPJ or CPAP) (Kirkham et al, 2003; Kleylein-Sohn et al, 2007; Leidel & Gonczy, 2003; Tang et al, 2009). The newly formed centrioles undergo maturation over two cell cycles to acquire appendages, become MTOCs and template cilia (Kong et al, 2014). Cilia formation relies on docking of the mother centriole to the plasma membrane through distal appendage proteins, such as CEP164 (Graser et al, 2007; Siller et al, 2017), and on intra-flagellar transport proteins, such as IFT88 (Haycraft et al, 2007).
During rodent development, and unlike the development of most organisms, the first cell divisions post-fertilization occur without centrioles (Courtois et al, 2012; Gueth-Hallonet et al, 1993; Howe & FitzHarris, 2013; Manandhar et al, 1998; Woolley & Fawcett, 1973). In the mouse embryo, centrioles first form by de novo biogenesis starting at the blastocyst stage around embryonic day (E) 3.5 (Courtois & Hiiragi, 2012). Before centriole formation, diffuse γ-tubulin signals, a PCM component and microtubule nucleator, appear at the morula stage around E3, and γ-tubulin signals become more focused as centrioles form; however, the newly formed centrioles do not seem to act as MTOCs in interphase cells (Howe & FitzHarris, 2013). In addition, the first cilia form almost two days post-implantation around E6.5 in cells of the epiblast (Bangs et al, 2015).
Mouse embryonic stem cells (mESCs) are a well-established in vitro model of embryo development that are derived from the pluripotent inner cell mass of blastocysts at E3.5 but molecularly resemble epiblast cells post-implantation (Nichols & Smith, 2011). To maintain uniform pluripotency, mESCs are cultured with leukemia inhibitory factor (LIF) and two other differentiation inhibitors abbreviated as 2i (Williams et al, 1988; Ying et al, 2008). In pluripotency, the transcription factor NANOG is highly expressed in mESCs and regulates self-renewal (Rosner et al, 1990). In this study, we used mESCs to complement our in vivo experiments by studying the growth dynamics of cells without centrioles.
We have previously shown that the genetic removal of SAS-4 in the mouse resulted in the loss of centrioles and cilia (Bazzi & Anderson, 2014a). The Sas-4−/− embryos arrested development around E9.5 due to p53-dependent cell death. The increase in p53 in Sas-4−/− embryos was not due the secondary loss of cilia, DNA damage or chromosome segregation errors. Also, these phenotypes are not specific to Sas-4−/− embryos because mutations in different genes, such as Cep152, that cause centriole loss show similar phenotypes (Bazzi & Anderson, 2014a, b). Notably, the fraction of mitotic cells was higher in Sas-4−/− embryos at E7.5 and E8.5, indicating a longer mitotic duration of cells without centrioles, which was also confirmed by time-lapse imaging of dividing cells. Because a short nocodazole treatment to prolong mitosis upregulated p53 in cultured wild-type (WT) embryos, the data suggested that the less efficient mitosis without centrioles activated a novel p53-dependent pathway (Bazzi & Anderson, 2014a). In cultured mammalian cell lines in vitro, a similar pathway that is activated by the loss of centrioles or prolonging mitosis leads to p53-dependent cell cycle arrest and is called the mitotic surveillance pathway (Lambrus & Holland, 2017; Lambrus et al, 2015; Wong et al, 2015). Recently, p53-binding protein 1 (53BP1) and ubiquitin specific peptidase 28 (USP28) have been shown to be essential for the conduction of this pathway in vitro (Fong et al, 2016; Lambrus et al, 2016; Meitinger et al, 2016). These studies showed that mutations in 53BP1 or USP28 rescued the growth arrest phenotype observed in cells without centrioles. However, whether a similar 53BP1- and USP28-dependent pathway operates in vivo and can cause the p53-dependent cell death phenotype in the mouse are still not known.
In this study, our data showed that the mitotic surveillance pathway is conserved in mice in vivo and that 53BP1 and USP28 are essential for its conduction upstream of p53. In order to explain the late onset of the phenotype upon the loss of centrioles, we also asked when during development this pathway is established. The data indicated that the newly formed centrioles around E3 are not fully mature and do not seem to be required for mitosis until around E7 of development, when the pathway is initiated. Our data suggest that once the cells start to depend on centrosomes as MTOCs in mitosis and ciliogenesis, then they sense the loss of centrioles and activate the p53-dependent mitotic surveillance pathway.
