extfieldsofstudy: Biology Medicine

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A novel microfluidic 3D platform for culturing pancreatic ductal adenocarcinoma cells: comparison with in vitro cultures and in vivo xenografts

The integration of microfluidics and cell biology has reached a significant milestone with the development of “organ-on-chips”, smart technological platforms that, once applied to the study of human diseases, such as cancer, might ultimately contribute to design personalised treatments and hence improve health outcomes. This paper reports that the combination of microfluidics and dielectrophoresis (DEP) allows to culture different pancreatic ductal adenocarcinoma (PDAC) human cell lines into a cyclic olefin polymer (COP) chamber (HepaChip®), enriched by the extracellular matrix (ECM) protein collagen. We show that PDAC cells cultured into the HepaChip® (1) are vital and grow, provided they properly attach to collagen; (2) show morphological appearance and growth characteristics closer to those of cells grown as spheroids than as classical 2 dimensional (2D) in vitro cultures. Finally, preliminary experiments show that PDAC cells respond to high doses of Cisplatin perfused through the chip. Overall, the present microfluidic platform could be exploited in the future for a personalised approach to PDAC.

as the development of a dormant tumour region 7,8 . Overall, 3D cultures allow to monitor cell growth dynamics and response to treatments more appropriately, and could hence fill the gap between in vitro and in vivo systems for preclinical oncological research. As a result, there has been increasing focus in developing 3D techniques, and many different platforms have been proposed, with different grades of complexity and expression of tumour microenvironmental conditions 5, 9 . A further improvement in this field could derive from the integration of microfluidics and cell biology, which has recently reached a significant milestone with the development of "organ-on-chip" technologies. What began at the turn of the millennium as simple demonstrations of biological cells being transported and manipulated in microchannels for basic short-term analysis, has now advanced to the point where we can engineer living cellular microsystems with controllable microenvironments that behave and function -with organ-level complexity -like their counterparts in vivo [10][11][12][13][14] . Organ-like features include continuous perfusion and physiological cell-matrix and cell-cell interactions. More recently, the "organ on chip" technology has been transferred to study human disease models, including cancer [15][16][17][18][19][20] . As a further step, the combination of microfluidics and dielectrophoresis (DEP) to assemble primary human cells, has enabled the automated in vitro construction of micro-organs, which mimic proper in vivo structures. As a unique feature of organ-on-chip technology, the use of DEP selectively assembles only viable cells 21,22 . For the HepaChip ® organ-specific 3D cell culture chambers are designed and validated by multiphysics simulations and realised by injection moulding of cyclic olefin polymer (COP) 21,23,24 . Proprietary surface functionalization enables selective deposition of ECM proteins in a simple perfusion process 25 . High resolution optical imaging of micro-organs along with the complete set of staining technologies is possible due to the exceptional optical properties of COP.
We applied these concepts to create a novel platform for studying pancreatic ductal adenocarcinoma (PDAC), one of the human cancers with worst prognosis, for which the design of novel therapeutic options is urgently needed. For these reasons, various model systems are being developed, from in vitro 2D and 3D cell cultures, to whole animal models 26 .
We here provide evidence that human PDAC cells can be cultured onto a novel microfluidic chamber, the HepaChip ® , maintaining cell vitality and displaying appropriate morphological appearance, growth characteristics and response to chemotherapeutic drugs.

Materials and Methods
Cell lines. The  and Images were taken using Nikon Eclipse TE300 florescence microscope.
Phalloidin staining. Thin glass cover slips were ethanol and flame sterilised. The cells were seeded on the glass cover slips for 24 h. The unattached cells were washed away with PBS and cells were fixed with 4% methanol-free formaldehyde (Thermo scientific) for 20 minutes at room temperature. Cells were permeabilised with 0.01% Triton-X for 5 minutes at room temperature, washed with PBS. Nonspecific sites were blocked using 10% BSA for 1 h. Then cells were stained using rhodamine conjugated Phalloidin (Sigma), followed by nucleus staining using 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen). Images were captured using confocal microscope (Nikon Eclipse TE2000-U).
Drug Treatment and analysis of drug efficacy. Ten thousand PANC1 cells were seeded per well (96 well plate). After 24 h of culturing, cells were treated with Cisplatin (Sigma) of 0, 1, 2.5,5,10,25,50 and 100 µM concentration. After 24 h of drug treatment, cells were detached using trypsin and counted manually using trypan blue as above.
Three dimensional cell culture (3D). Three dimensional spheroids were generated using liquid overlay technique (U tube technique). Cells were treated with trypsin and counted using trypan blue. Subsequently cells were seeded onto agarose-coated (50 µL, 1.5% agarose/well) 96 well plate in 100 µL of medium 28 . After 72 h, 100 µL medium was added and afterwards, every 48 h, 50% of the supernatant was replaced with fresh medium. Spheroids were cultured at 37 °C in a humidified atmosphere containing 5% CO 2 in air.
Growth Curve and Live/Dead cell imaging. Five hundred cells were seeded on agarose coated 96 well plate. Spheroids were imaged using bright field microscopy (Nikon) every day with a 10x objective. Image resolution was 0.757 µm/pix. Spheroid volume was calculated using SpheroidSizer program as reported by Chen W. et al. 28 .
After 72 h of cell seeding, cells were stained by adding fresh medium containing Calcein AM (2 µg/mL) and PI (10 µg/ mL), incubated for 20 minutes and then washed with PBS. Images were taken using Nikon Elipse TE300 florescence microscope.
Drug Treatment and analysis of drug efficacy. For drug treatment, 1000 PANC1 cells were seeded per well (96well plate). After 72 h, spheroids were treated with Cisplatin of 0, 5,10,20,50 and 100 µM concentration. Untreated spheroids were also cultured in parallel. Spheroids were maintained without changing medium for the whole experiment. After 72 h of drug treatment, 20 to 30 spheroids were collected, washed with PBS, dissociated using 5 mM Ethylenediaminetetraacetic acid (EDTA) and incubated with PI (0.05 µg/mL) for 5 min. Stained cells were analysed using flow cytometer.
Briefly, the device comprises eight identical cell chambers, containing three cell culture regions each, sized 1 mm × 60 µm, i.e. 24 cell culture regions per chip. These culture regions were selectively coated with collagen to facilitate cell adhesion as reported before 25 . In brief, the regions were irradiated through a shadow mask by UV light to induce acid groups on the surface. Collagen was incubated from a 20 µM solution for 90 min and bound to these acid groups. After rinsing the chip was sealed with an adhesive foil (polyolefin cover foil, HJ-Bioanalytik Moenchengladbach). Chips (without cover foil) were manufactured by microfluidic ChipShop, Jena, by micro injection moulding.
On the side walls of each cell chamber electrodes are integrated which are used to generate an inhomogeneous high frequency electrical field. This gives rise to dielectrophoretic forces by which cells entering the cell chamber are drawn into the assembly areas. Only viable cells will be selectively assembled as only they exhibit intact cell membranes with their intracellular permittivity differing from that of the extracellular medium they are suspended in.
The microfluidic devices were mounted in a fixture providing for fluidic, electrical, and thermal contact and control of the chip by an external periphery unit containing Polytetrafluoroethylene (PTFE) tubes and syringe pumps as well as function generator, amplifier and electronic heat control.
Cell assembly and culture in HepaChip ® . Cells were suspended in a DEP medium exhibiting an especially low conductance of 80 to 200 µS/cm. It comprised 95 g/L sucrose, 1 g/L glucose, 57.9 mg/L sodium pyruvate, 28.4 mg/L calcium chloride and 24.6 mg/L MgSO 4 (aq). From this suspension cells were assembled by DEP at a flow rate of 100 µl/min, a peak to peak voltage between 140 and 150 V and a frequency of 350 kHz, for durations of 3 to 5 min.
After cell assembly, culture medium was perfused through the chip for 3 to 9 days at a perfusion rate of 3.125 µl/min. During culture the chips were maintained at temperatures between 35 °C and 37 °C while the medium reservoirs were heated to between 37 °C and 39 °C. To make sure that no gas or air bubbles enter the microfluidic chips, a bubble trap was inserted in the inlet tubing of each chip. In this trap the medium flows underneath a gas permeable PTFE membrane where bubbles can exit but the medium is kept inside the system.
Viability assay in HepaChip ® . Each day the cells in the microfluidic chip were stained with green fluorescent Calcein AM (2 µg/mL) and red fluorescent PI (10 µg/mL) in order to visualise viable cells in green and nuclei of dead or dying cells in red. After incubating with the staining solution for 20 min at 7 µL/min the chip was washed with culture medium before the perfused culture restarted. Images were captured with Nikon DS 2MBWc microscope camera using Nikon Eclipse Ti.
Phalloidin staining in HepaChip ® . The chip was flushed with 4% paraformaldehyde for 20 min at 15 µL/min to fix the cells. Next the cells were permeabilised by pumping phosphate buffered saline (PBS) with 1% triton X through the chip for 10 min at a flow rate of 50 µL/min. Then cells were stained with green fluorescent Phalloidin and DAPI in PBS for 20 min at 15 µL/min and finally washed with PBS at 50 µL/min. The cells in the chips were then imaged as those stained in the viability assay.