Results and Discussion
Mutations in 53bp1 or Usp28 rescue the Sas-4 mutant phenotype in vivo
To test the conservation of the mitotic surveillance pathway and the involvement of 53BP1 and USP28 in its activation in vivo, 53bp1+/− and Usp28+/− null mouse alleles were generated using CRISPR/Cas9 gene editing (see Methods and Fig. EV1) and crossed to Sas-4+/− mice (Bazzi & Anderson, 2014a). Both Sas-4−/− 53bp1−/− and Sas-4−/− Usp28−/− embryos showed remarkable rescues of the morphology and size compared to the Sas-4−/− embryos at E9.5 (Fig. 1A). Sas-4−/− 53bp1−/− and Sas-4−/− Usp28−/− embryos both underwent body turning, had visible somites and open heads, and were similar to Sas-4−/− p53−/− mutants (Bazzi & Anderson, 2014a). At the molecular level, Sas-4−/− 53bp1−/− and Sas-4−/− Usp28−/− embryos showed highly reduced levels of p53 and cleaved-Caspase 3 (Cl-CASP3) compared to Sas-4−/− embryos (Fig. 1B, C). The data indicated that mutating 53bp1 or Usp28 suppressed both p53 stabilization and p53-dependent cell death upon centriole loss in vivo and established the conservation of the mitotic surveillance pathway in the mouse.
The mitotic surveillance pathway is activated around E7
In order to determine when the mitotic surveillance pathway is activated in Sas-4−/− embryos, we used immunostaining and quantified nuclear p53 levels during development. At E7.5, Sas-4−/− embryos were smaller than control embryos (WT or Sas-4+/−) with around 1.5-fold higher nuclear p53 in the epiblast (Fig. 2A, B) (Bazzi & Anderson, 2014a). Earlier in development at E6.5, Sas-4−/− embryos were morphologically indistinguishable from control embryos, and nuclear p53 was not detectably different (Fig. 2A, B). Ift88 null (cilia mutant) embryos were used as controls for Sas-4−/− centriole mutant embryos, and were similar to WT embryos both morphologically and in terms of p53 nuclear levels at E7.5 and E6.5 (Fig. EV2), confirming our earlier finding that p53 upregulation was due to centriole loss and not the secondary loss of cilia (Bazzi & Anderson, 2014a). The data suggested that the increased level of nuclear p53 in Sas-4−/− embryos starts around E7 of development and is independent of cilia loss per se.
USP28 and 53BP1 are expressed in the epiblast before E6
We next asked whether the upregulation of p53 in Sas-4−/− embryos around E7, and not before, coincided with the onset of expression of either 53BP1 or USP28, the upstream regulators of p53. We performed immunostaining of 53BP1 and USP28 in control and Sas-4−/− embryos at E5.5 and E6.5. Both 53BP1 and USP28 were expressed in WT embryos at E5.5 (Fig. 2C) and E6.5 (Fig. 2D). Of note, USP28 expression was clearly detectable in the embryonic epiblast but not in the surrounding visceral endoderm. Sas-4−/− embryos also expressed both 53BP1 and USP28 at E6.5 (Fig. EV2C). The data indicated that the regulation of the onset of 53BP1 or USP28 expression does not seem to be responsible for p53 upregulation and activation of the mitotic surveillance pathway in Sas-4−/− embryos, suggesting that other mechanisms establish the pathway around E7.
The proper growth of Sas-4−/− mESCs is dependent on p53
To study the dynamics of the mitotic surveillance pathway activation, we derived primary mESCs from WT and Sas-4−/− blastocysts at E3.5. Sas-4−/− primary mESCs were successfully derived and propagated in vitro and lacked detectable centrosomes in interphase cells, as judged by γ-tubulin (TUBG) staining, compared to WT cells, which had centrosomes in every cell (Fig. 3A). TUBG aggregates were seen only at the poles of mitotic cells in Sas-4−/− mESCs, consistent with our findings in Sas-4−/− embryos where these PCM aggregates lacked centrioles (Bazzi & Anderson, 2014a). Both WT and Sas-4−/− primary mESCs showed high levels of nuclear NANOG in media containing LIF and 2i, indicating their pluripotent potential (Fig. 3B). In pluripotent conditions (LIF and 2i), WT and Sas-4−/− primary mESCs had seemingly similar levels of p53, as judged by immunofluorescence (Fig. 3B). Because Sas-4−/− embryos upregulated p53 starting after E6.5 (Fig. 2), we reasoned that Sas-4−/− mESC partial differentiation may trigger a similar response in vitro. Thus, we removed the pluripotency factors (LIF and 2i) for three days, and the pluripotency potential declined as shown by the decrease in NANOG nuclear signal in both WT and Sas-4−/− mESCs (Fig. 3B). The partially differentiated mESCs are not likely to represent a specific lineage because mESCs first move into a transitional state as they exit self-renewal (Martello & Smith, 2014). Importantly, upon partial differentiation, nuclear p53 levels decreased in WT but not in Sas-4−/− mESCs (Fig. 3B). Quantification of the normalized nuclear p53 levels revealed that they were slightly, but significantly, higher in Sas-4−/− mESCs compared to WT mESCs in pluripotent conditions, and this difference appeared more pronounced upon partial differentiation (Fig. 3C). Also, the decrease in p53 in WT mESCs upon partial differentiation was also significant (Fig. 3C).
Although Sas-4−/− mESCs could be derived and propagated in pluripotent condition cultures, we noticed that they grew slower than WT mESCs (Fig. 3D). The growth defect became more obvious upon partial differentiation (Fig. 3D). To check whether the slower growth in Sas-4−/− mESCs was dependent on p53 and the possible activation of the mitotic surveillance pathway, we generated Sas-4−/− p53−/− and p53−/− control mESCs using CRISPR/Cas9 (see Methods and Fig. EV3). The data showed that Sas-4−/− p53−/− completely rescued the growth delay phenotype relative to p53−/− and WT mESCs under pluripotent and partially differentiated conditions (Fig. 3D).
Mitotic surveillance pathway activation is associated with prolonged mitosis in vivo and in vitro
We have previously shown that prometaphase was prolonged in Sas-4−/− embryos at E7.5 and at E8.5 (Bazzi & Anderson, 2014a). To address whether the activation of the mitotic surveillance pathway around E7 coincided with the onset of prolonged mitosis in Sas-4−/− embryos, we performed immunostaining for the mitotic marker phospho-histone H3 (pHH3) at E6.5. We calculated the mitotic index, the percentage of pHH3-positive cells in the epiblast, as an indirect measure of mitotic duration and detected no difference between control and Sas-4−/− embryos at E6.5 (Fig. 4A). In contrast, our previous data showed that the mitotic index of Sas-4−/− embryos at E7.5 was significantly higher than that of control embryos (Fig. 4A) (Bazzi & Anderson, 2014a). The data indicated that the mitotic surveillance pathway activation through p53 upregulation temporally correlates with prolonged mitosis in vivo.
In line with the embryo data in vivo, the mitotic indices of WT and Sas-4−/− mESCs in vitro were similar in pluripotency. However, upon partial differentiation, the mitotic index of Sas-4−/− mESCs was significantly higher than that of WT mESCs (Fig. 4B). These findings indicated that the enhanced activation of the mitotic surveillance pathway in mESCs also correlates with prolonged mitosis upon partial differentiation, and that the growth dynamics of Sas-4−/− mESCs largely resemble those of Sas-4−/− embryos.
Gradual centriole maturation correlates with the establishment of the mitotic surveillance pathway in vivo
We next hypothesized that the centrioles that are first formed by de novo biogenesis around E3 were not fully mature yet and that their maturation correlates with the delayed response to centriole loss in Sas-4−/− embryos around E7. Therefore, we performed immunostaining for TUBG and the distal appendage protein CEP164, as a marker of the more mature mother centrioles, on developing embryos between E3.5 and E6.5. Starting at E3.5, almost all the cells contained centrosomes marked by TUBG foci (Fig. 4C, D). Intriguingly, CEP164 did not localize to these centrosomes at E3.5, supporting our hypothesis that the centrioles were not mature (Fig. 4C, D). At E5.5, around 65% of the centrosomes in the epiblast colocalized with CEP164, and the percentage increased to 85% at E6.5 (Fig. 4C, D). We concluded that the newly formed centrioles in mouse embryos gradually mature to participate in mitosis and cilia formation overlapping with the activation of the mitotic surveillance pathway in Sas-4−/− centriole mutant embryos around E7 (Fig. 5).