In vivo experiments on nu/nu mice. In vivo experiments were performed at Dival Toscana Srl, inside
the Animal Facility of the University of Florence. All experiments on live vertebrates were performed in accordance with relevant guidelines and regulations. We confirm that the Ethical Committee of the University of Firenze approved all the experiments described in this paper. Female, 9 weeks aged, athymic nude nu/nu mice (Envigo-Harlan Italy; Udine) were subcutaneously (s.c.) injected with 3 × 10 6 PANC1 cells per mouse. Mice were treated daily intraperitoneum (i.p.) with Cisplatin (10 mg/kg) (n = 4 masses) or with saline (n = 4 masses) for one week, starting the day the masses reached the measure of (0,2 × 0,2 cm). Tumour growth was monitored daily by measuring two perpendicular diameters; the volume of the tumour masses was calculated using the ellipsoid equation. At the end of the treatment, all mice were sacrificed, and tumour masses were collected measured and weighted.
Statistical analysis. Data are generally given as mean values ± standard error of the mean (SEM), with n indicating the number of independent experiments. When the mean ± standard deviation (SD) was calculated, it is indicated in the figure legend. Statistical comparisons between two groups of data were performed with the Mann-Whitney test.

Results
The aim of the present study was to demonstrate the feasibility of obtaining healthy and reproducible cultures of PDAC cells in a microfluidic chamber, which enables active assembly of cell aggregates and cultivation under continuous and controlled perfusion. For this purpose, we used three different human PDAC cell lines (BxPC3, PANC1 and MiaPaCa2), whose growth characteristics were first analysed in 2D (Fig. 1) and spheroid 3D (Fig. 2) cultures. In 2D culture conditions, all the PDAC cells proliferate roughly at the same extent and with similar kinetics, reaching a plateau at about 3 × 10 5 cells/ml after 72 hours of incubation (Fig. 1A). As evident from phase contrast pictures in Fig. 1B, BxPC3 cells show a flattened morphology, with the tendency to grow as small "islands". MiaPaCa2 cell cultures show two types of cells (1) Adherent cells with a mesenchymal morphology, with round body and more or less long and substrate adherent cytoplasmic extensions and (2) Round, less adherent cells. PANC1 display an intermediate morphology, not fully epithelial, more mesenchymal, but more adherent to the substrate compared to MiaPaCa2 cells. Roughly all the PDAC cells in 2D cultures are vital after 48 hours of incubation, as witnessed by the Calcein/PI staining (Fig. 1C). One of the functional characteristics of cancer is their propensity to migrate, an essential step for further metastatisation. Hence cancer cells are characterised by a peculiar organisation of their actin cytoskeleton which can underlie the formation of lamellipodia 29 . We then chose to determine the state of the actin cytoskeleton, determined by the Phalloidin staining, in the different PDAC cell lines as a functional indication of their pro-migratory propensity, and hence "cancerous" behaviour. Figure 1D shows that all PDAC cells have a cancer-type organisation of their cytoskeleton, with dendritic arrays of actin filaments.
When the cells were cultured as 3D, employing the U tube technique, PANC1 and MiaPaCa2 cells grew to form big spheroids, with a comparable growth rate over the 10 days' culture period. On the other hand, BxPC3 showed very low or null growth rate ( Fig. 2A). As better evident from Fig. 2B, the three different PDAC cells formed spheroids with varying degree of compactness: both PANC1 and MiaPaCa2 cells gave rise to big and loosely compact spheroids. PANC1 spheroids were also characterised by branches of cells coming out from the originary spheroid mass. Moreover, they showed a clear distinction between tightly formed round central dormant and loosely ruffled peripheral cells. On the contrary, BxPC3 formed very small and highly compact spheroids, with only few scattered cells outgrowing from the originary mass, with no distinction between central and peripheral region. Calcein/PI staining (Fig. 2C) showed that the majority of cells in the spheroid were viable after 72 h of incubation. Furthermore, the PDAC cells in the spheroids showed an actin cytoskeleton compactly arranged, with short actin filament distributed around the periphery of cells, especially where cell-cell interaction and aggregation took place (Fig. 2D). The same PDAC cell lines were then cultured in a novel type of microfluidic cell culture chamber, the HepaChip ® . Figure 3A shows the HepaChip ® , with fluid inlet and outlet, microchannels addressing eight cell chambers and electrodes contacting the lateral faces of each cell chamber (Fig. 3B). When energised with a high frequency (typically 100 kHz to 1 MHz) voltage, an inhomogeneous electric field is generated as local field strength is modulated by the assembly ridges positioned between opposite faces of the cell chambers. Under the media conditions chosen (low conductivity medium) cells are attracted from the cell suspension onto the assembly ridges. Figure 3C shows a multiphysics simulation of hydrodynamic and electrical forces acting on cells. Each line represents the calculated trajectory of a cell, part of which are ending on assembly ridges. Simulations as shown here enable assessment and optimisation of assembly efficiency for a given 3D microstructure of the chamber, voltage amplitude, and medium composition 14,30 . A video of PANC1 cell assembly is provided as Supplementary  Information (Supplementary Video S1). Figure 3D and E show typical examples of PANC1 cells on assembly ridges (immediately after assembly and after 146 h of culture under continuous perfusion, respectively).
Viable BxPC3, MiaPaCa2 and PANC1 cells were assembled inside the HepaChip ® using DEP. When they were cultured inside the chip, all three cell types initially adhered to the chip surface. Both BxPC3 (Fig. 4A) and PANC1 (Fig. 4B) cells spread on the microstructure and remained viable for several days. Cells grew inside the HepaChip ® under perfusion with culture medium, as shown by the increase in cell number and density.
We observed mitosis of both BxPC3 and PANC1 cells, as a clear index of their functionality. Figure 4C shows a representative example thereof obtained with BxPC3 cells. On the contrary, MiaPaCa2 cells showed very weak adhesion to the collagen-coated surfaces. This fact could be related to the expression, on the plasma membrane of these cells, of integrin adhesion receptors with lower affinity for collagen (Manoli S., personal communication). While some of them spread on the surface during the first 24 hours, others remained in a spherical shape  Supplementary Information Fig. S1).
Hence we focused our attention to BxPC3 and PANC1 cultures only ( Fig. 4A and B). When comparing BxPC3 and PANC1 cultured within the microfluidic chip, we observed the following behaviours: (1) BxPC3: during DEP BxPC3 cells adhered and spread on the collagen coated ridges until they formed a dense layer. During perfused culture of several days they slowly extended up the ramp and formed a thick rim. This rim grew thicker with culture time rather than further extending into the channel region (Fig. 4A). After 24 and 48 h, we observed mainly adherent, flattened cells, which continuously changed their shape. Furthermore, we observed small spheroids growing on top of the adherent cells. (2) PANC1: during DEP PANC1 cells showed less adhesion to collagen coated areas but tended to slide over the surface before adhering. They were incubated for 10 to 15 min in DEP medium and left to sink onto the micro structured surface before the DEP medium was exchanged for culture medium. After that, PANC1 adhered everywhere in the chip, on collagen coating as well as on pluronic coated ramps of the ridges (Fig. 4B-0), on channel walls and on gold electrodes. They spread on any of these surfaces and grew one on top of others (Fig. 4B-1 to B-6). In order to demonstrate the cell growth inside the 3D microstructure, Fig. 4B-6 was taken in two focal planes: that of the channel (left) as well as that of the collagen coated ridge (right). It shows that PANC1 also formed small aggregates on any of these surfaces after 6 days of cell culture. Overall, PANC1 cells behave inside the chip as a spheroid forming type. Actin stained with Phalloidin extended over the entire cytoplasm of both BxPC3 and PANC1 cells cultivated in the chip. As expected, it appeared denser along the cell membrane ( Fig. 4D and E). PANC1 showed additional short actin filaments that extend from the membrane both into the cell and outside, a sign of the maintenance of their pro-migratory, cancerous features, even when cultured on the chip. The morphological and functional features of the three cell lines cultured as 2D, 3D and inside the chip are summarised in Table 1.
Finally, we tested whether the microfluidic chip could be a useful device for testing drugs in PDAC cells. In particular, we tested the effects of Cisplatin, a widely used drug for many cancer types including PDAC, on PANC1 cells. Cisplatin exerted cytotoxic activity on PANC1, with IC 50 values of 3.25 ± 0.2 μM (n = 2) and 14.6 ± 1.6 μM (n = 2) in 2D and 3D cultures, respectively. In 3D cultures 100 μM Cisplatin caused a decrease in cell viability (roughly 80%) after 72 h of incubation, and caused the spheroids to disaggregate after 72 h of treatment (Fig. 5B). We also tested the effects of Cisplatin in vivo, in immunocompromised mice subcutaneously injected with PANC1 cells, at 10 mg/kg 31 Table 1. Morphological and functional features of PDAC cells cultured in 2D, 3D and inside the microfluidics HepaChip device. * Maximum aggregate thickness in the HepaChip ® culture chamber was in the range of the measured width of the aggregates on the ramps. It is limited by the 3D geometry of the chamber between 40 µm on the assembly ridges and 190 µm inside the channels (see Fig. 3C) if assuming that at maximum aggregates can fill the height between the lid of the chamber and this ramp at the channel bottom. days (Fig. 5C). The effects of Cisplatin on PANC1 cells cultured inside the microfluidic chamber were also analysed (Fig. 5D). After 72 h of perfusion with 25 µM Cisplatin in culture medium, a certain reduction of viability from 95 to 83% in both chips and a slight decrease of cell density were observed. The highest concentration of 100 µM Cisplatin perfused through the cell culture chip started to reduce viability from 92 to 85% after 24 h incubation, reaching 68% after 3 days. At this time an overall reduction of the cell density in the chip was observed as shown in the yellow framed area of Fig. 5D on the right compared to the inset below that shows the same area of this chip before incubation with Cisplatin. Viabilities in the control chips without Cisplatin remained between 90 and 78% over a culture time of 5 days in total. Overall a trend in concentration dependence of Cisplatin effects is evident in the graph in Fig. 5E. The higher Cisplatin concentration needed to obtain reduction in cell vitality in the chip compared to conventional 2D in vitro cultures could be due either to differences in cellular functions (e.g. presence of a structured tridimensional ECM in the chip) or to an unfavourable adsorption of the drug onto the microfluidic device which might then result in a lower effective concentration acting on the cells. To test this latter hypothesis, and taking into account the fact that Cisplatin is relatively hydrophobic, we tested the recovery of a number of compounds covering a wide range with respect to hydrophobicity. Uncoated chips showed a considerable degree of adsorption, so that that the most hydrophobic compounds tested (Bupropion, Amodiaquine) were recovered at below 50% even after 3 hours of perfusion. On the contrary, coated chips identical to those used in this study, exhibited almost complete recovery after only 1 hour of perfusion ( Supplementary Information Fig. S2). Therefore, considering that Cisplatin was applied over a period of 72 h, adsorption to polymer surfaces can be ruled out as the cause for the high doses of the drug needed to affect viability of PANC1 cells.

Discussion
In the present paper we examined the possibility of using a microfluidic platform suitable for studying biomolecular characteristics of PDAC cells. Such platform could be further employed to improve diagnosis and prognosis of PDAC as well as for personalised pharmacological testing, in the future. We chose to study PDAC cells since PDAC is one of the human cancers with worst prognosis, with a 5-year survival rate less than 6%. PDAC malignancy can be traced back to the limited capabilities to diagnose (and treat) the disease in its earliest stages, its propensity to migrate and invade surrounding tissues and hence give rise to local and distant metastases, as well as to its substantial chemo-and radio-resistance. Overall new strategies are urgently needed to impact PDAC treatment and to extend and save PDAC patients' lives. For these purposes, various cancer model systems 26 have been developed, from in vitro 2D and 3D cell cultures to whole animal models.
We here provide evidence that PDAC cells can be cultured onto a novel microfluidic chamber, the HepaChip ® , maintaining cell vitality, morphological appearance and growth characteristics that more closely resemble 3D cultures. Furthermore the actin organisation, taken as a functional marker of a proper pro-migratory, neoplastic phenotype, showed a typical cancer-type assembly. Several cultures of PDAC cells in microfluidic channels have been published that culture one or more cell types entrapped in a hydrogel scaffold containing ECM proteins 26,32 . Although the microfluidic structures used so far, allow microscopic image acquisition similar to 2D in vitro assays 32, 33 , they do not mimic the flow of nutrients as occurs inside blood vessels and around them. Such a flow produces shear forces and pressure, which closely resemble in vivo tissues 34,35 . The microfluidic system (HepaChip ® ) we have developed allows for continuous perfusion of the cell culture. Furthermore, viable cells are actively assembled in a dense layer inside the HepaChip ® culture chamber without entrapping them inside a hydrogel that might slow down the transport of nutrients to and metabolites from the cells. The 3D structure of the HepaChip ® enables the formation of cell aggregates especially in areas of reduced flow between the pillars.
These aggregates are still in direct contact to the medium flow and hence delivered a continuously high concentration of nutrients. Media perfused through the chip was assumed to be saturated by oxygen upon entering the chip as the tubing employed was permeable to oxygen. At a perfusion rate of 3.125 µl/min oxygen was supplied at a rate of 1.2 nmol/min to the chip, by far in line with common oxygen perfusion and pressure inside pancreatic cancers in vivo 36 . This means they can also be exposed to a continuously high dose of drug during toxicity tests.
Indeed, we also tested the suitability of the platform for testing chemotherapeutic drugs. We chose to test Cisplatin as a model drug, since Platinum-based drugs are used in both the neo-adjuvant and the adjuvant settings for PDAC treatment. One major hindrance when testing chemotherapeutic drugs in vivo (both in preclinical models and in patients) is the frequent lack of response applying drug concentrations derived from in vitro data, obtained in standard 2D cultures [37][38][39] . We confirmed these findings showing that the IC 50 s of Cisplatin in PANC1 cells cultured in vitro are around 3 μM in 2D cultures, and only slightly higher, 15 μM, in spheroid 3D cultures. On the contrary, the dose used to obtain complete growth inhibition (10 mg/kg) in vivo is much higher, corresponding to an estimated plasma concentration of 240 μM 40 . When tested on cells cultured onto the chip, Cisplatin decreased PANC1 cell vitality (about 30%) after 72 hours of incubation with high doses (100 μM). This dose is much higher than the IC 50 s determined in in vitro 2D and 3D cultures. Although these are still singular experiments and a dose-response assay was not performed, the necessity of higher doses of a chemotherapeutic drug to decrease cell viability when applied to a chip are consistent with those previously reported 32,33 . Moreover, although we cannot compare the results obtained in the chip with those found in mice, our data suggest that the effects of Cisplatin perfused in the chip are more similar to those exerted in vivo, e.g. in mice (Fig. 5C). This is consistent with data reported in 41 showing that the IC 50 values of five model drugs, tested in a microfluidic chip for in vitro hepatocyte drug toxicity tests, correlate with the IC 50 s determined in vivo. Overall our data suggest that PDAC cells, when cultured in the microfluidic chip under continuous perfusion, withstand incubation with Cisplatin at higher doses compared to classical in vitro cultures. Our data showing no adsorption of the drug to polymer surfaces, lead to exclude drug adsorption as the cause for this chemo-resistance. The proper culture in the chip needs cell adhesion to collagen, that can re-establish the cell-cell and cell-ECM interactions that mimic the real tissues 42 . Hence we hypothesise that when PDAC cells are cultured in the chip behave differently compared to 2D. For example they could form a thin layer of ECM around them under flow conditions which could shield from perfused compounds. Consistently, the ECM is known to sustain tumour chemoresistance 43 , through the triggering of different molecular mechanism, including an up regulation of the Multi Drug Resistance p-glycoprotein 44 . Although we will deepen these points in the future, from the present paper it appears that tumour cell cultures onto the HepaChip ® provide a more realistic and predictive response towards chemotherapeutic agents when compared to 2D cultures of the same cell type.
Overall, we provide a novel microfluidic platform that could be fruitfully transferred to both clinical and pharmaceutical testing, in order to provide biological characteristics as well as responses to drug treatments, to improve a personalised approach to PDAC.