Although mitosis is usually the shortest phase of the cell cycle and lasts only around half an hour, it is an essential phase where the segregation of DNA and other cellular components must be precisely accomplished. In addition to the well-studied spindle assembly checkpoint (SAC), mammalian cells have developed a newly discovered pathway to monitor mitosis termed the mitotic surveillance pathway that is independent of the SAC (Lambrus & Holland, 2017). This pathway seems to be limited to mammalian systems because organisms such as Drosophila melanogaster lack 53PB1 and USP28 homologs (Lambrus & Holland, 2017), and zygotic Sas-4 mutant flies survive until adulthood (Basto et al, 2006).
Both control and Sas-4−/− embryos show relatively high nuclear p53 around E6.5, which has been reported in WT embryos at E5.5 and E6.5 (Bowling et al, 2018). It has been suggested that p53 may be involved in cellular competition during this stage of development to eliminate less fit cells before the germline is selected (Zhang et al, 2017). Higher p53 levels in Sas-4−/− embryos around E7 coincide with the window of the initiation of gastrulation as well as the appearance of cilia on epiblast-derived lineages (Bangs et al., 2015). Our data largely exclude the lack of cilia per se (Fig. EV2) or the expression of the mitotic surveillance pathway components (Fig. 2C, D) as determinants of pathway activation after a lag period and at a specific developmental window. In line with this, 53BP1 expression has been reported throughout mouse pre-implantation development (Ziegler-Birling et al, 2009). In addition, USP28 expression was restricted to the epiblast, which may explain why the fast proliferating epiblast cells seem to be more affected by centriole loss compared to the visceral endoderm (Bazzi & Anderson, 2014a). Collectively, our data support a model whereby the newly formed centrioles around E3 gradually mature during development until around E7, when they are competent to participate in cilia formation as well as act as efficient MTOCs during mitosis (Fig. 5). As such, Sas-4−/− centriole mutant embryos may not activate the p53-dependent mitotic surveillance pathway until centrioles are more mature and required for mitosis. This gradual transition is reminiscent of the earlier transition from meiotic- to mitotic-like divisions during pre-implantation and may be a general phenomenon in development including, for example, cilia formation, elongation and function (Bangs et al., 2015; Courtois & Hiiragi, 2012).
Materials and Methods
Animals and genotyping
The Sas-4−/− mice (Cenpjtm1d(EUCOMM)Wtsi/tm1d(EUCOMM)Wtsi) (Bazzi & Anderson, 2014a) and the Ift88−/− null mouse allele generated from the Ift88fl/fl allele (Ift88tm1Bky) (Haycraft et al., 2007) were used in this study. The CRISPR/Cas9 endonuclease-mediated knockouts of 53bp1−/− and Usp28−/− were generated by the CECAD in vivo Research Facility using microinjection or electroporation of the corresponding gRNA, Cas9 mRNA and Cas9 protein into fertilized zygotes (Table 1) (Chu et al, 2016; Troder et al, 2018). The gRNA target sequence predictor tool developed by the Broad Institute was used to design gRNAs (Doench et al, 2016).
The animals were housed and bred under standard conditions in the CECAD animal facility, and the allele generation (84-02.04.2014.A372) and experiments (84-02.05.50.15.039) were approved by the Landesamt für Natur, Umwelt, und Verbraucherschutz Nordrhein-Westfalen (LANUV-NRW) in Germany. All the phenotypes were analyzed in the FVB/NRj background. Genotyping was carried out using standard and published PCR protocols. The PCR products for 53bp1- and Usp28-mutant mice were digested with KpnI and ApoI restriction enzymes (New England BioLabs; Ipswich, MA, USA), respectively, to distinguish the WT and mutant alleles.