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First complete female mitochondrial genome in four bivalve species genus Donax and their phylogenetic relationships within the Veneroida order

Background Four species of the genus Donax (D. semistriatus, D. trunculus, D. variegatus and D. vittatus) are common on Iberian Peninsula coasts. Nevertheless, despite their economic importance and overexploitation, scarce genetic resources are available. In this work, we newly determined the complete mitochondrial genomes of these four representatives of the family Donacidae, with the aim of contributing to unveil phylogenetic relationships within the Veneroida order, and of developing genetic markers being useful in wedge clam identification and authentication, and aquaculture stock management. Principal findings The complete female mitochondrial genomes of the four species vary in size from 17,044 to 17,365 bp, and encode 13 protein-coding genes (including the atp8 gene), 2 rRNAs and 22 tRNAs, all located on the same strand. A long non-coding region was identified in each of the four Donax species between cob and cox2 genes, presumably corresponding to the Control Region. The Bayesian and Maximum Likelihood phylogenetic analysis of the Veneroida order indicate that all four species of Donax form a single clade as a sister group of other bivalves within the Tellinoidea superfamily. However, although Tellinoidea is actually monophyletic, none of its families are monophyletic. Conclusions Sequencing of complete mitochondrial genomes provides highly valuable information to establish the phylogenetic relationships within the Veneroida order. Furthermore, we provide here significant genetic resources for further research and conservation of this commercially important fishing resource.

Principal findings
The complete female mitochondrial genomes of the four species vary in size from 17,044 to 17,365 bp, and encode 13 protein-coding genes (including the atp8 gene), 2 rRNAs and 22 tRNAs, all located on the same strand. A long non-coding region was identified in each of the four Donax species between cob and cox2 genes, presumably corresponding to the Control Region. The Bayesian and Maximum Likelihood phylogenetic analysis of the Veneroida order indicate that all four species of Donax form a single clade as a sister group of other bivalves within the Tellinoidea superfamily. However, although Tellinoidea is actually monophyletic, none of its families are monophyletic.

Conclusions
Sequencing of complete mitochondrial genomes provides highly valuable information to establish the phylogenetic relationships within the Veneroida order. Furthermore, we provide here significant genetic resources for further research and conservation of this commercially important fishing resource. PLOS

Introduction
Bivalve molluscs of the genus Donax (Donacidae family) are an important constituent of the macrofauna of sandy beaches in temperate, tropical and subtropical zones, being the dominant organisms in this type of environment [1]. In the littoral of Iberian Peninsula, the five European species of Donax live sympatrically in the same beaches [2,3] [4,5,6,7]. Nevertheless, D. venustus is practically non-existent in the Iberian Peninsula as a single individual has been found between the years 2000 and 2006 along the south coast of Portugal [3]. Few species of the genus Donax are commercially exploited, but some are consumed locally or used as fishing bait. D. trunculus is exploited in many countries bordering the Mediterranean Sea and Atlantic Ocean, including Portugal [8,9], Italy [10], France [11], and Spain [12,13]. Only in Iberian Peninsula, the recorded captures since 1999 to 2014 equal 10,156 tons, with a maximum production of 1,042 tons in 2005 followed by an incessant decline reaching only 250 tons in 2014 [14]. Although this data only reflects production since fishermen were obliged to declare their captures [8], the species has been subjected to intense exploitation over the last decades and, currently, some D. trunculus populations seem to be at high long-term risk of extinction [15]. Furthermore, this species constitutes an important shellfish resource due to its high economical value. For instance, in Galicia (northwest of Spain), D. trunculus is a species with a high contribution rate, being the bivalve with greater commercial value (38.52 €/kg in the year 2016) [16] in markets during last years. Due to the similarity in size, shape and colour of the Donax clams in different species, captures of D. trunculus in natural beds may contain other species of the genus with lesser economical value and may be marketed together. However, despite their overexploitation and economic importance, relatively few genetic resources are available for this species [15,17] and the whole genus [18,19].
In order to preserve this important fishing resource, genetic tools should be employed. Molecular genetics has proven highly informative to determine the level of genetic variability, which is an essential feature to consider when defining conservation priorities, as well as to better understand the (recent) evolutionary history of species groups. Within the molecular resources, mitochondrial (mt) genome stands out to be considered a useful tool for population genetic and phylogenetic studies, not only because complete mt genomes are often more informative than single genes, but also because they reveal some genome-level details, such as the rearrangement of genes, which are valuable information for studies of evolutionary relationships among species [20,21,22,23]. Moreover, mitochondrial DNA (mtDNA) is particularly important in helping to differentiate species that are morphologically similar, contributing to the identification and authentication of commercial food species to detect and avoid fraud, to protect consumer rights and to achieve other quality objectives, such as certificate of origin.
Most metazoan mitochondrial genomes are typically closed circular molecules of~16 kb, enconding 37 genes: 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes and two ribosomal RNA (rRNA) genes [24]. In addition, at least one extensive non-coding sequence is present which contain elements that control the initiation of replication and transcription [25]. Mitochondrial genome has several valuable features that make it exclusive, including its small size, high evolutionary rates, limited recombination, relatively conserved gene content and organization, and maternal inheritance [22,26]. Though, an extreme exception to the paradigm of strict maternal inheritance of animal mtDNA (SMI) is found in some bivalve lineages, which possess an unusual system known as doubly uniparental inhertince (DUI) ( [27,28,29] for reviews). Species showing DUI display two different kinds of mitochondrial genomes, i.e. male (M) and female (F) mitogenomes. While females have only the F genome, males are heteroplasmic and possess F and M genomes, which the F type predominating in somatic tissues and the M one in gonads [30,31]. To date, the vast majority of species with DUI which have been reported belong to the orders Mytiloida, Nuculanoida, Unionoida and Veneroida [32], including the wedge clam D. trunculus [33].
In this study, we determine, for the first time, the complete female mitochondrial (mt) genome sequences in four species of Donax from the Iberian Peninsula, and compare them with those of other marine bivalves. In addition, the four newly sequenced mitogenomes, together with the veneroids mt genomes available in GenBank, were used to construct the phylogenetic relationships in the Veneroida order. This work should be of importance not only for better understanding the phylogenetic relationships within the Veneroida order, but also for the development of genetic markers useful in wedge clams aquaculture and restoration effects, as well as for the identification and authentication of commercial species.

Ethics statement
All clams handling was conducted in accordance with the guidelines and regulations established by the University of A Coruña and the local governments. Field sampling did not require specific permissions but was in accordance with general governmental regulations. No endangered or protected species were involved.

Samples collection and DNA extraction
Given that DUI has been described in D. trunculus [33] and we have found evidence for it in D. vittatus and D. semistriatus [34], and since the goal of our work was on female mtDNA, we used somatic cells of female specimens as the only source for mtDNA sequencing. Therefore, each of the four Donax complete mt genomes sequenced here was obtained from a single female specimen in each species, sampled at natural beds. The D. trunculus sample was collected at Corrubedo (A Coruña, northwestern Spain) while the D. semistriatus, D. variegatus and D. vittatus samples came from the Portuguese coast (Table 1). Gender determination was performed on each individual by microscopic examination of gametogenic tissue from the visceral mass, and was based on the presence of eggs or sperm. Specimens were taxonomically identified using Pereira et al. 2012 [18] and Nantón et al. 2015 [19] molecular protocols developed in our laboratory. Voucher specimens and their shells were deposited at the malacology collections of the Museo Nacional de Ciencias Naturales (MNCN), Madrid (Spain) ( Table 1).
Total genomic DNA was extracted from about 40 mg of ethanol-preserved foot muscle tissue of female specimens using DNAeasy Blood and Tissue Kit (Qiagen, Germany) following manufacturer´s instructions with only a minor modification, namely EB (10mM Tris-Cl, pH 8.5) rather than AE (10mM Tris-Cl, 0.5 mM EDTA, pH 9.0) buffer was used to avoid possible interference of EDTA with Nextera enzyme.

Mitogenome assembly and annotation
The mt genomes were reconstructed using 2x1,000,000 reads per species with the MITObim assembler [35]. We performed a first assembly with the -quick option, which resulted in a partial mt genome sequence of about 10,000 bp. In order to get the complete sequence, we extracted the sequence of the COI gene from the previous assembly to be used as starting sequence in MITObim with the -seed option. This yielded sequence of about 17,000 bp whose quality and completeness were assessed on the basis of their average coverage along their whole length, by mapping, in each species, the same 2x1,000,000 reads used in the assembly against the inferred mitogenome sequence. For this purpose, we used the SSAHA2 software [36] with a minimum score of 100. Then we extracted coverage information from these mapping using pysamstats (available at: http://github.com/alimanfoo/pysamstats).
The mt genomes were annotated using the MITOS Web Server [37] applying the invertebrate mitochondrial genetic code and followed by manual validation of the coding regions using the NCBI ORF Finder (https://www.ncbi.nlm.nih.gov/orffinder/). Based on ORF Finder result, the sqn files generated from MITOS were edited and submitted to NCBI. The annotations of PCGs were refined, while the annotations of tRNA genes were kept unchanged. tRNA genes were detected using MITOS, tRNAScan-SE v.2.0 [38] and ARWEN v.1.2 [39]; and secondary structures of tRNAs were inferred using MITOS in default search mode. Mitogenome maps were drawn using GenomeVx online tool [40] followed by manual modification. Repeat sequence patterns in the longest non-coding region (NCR) were checked using the web-based software server Tandem Repeats Finder (http://tandem.bu.edu/trf/trf.html) [41].

Phylogenetic analyses
To investigate the phylogenetic relationships between species of the Veneroida order, we used the 33 mitogenomes currently available in GenBank (last accessed 17 January 2017), in addition to the four newly determined in this work. Lucinella divaricata and Loripes lacteus, belonging to the order Lucinoida, were used as outgroups ( Table 2). Owing to the fact that a lack of the Atpase subunit 8 (atp8) gene has been reported in some bivalve species, we investigated the possibility that its presence might have gone unnoticed in these species by actively searching for atp8 sequence in an annotation with MITOS and aligning with other mitogenomes using Geneious Pro v.4.8.5 [42]. We found the atp8 gene in eight species where previous analyses had concluded the absence of this gene. The alignment of the amino acid sequences for each of the 13 mitochondrial PCGs was performed with the MUSCLE plug-in in Geneious Pro v.4.8.5 [42] with default parameters. We removed poorly aligned regions with Gblocks v.0.91b [43], with options allowing gaps for all positions and 85% of the number of sequences for flanking positions. The 13 separate amino acid sequence alignments were then concatenated into a single large dataset consisting of 2617 sites (S1 File).
Phylogenetic analyses were performed under Maximun Likelihood (ML) using RaxML [44] in a web server (http://embnet.vital-it.ch/raxml-bb/) and Bayesian inference (BI) using MrBayes v3.2.6 [45] and PhyloBayes [46]. The best fit models of amino acid evolution were LG + G + F for cox2, atp6, nad6 and atp8; and LG + G for nad4l. The ML analyses consisted of 1000 bootstrap iterations using the CAT model for each partition. BI analysis consisted of two independent Markov chain Monte Carlo (MCMC) runs, each comprising four linked chains (one cold and three heated; as default settings). They were performed for 1,000,000 generations, sampling every 100 generations to allow adequate time for convergence. The convergence of the two runs was assessed by stopping the analysis when the average standard deviation was below 0.01 (stoprule = yes and stopval = 0.01 in the mcmc command). 1,000,000 generations were enough to reach adequate average standard deviation (<0.01). By default, the first 25% trees were discarded as burn-in. BI analyses were also conducted at the amino-acid level using the CAT + GTR model in PhyloBayes [46]. Two independent MCMC analyses were run in parallel for 4,000 generations. The first 1,000 samples were discarded as burn-in. From the remaining samples, we sampled a tree every 10 cycles to compute a consensus tree. The convergence between the two chains were considered acceptable when the maxdiff parameter was below 0.3 (maxdiff = 0.218586) and the minimum effective size (MES) was >50 (MES = 64).

Genome composition
The mitogenomes of the four Donax species sequenced in this study were circular molecules, as revealed by the MITObim assembly. They are composed of 37 genes: 13 PCGs (including the atp8 gene), two ribosomal RNA genes and 22 transfer RNA genes (Fig 1). Their main structural features are summarized in Table 3 Although gene organization is known to vary extensively, even among species from the same genus [22,48,49], all four complete Donax mt genomes showed the same gene order and they are located on the "+" strand, likewise in Macoma balthica, other member of the Tellinoidea superfamily for which the whole mt genome is available [50]. The only difference was noted in the location of the longest NCR which, in M. balthica, is situated between rrnS and tRNA-Met, whereas in Donax clams it is located between cob and cox2 genes (Fig 1). Therefore, in consistency with the highly rearranged gene order in bivalves, the longest NCR is not conserved at the same position among bivalve mt genomes [51,52].