Mouse embryonic stem cell culture
Primary mESCs were derived from E3.5 blastocysts as previously described (Bryja et al, 2006), cultured on feeder cells that were proliferation-inactivated with mitomycin C (Sigma Aldrich; St. Louis, MO, USA) and 0.1% gelatin-coated plates (Sigma Aldrich). They were maintained in media containing Knock-Out DMEM (Thermo Fisher Scientific; Waltham, MA, USA), supplemented with 15% Hyclone fetal bovine serum (FBS; VWR; Radnor, PA, USA), 2 mM L-glutamine (Biochrom; Berlin, Germany), 1% penicillin/streptomycin (Biochrom), 0.1 mM MEM non-essential amino acids (Thermo Fisher Scientific), 1 mM sodium pyruvate (Thermo Fisher Scientific), 0.1 mM β-mercaptoethanol (Thermo Fisher Scientific), 1000 U/ml leukemia inhibitory factor (LIF; Merck; Darmstadt, Germany), and with 1 μM PD0325901 (Miltenyi Biotec; Bergisch Gladbach, Germany) and 3 μM CHIR99021 (Miltenyi Biotec), together abbreviated as 2i. Primary mESCs were gradually weaned off feeder cells and maintained in feeder-free conditions. To induce partial differentiation, feeder-free primary mESCs were split and cultured in media without LIF and 2i for three days.
Generating CRISPR-modified primary mESCs
The gRNA sequence for targeting p53 was cloned as double-stranded DNA oligonucleotides into the BbsI restriction site of the pX330-U6-Chimeric_BB-CBh-hSpCas9 vector (Addgene; Watertown, MA, USA) modified with a Puro-T2K-GFP cassette containing puromycin-resistance and eGFP expression by Dr. Leo Kurian’s research group (Center for Molecular Medicine Cologne).
p53−/− and Sas-4−/− p53−/− mESCs (Table 2) were generated by lipofection of the modified pX330 vector containing the gRNA target sequences using Lipofectamine 3000 (Thermo Fisher Scientific). One day after transfection, 2 μg/ml puromycin (Sigma Aldrich) was added to the medium for two days, and the cells were allowed to recover in regular medium up to one week after transfection. Single colonies were picked under a dissecting microscope and were expanded. p53 null cell lines were confirmed with sequencing (primers in Table 2), immunofluorescence, and western blotting.
Growth assay
To determine the growth kinetics of mESCs over three days, WT, Sas-4−/−, p53−/−, and Sas-4−/− p53−/− mESCs were seeded at 105 cells per well of a 6-well plate in media with or without LIF and 2i in triplicate. One set was counted every day for three days using a hemocytometer. Two pairs of each genotype from separate derivations were counted twice and constituted four biological replicates.
Embryo dissection, immunofluorescence and imaging
Pregnant female mice (E3.5 and E9.5) were sacrificed by cervical dislocation for embryo dissections under a dissecting microscope (M165C or M80, Leica Microsystems; Wetzlar, Germany) as previously described (Behringer et al, 2014; Bryja et al., 2006). The embryos were fixed in 4% paraformaldehyde (PFA; Carl Roth; Karlsruhe, Germany) for 2 h at room temperature or overnight at 4°C. Embryos at E3.5 were fixed in 4% PFA for 30 min and in ice cold methanol for 15 min. After fixing, the embryos were washed with phosphate buffered saline (PBS; VWR), and then either used for whole-mount immunostaining or cryoprotected in 30% sucrose overnight at 4°C. The embryos were transferred from 30% sucrose and embedded in optimum cutting temperature (OCT) compound (Sakura Finetek; Alphen an den Rijn, Netherlands) for cryosectioning.
Whole-mount immunofluorescence staining of intact mouse embryos was performed as previously described (Xiao et al, 2018). The embryos were then mounted in 1% low-melting agarose (Lonza; Basel, Switzerland) on a glass bottom dish (Thermo Fisher Scientific), covered in VectaShield mounting medium (Linaris; Dossenheim, Germany), and kept cold and protected from light until imaging. After imaging, the embryos were removed from the agarose, washed and digested for genotyping. Embryos at E3.5 were directly imaged in PBS in a glass bottom dish.
OCT-embedded embryos were cryosectioned at 8 μm thickness and the slides were fixed with ice-cold methanol for 10 min at −20°C, then washed two times with wash buffer containing 0.2% Triton-X in PBS while shaking and blocked with wash buffer with 5% heat-inactivated goat serum for 1 h at room temperature. The slides were incubated with primary antibodies diluted in blocking solution overnight at 4°C. After washing two times with wash buffer, the slides were incubated with secondary antibodies and DAPI in blocking solution for 1 h at room temperature. After washing, the slides were mounted with coverslips using Prolong Gold (Cell Signaling Technology; Danvers, MA, USA).