Protein coding genes
The typical 13 PCGs were identified in the four new mitogenomes analyzed here, including the atp8 gene, which had been reported as missing in several bivalve species [51,53,54,55,56,57,58], but subsequent analysis found its presence in several of them [48,50,52,59,60,61,62]. It was suggested that the short and variable length of this protein, along with its high variation in amino acid composition, might hinder the finding of this gene due to annotation difficulties [22]. However, using the same bioinformatic approach employed in Donax species, we found the atp8 gene in publicly available mitogenome sequences of most Veneroida order species available in the databases (Table 4). Moreover, we found other discrepancies with Gen-Bank annotations.    [63]. However, in Donax species atp6 and atp8 genes are physically separated by 1,917 (D. trunculus)-1,928 bp (D. vittatus). Likewise, these two genes also fail to be adjacent in the mitogenome of other heterodont bivalves, such as Hiatella arctica [59], M. balthica [50] and Meretrix lamarckii [64]. On the contrary, they are adjacent in the Unionidae [65] and Solemydae [66], as well as in basal molluscs like Chaetoderma nitidilum (EF211990) and Katharina tunicata [67]. This suggests that the association of these genes might be an example of an ancestral state that has later been lost in derived bivalves. Total length of the 13 PCGs ranged from 11,718 bp (D. variegatus) to 11,829 bp (D. trunculus), accounting for 68.1-69.2% of its total mt genome length. The longest PCG is nad5, with a size of 1,734 bp (577 aa), whereas nad2, cox1, nad4 and cob exceed 1,000 bp. However, nad3 and nad4l genes are shorter than 400 bp and atp8 gene is the shortest PGC with 126 bp (41 aa). These features are similar to those previously reported in M. balthica [50] and five other species of the Tellinoidea superfamily (Moerella iridescens, Sanguilonaria diphos, Sanguinolaria olivacea, Semele scabra and Solecurtus divaricatus) [51].
The ATN conventional start codon is used in most PCGs (ATG, N = 41; ATA, N = 2; the last codon being classically found in the invertebrate mitochondrial genetic code, particularly in bivalves [50]). However, like most invertebrate mt genomes, Donax mtDNA shows alternative start codons, and some PCGs start with NTG codons (TTG, N = 8; GTG, N = 1). In contrast, the observed stop codons are TAA (N = 32) and TAG (N = 20), and all 13 PCGs of the four mt genomes end in a full termination codon.
Twenty-two discrete nucleotide sequences (ranging from 62 to 69 bp) were predicted to fold into the typical secondary structures of tRNAs (see S2-S5 Figs). The predicted structures of tRNA genes showed cloverleaf shape with four arms in the four species, although some of them exhibited folding differences. Sixteen tRNAs showed a small supplemental stem loop (four in D. semistriatus: tRNA-Pro, tRNA-Phe, tRNA-Ile and tRNA-Leu2; two in D. trunculus: tRNA-Ile and tRNA-Thr; six in D. variegatus: tRNA-Val, tRNA-Pro, tRNA-Gln, tRNA-His, tRNA-Ile and tRNA-Arg; and four in D. vittatus: tRNA-Pro, tRNA-Phe, tRNA-Ile and tRNA-Leu2). Seven tRNAs showed no terminal TCC loop (three in D. semistriatus: tRNA-His, tRNA-Thr and tRNA-Arg; one in D. trunculus: tRNA-Asn; and three in D. vittatus: tRNA-His, tRNA-Thr and tRNA-Asp). In addition, tRNA-Ser2 in D. trunculus showed the dihydrouracil (DHU) stem replaced by a big DHU loop. Finally, the single unpaired nucleotide, which is usually present at the 5´end in other tRNAs, appeared at the 3´end in tRNA-Tyr, with the only exception of D. variegatus where this tRNA lacks this unpaired nucleotide. These features have previously been found in mtDNAs of other bivalve species, such as M. balthica [50] and M. lamarckii [64].

Non-coding regions
As in most bivalves, the four species of the genus Donax analyzed here contained a large number of NCRs. The number of intergenic sequences varied from 17 (D. trunculus and D. vittatus) to 22 (D. variegatus), with 1,679 bp (representing 9.9% of the whole mitogenome) in D. semistriatus to 1,985 bp (11.4% of the mt genome) in D. trunculus ( Table 5). The longest NCR was First complete female mitogenome in four Donax species located between cob and cox2 genes in the four species, with length ranging from 1,549 bp (D. semistriatus) to 1,863 bp (D. trunculus). The other NCRs ranged fom 1 to 21 bp. The longest NCR is thought to contain the Control Region (CR) because it presents some peculiar patterns, such as AT-rich or tandem repeats, believed to play a role in initiating and/or regulating mitochondrial transcription and replication [24,68,69]. The A+T content of the longest NCR in each mt genome was higher (D. semistriatus, D. variegatus and D. vittatus) or slightly lower (D. trunculus) than that of the whole mt genome (Table 5).
Six tandem repeats were also found in the longest NCRs of the four mt genomes, four of which were distinct tandem repeat units.  [51,55,59,62]. The study of tandem repeats in the CR is important for the light it sheds on a variety of processes, including the molecular mechanisms arising them and their possible functional implications [70].

Phylogenetic analysis in Veneroida
To further study the relationships among Donax species and its position within the Veneroida order, ML and BI trees based on amino acid sequences of 13 concatenated PCGs belonging to 37 species were performed (Fig 2). Tree topologies were congruent and received high support in most nodes, with the exception of S. scabra, which showed a less basal position in the Phylo-Bayes phylogeny ((M. balthica + M. iridescens) + S. scabra) with 0.57 posterior probability as branch support.
We perform here the first phylogeny including the species of the genus Donax from the Iberian Peninsula (D. trunculus, D. semistriatus, D. variegatus and D.vittatus). Our analysis has shown that the four species form a single clade as a sister group to other bivalves of the superfamily Tellinoidea. All ten species of this superfamily belong to five different families and form a strongly supported clade, thus corroborating the monophyly of this superfamily [71,72]. Nevertheless, our phylogenetic tree indicated, with high support by BI and ML, that S. diphos (Psammobiidae) shows closer relationship with S. divaricatus (Solecurtidae), M. balthica and M. iridescens (Tellinidae), S. scabra (Semelidae) and Donax species (Donacidae) rather than with N. olivacea (Psammobiidae), which implies that these two species (S. diphos and N. olivacea) do not form monophyletic groups. This result is also reported by Yuan et al. 2012 [51] and Ozawa et al. 2017 [73], and it is in agreement with the conclusion put forward by Taylor et al. 2007 [71] when analysed familial relationships within Tellinoidea, as Semelidae, Donacidae and Tellinidae do not form monophyletic groups. Tellinoidea is actually monophyletic, but none of its families are monophyletic [72], suggesting the need for a more exhaustive study within this commercially important marine bivalve clade. Gene arrangement within mitogenomes is highly conserved in many taxonomic groups. For instance, most vertebrates share the same gene order [74]. However, in other animal groups, like the class Bivalvia, the mitochondrial genome arrangement is more variable [51,52]. We compare here the gene arrangements of four newly sequenced mitogenomes to other closed related species belonging to Tellinoidea superfamily. This comparison was previously done by by , without taking into account the atp8 gene and without including Donax species and M. balthica, and their results supported the conclusion that comparisons of mitochondrial gene order rearrangements are, to some extent, a useful tool for phylogenetic studies. Seven out of the ten Tellinoidea mitogenomes hitherto analyzed (including the four Donax species analyzed by us, M. balthica, M. iridescens and S. divaricatus) show completely identical gene order, and S. diphos only differs in lacking a tRNA-Phe. Remarkably, the atp8 gene shows the same location within the mitogenome of these eight species of the Tellinoidea superfamily, specifically between tRNA-Met and tRNA-Ser1. This result is consistent with the main phylogenetic conclusions from the 37 mitogenomes analyzed here (see above), and remarks the interest of performing additional full mitogenome sequencing, especially including more veneroid families and subfamilies, with gene order being a useful hallmark helping to clarify phylogenetic relationships within the order.

Future implications
This is a basic research work where we describe and characterize, for the first time, the female mitochondrial genome in four bivalve molluscs belonging to the genus Donax. This has provided new interesting information for the scientific community which can be feasible for First complete female mitogenome in four Donax species application in aquaculture. In fact, the mtDNA sequences contributed here add significantly useful genetic markers for i) helping to differentiate these commercial food species being morphologically similar, ii) detecting and avoiding fraud, iii) protecting consumer rights and achieving other quality objectives, such as certificate of origin, and iv) for using in population genetics studies and aquaculture stock management in Donax species. However, this possible applicability requires a broader work, where the different markers will be tested in a higher number of individuals, not only fresh individuals but also processed, packaged or frozen ones, as well as in a high number of females and males given that male genomes are still not available.

Conclusions
In this study, we determined the complete mt genomes of four bivalve species of the genus Donax, which are the first representatives from the family Donacidae being analyzed at this respect. Not only we have increased the number of complete mt genomes sequenced within Veneroida order, but also, we have illustrated the phylogenetic relationships among Donax species and their position within this order. Our results demonstrate that the sequencing of complete mitogenomes provides highly valuable information for phylogenetic analysis in bivalves. Furthermore, the mtDNA sequences contributed here add significantly useful genetic markers for use in species identification and authentication, phylogeny, population genetics, and aquaculture stock management in species of Donax.


extfieldsofstudy: Chemistry Medicine

text:
Phosphorylation of STAT proteins by recombinant human IL-6 in immortalized human chondrocyte cell lines, T/C28a2 and C28/I2

Two immortalized human juvenile chondrocyte cell lines, T/C28a2 and C28/I2, were employed to determine the extent to which recombinant human (rh) IL-6, a known cytokine activator of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway in many cell types, caused STAT proteins to be phosphorylated. The results showed that STAT3 was constitutively phosphorylated in the absence of rhIL-6 in T/C28a2 chondrocytes. However, C28/I2 chondrocytes treated with rhIL-6 caused STAT1, STAT3, and STAT5 to be phosphorylated without altering total unphosphorylated STAT proteins. STAT3 phosphorylation in response to rhIL-6 in T/C28a and C28/I2 chondrocytes was efficiently blocked by the JAK3-selective inhibitor WHI-P131 (Janex-1) and by soluble IL-6 receptor-α (sIL-6R). However, the combination of rhIL-6 and ruxolitinib, a JAK1/JAK2-selective inhibitor, was a less effective inhibitor of STAT protein activation. These findings showed that rhIL-6 activated STAT proteins in the C28/I2 chondrocyte cell line. STAT protein phosphorylation could be blocked by a JAK3-selective inhibitor or by the combination of rhIL-6 and sIL-6R.

Introduction
Several proinflammatory cytokines, including IL-6, activate the Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway where phosphorylation of STAT proteins is critical for maintaining normal cellular homeostasis. Seven STAT proteins, STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6, have been identified and localized to the cytoplasm, 1 with STAT1, STAT3, and STAT5 proteins being the most prominent in various musculoskeletal tissues.
STAT proteins contain Src homology-2 regions which are the sites for STAT protein phosphorylation by cytokines or growth factors. 2 In the first step in this cascade, one of several JAK isoforms, JAK1, JAK2, JAK3, JAK5A, JAK5B, or JAK6, is phosphorylated (ie, activated) following the binding of cytokine(s) and/or growth factor(s) to their specific receptors. [3][4][5] After this step is completed, activated JAK phosphorylates U-STAT proteins. Once STAT proteins are phosphorylated, they form p-STAT homodimers and/or p-STAT heterodimers. These STAT dimers undergo nuclear translocation, with p-STAT proteins acting as potent transcription factors by interacting with gene promoter regions containing STAT-responsive DNA motifs. 6 The repertoire of human chondrocyte STAT proteins undergoing phosphorylation in response to rhIL-6 or other proinflammatory cytokines has not been extensively studied. Therefore, to achieve this objective, the extent to which rhIL-6 activated STAT1, STAT3, and STAT5 was assessed in two cell lines of immortalized human juvenile chondrocytes. The results of this analysis with rhIL-6 were compared to STAT proteins which were phosphorylated in response to another proinflammatory cytokine, rhTNF-α.
We employed the immortalized human juvenile T/C28a2 and C28/I2 chondrocyte lines for these analyses. These immortalized human chondrocyte lines have proven to be useful cell culture models for human articular chondrocyte responses to proinflammatory cytokines. 7 Furthermore and most importantly, these chondrocyte lines were also previously shown to express cartilage-specific extracellular matrix protein genes 7,8 as well as several other molecular characteristics of authentic human chondrocytes, most notably the molecular signature SOX9 gene, considered to be the "master" transcriptional regulator of several cartilage-specific genes, including type II collagen (COL2A1) and aggrecan (AGRN). [7][8][9] Recently, JAK1, JAK2, and JAK3 have been identified as targets for pharmacologic intervention in rheumatoid arthritis (RA) primarily because several of the cytokines, growth factors, and other soluble mediators of inflammation known to activate these JAK isoforms were significantly increased in the sera and/or synovial fluid of RA patients. 10 On this basis, the proinflammatory cytokines that were considered to be most relevant to RA and that also activate the JAK/ STAT pathway included INF-γ, IL-7, IL-17, and IL-23, 11 but most prominently, members of the IL-6 cytokine family, IL-6, OSM, ciliary neutrotrophic factor, leukemia inhibitory factor, and cardiotrophin-1. 12 STAT1 and STAT3 were reported to be preferentially phosphorylated by the classical or "canonical" pathways involving IL-6/IL-6Rα/gp130-receptor-mediated signaling. 12 However, additional evidence also indicated that activation of STAT1 by IL-6 could occur via membrane-bound IL-6R (mIL-6R) as well as through the IL-6 trans-signaling pathway involving soluble IL-6R (sIL-6R). The analysis of STAT5 phosphorylation in response to IL-6 has generally been limited to that involved with T-cell activation with little or no focus on human articular chondrocytes. For example, with respect to cells of the immune system, Tormo et al 13 showed that the treatment of CD4 + and CD8 + T cells as well as natural killer cells with rhIL-6 in vitro caused activation of STAT5.
In the present study, we also probed the extent to which neutralizing the interaction between IL-6 and the soluble IL-6 receptor-α (sIL-6Rα) 14 or with JAK-selective small-molecule inhibitors (SMIs) altered STAT protein phosphorylation in response to rhIL-6 or rhTNF-α.

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Human chondrocyte cell line STAT proteins (FBS). For the measurement of total U-STAT and p-STAT proteins, T/C28a2 or C28/I2 chondrocytes were subcultured into 60 mm culture dishes containing 3 mL of DMEM-F12-10% FBS (v/v) at an initial cell density of 3×10 5 cells. The culture medium was changed every 2-3 days until the cells became confluent, generally within 3-5 days after subpassage. At that time, the medium was aspirated, chondrocytes washed once with phosphate-buffered saline (PBS), and DMEM-F12 supplemented with 0.1% FBS (v/v) added to each culture dish.
The 30-and 60-min time points were employed for the Western blot analyses of U-STAT and p-STAT proteins based on our previously published Western blot results, 17 which showed that p-STAT3 protein accumulated in human chondrocyte protein lysates in response to rhTNF-α for up to 30 min after which, at 60 min, the anti-p-STAT3 antibody reactive band was undetectable on Western blots.
Thus, at baseline (0 min) and after 30 and 60 min, the culture medium was aspirated; cells were washed once with ice-cold PBS, and then scraped into fresh ice-cold PBS or ice-cold PBS containing a protease/phosphatase inhibitor "cocktail". The cell pellet was collected by centrifugation and a cell lysate produced by sonication of the cell pellets. Cell lysates were stored at −80°C until used for the various assays.