For immunofluorescence of mESCs, 2×104 cells were seeded per chamber onto pre-gelatinized Lab-Tek II chamber slides (Thermo Fisher Scientific). After three days of culturing in corresponding media, the cells were washed with PBS, fixed with 4% PFA for 10 min at room temperature and washed three times with PBS. Next, the cells were fixed with ice-cold methanol for 10 min at −20°C, permeabilized with 0.5% Triton-X in PBS for 5 min at room temperature, and blocked with 5% heat-inactivated goat serum (Thermo Fisher Scientific) for at least 15 min at room temperature. The cells were incubated with primary antibodies diluted in blocking solution overnight at 4°C. After washing three times with wash buffer, the cells were incubated with secondary antibodies and DAPI diluted 1:1000 in blocking solution for 1 h at room temperature. After washing, the chamber was removed from the glass slide and coverslips were mounted using Prolong Gold anti-fade reagent (Cell Signaling Technology). The images were obtained using an SP8 confocal microscope (Leica Microsystems).
Antibodies
Primary antibodies used in this study and their dilutions and sources are listed in Table 3. The secondary antibodies used were Alexafluor® 488, 568, or 647 conjugates (Life Technologies) and diluted at 1:1000, in combination with DAPI (AppliChem) at 1:1000.
Image analyses
Images of whole E6.5 or E7.5 embryos or mESCs stained with p53 or pHH3 and DAPI were quantified using ImageJ (NIH, Maryland, USA). A maximum projection image of mESCs or the middle five slices of the embryo were generated. The DAPI channel was used to set a threshold to obtain a region of interest. p53 and DAPI fluorescence intensities were measured, and the p53 intensity was normalized to the DAPI intensity. The average p53 intensity of controls was set to 1.0 and a fold-change p53 intensity was calculated. Centrosomes (defined as one TUBG focus or two close TUBG foci) and CEP164 foci were manually quantified using ImageJ (NIH). The number of nuclei were quantified using the image-based tool for counting nuclei (ITCN) ImageJ plug-in.
Western Blotting
mESCs were scraped in radioimmunoprecipitation assay (RIPA) buffer containing 150 mM NaCl, 50 mM Tris pH 7.6, 1% Triton X-100 (Sigma-Aldrich), 0.25% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS; AppliChem; Darmstadt, Germany) with an ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor cocktail (Merck), phosphatase inhibitor cocktail sets II (Merck) and IV (Merck), and phenylmethylsulfonyl fluoride (PMSF; Sigma-Aldrich). Protein concentration was measured with an RC DC protein assay kit (Bio-Rad; Feldkirchen, Germany). 10 μg protein per sample was loaded. SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotting were performed following standard procedures (Kurien and Scofield, 2006; Towbin et al., 1979). Following SDS-PAGE, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Merck) that were activated in methanol (Carl Roth) for 1 min, blocked in 5% milk (Carl Roth) for 1 h, and incubated with an anti-p53 antibody (1:5000; Leica Biosystems; Buffalo Grove, IL, USA) or an anti-GAPDH antibody (1:104; Merck) overnight at 4°C. The membranes were washed with Tris buffered saline containing Tween 20 (AppliChem; TBST) and incubated with secondary anti-rabbit (GE Healthcare; Chicago, IL, US) or anti-mouse (GE Healthcare) antibodies linked with horseradish peroxidase (HRP) at 1:104 for 1 h at room temperature. Finally, the membranes were washed with TBST and incubated with enhanced chemiluminescence (ECL; GE Healthcare; Chicago, IL, USA) and signals were detected with on film (GE Healthcare) in a dark room.
Statistical Analyses
Statistical analyses comparing two groups of data using a two-tailed Student’s t-test with a cutoff for significance of less than 0.05 and graphic representations with standard deviations were performed using GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA).
Author contributions
Conceptualization: C.X. and H.B.; Methodology: C.X., M.G., C.G., M.M., R.F. and H.B.; Software: C.X., M.G., C.G.; Formal Analysis: C.X, M.G., C.G.; Investigation: C.X., M.G., C.G., M.M., R.F. and H.B.; Writing: C.X. and H.B.; Visualization: C.X., M.G., C.G.; Supervision, Project administration and Funding Acquisition: H.B.
Competing Conflict of interest
No competing interests declared.
Expanded View Figure Legends
Acknowledgements
We thank the CECAD in vivo research facility for generating and maintaining our mouse lines and the CECAD imaging facility for microscopy support. The work was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) [BA 5810/1-1 to H.B]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.