Western blots
Western blots were produced essentially as previously described 17 with one essential modification, which was required for quantifying the Western blots. Thus, for this purpose, we employed a sequential antibody-probing/ antibody-stripping technique to localize either U-STAT or p-STAT proteins on the Western blots after which these Western blots were restripped and blotted with the β-actin antibody. The various antibody-reactive bands corresponding to the migration rate (M r ) of authentic U-STAT or p-STAT proteins were identified. These antibody-reactive bands were assigned a relative molecular size consistent with the M r of standard proteins on SDS-PAGE.
However, for the initial qualitative assessment of STAT3 activation, the protein band reactive with the anti-U-STAT3 or anti-p-STAT3 antibody was identified by their respective M r . Then the ratio of p-STAT3 to U-STAT3 was used to determine the extent to which U-STAT3 was phosphorylated in response to the various incubation conditions. For all of the subsequent quantitative assessments of total U-STAT1, U-STAT3, or U-STAT5, and STAT1, STAT3, or STAT5 phosphorylation, anti-U-STAT antibody or P-STAT antibody-reactive bands were identified on Western blots. The average signal intensity of U-STAT and p-STAT proteins, as well as their associated anti-β-actin antibody signal intensity, was determined from computer-scanned images of autoradiographs using Metamorph software (MetaMorph Inc., Nashville, TN, USA). The average intensity values from each β-actin Western blot were normalized to the highest value measured in control and treatment groups. These values were then multiplied by the relative intensity values obtained for antibody-reactive bands in their respective Western blot (ie, blots probed with U-STAT or p-STAT antibodies) in order to normalize each antibody-reactive band to its corresponding antibody-reactive band probed with the anti-β-actin antibody. Finally, each of these calculated values were normalized to the experimental treatment group (ie, generally normalized to either lane 1 or lane 2 of each Western blot) to obtain the relative intensity value. Bar graphs were produced using Excel.
A "no additions" control group was employed for comparing all of the other incubation conditions except those treatment groups which contained Janex-1 or ruxolitinib. For those treatment groups, a control group containing dimethyl sulfoxide (DMSO; ie, generally lane 2 of the Western blots) was employed for comparison purposes because Janex-1 or ruxolitinib was initially dissolved in DMSO, and a Janex-1 or ruxolitinib stock solution was produced after diluting the stock solution 1:1 with DMEM-F12 (1:1). Thus, treatment groups containing Janex-1 or ruxolitinib had the same concentration of DMSO as did the corresponding DMSO control group.

Data analysis
The relative differences in antibody-reactive band intensities between control and various treatment groups on individual Western blots were expressed as ±Δ%. The significance of these single-point values were analyzed by χ 2 analysis, with the expected difference between control and treated groups set at Δ = ±20%, where p<0.05 was considered significant.

Results
Treatment with rhIL-6 results in the phosphorylation of STAT3 by T/C28a2 and C28/I2 human chondrocytes Confluent cultures of T/C28a or C28/I2 chondrocytes were treated with "no additions" plus DMSO, rhIL-6 (50 ng/mL), or rhIL-6 plus Janex-1 (100 μM) and held for ~10 min at 25° ("no incubation") or incubated at 37°C for 30 min with either "no additions" or "no additions" plus DMSO rhIL-6 (50 ng/mL), or rhIL-6 plus sIL-6R (30 ng/mL), or rhIL-6 plus Janex-1 (100 μM). The Western blot, as can be seen in Figure  1, showed that STAT3 was constitutively phosphorylated in T/C28a2 chondrocytes in both the "no additions" control group and the "no additions" plus DMSO control group ( Figure 1A). However, incubation of T/C28a2 chondrocytes with rhIL-6 at 37°C for 30 min increased the intensity of the anti-p-STAT3 antibody-reactive band without altering total U-STAT3 ( Figure 1A). Importantly, the p-STAT3 band was not detected in C28/I2 chondrocytes in the "no additions" or "no additions" plus DMSO group under the "no incubation" condition or after 30 min at 37°C (Figure 1B), indicating that STAT3 was not constitutively phosphorylated in the C28/I2 chondrocyte line. Of note, as with the T/C28a2 chondrocyte line, the incubation of C28/I2 chondrocytes with rhIL-6 increased the signal intensity of p-STAT3 without altering total U-STAT3 ( Figure 1B). The combination of rhIL-6 and Janex-1 resulted in a complete loss of the anti-p-STAT3 antibody-reactive band when compared to the "no additions" plus DMSO control group and, in addition, without altering the signal intensity of the anti-U-STAT3 antibody-reactive band in both T/C28a2 ( Figure 1A) and C28/I2 chondrocytes ( Figure 1B). The appearance of U-STAT3 antibody-reactive bands in all of the lanes of this Western blot provided a good indication that each lane was loaded with the same amount of protein lysate. With this in mind, the result of this experiment provided ample evidence that Janex-1 or the combination of rhIL-6 and sIL-6R dampened the phosphorylation of STAT3 in both of these immortalized human chondrocyte lines ( Figure 1A and B).
We also determined whether or not JAK1/JAK2 activation in response to rhTNF-α or rhIL-6 contributed to STAT3 phosphorylation by C28/I2 chondrocytes. However, for this analysis, we employed a quantitative Western blot analysis to determine the effect of the JAK1/JAK2-selective SMI, ruxolitinib, on STAT3 activation (Figure 2). In general, none of the incubation conditions altered the ratio of total U-STAT3  When the quantitative Western blot results of STAT3 in Figure 2 were considered in conjunction with the results shown in Figure 1, we saw that the combination of rhIL-6 plus sIL-6R reduced the ratio of p-STAT3 to β-actin ( Figure 2). This result, in turn, supported the results of our initial qualitative analysis on the effect of sIL-6R on rhIL-6-induced p-STAT3 ( Figure 1).
Next, we examined the profile of STAT1 and STAT5 proteins produced by C28/I2 chondrocytes. The Western blot for this analysis is shown in Figure 3. With respect to STAT1 and p-STAT1, 3 bands were identified that reacted with the STAT1 or p-STAT1 antibody. One band, identified as STAT1A (Figure 3B) or p-STAT1A ( Figure 3B), comigrated with authentic STAT1A, whereas the bands provisionally labeled STAT1B, STAT1C ( Figure 3B) or p-STAT1B, p-STAT1C ( Figure 3D) migrated faster than STAT1A/p-STAT1A. Thus, the ratio of STAT1A or p-STAT1A to β-actin was only performed on these immunoreactive bands (Figure 3).
A single anti-STAT5 antibody-reactive band (arrow) which migrated with authentic STAT5A was also identified by its reactivity with the anti-STAT5 antibody ( Figure 3E and G). Similar patterns of STAT1 ( Figure 3A and C) and STAT5 ( Figure 3E and G) were identified at 30 and 60 min.
To determine whether or not the faster migrating anti-STAT antibody-reactive bands resulted from STAT protein degradation, C28/I2 chondrocyte protein lysates were prepared in PBS containing a protease/phosphatase inhibitor "cocktail" used at the concentration recommended by the manufacturer. The results from Western blots employing this alternative protein lysate preparatory method indicated that the faster migrating anti-STAT antibody-reactive protein bands remained detectable, thus making it less likely that the faster migrating anti-STAT antibody-reactive bands were products of STAT protein degradation (data not shown).

Phosphorylation of STAT1 and STAT5 proteins by rhIL-6 or rhTNF-α
We then examined the extent to which the U-STAT1 and U-STAT5 proteins identified in these Western blots were phosphorylated in response to rhIL-6 or rhTNF-α at 30 and 60 min (Figure 3). Three forms of p-STAT1 corresponding to p-STAT1A (arrow), p-STAT1B (arrow), and most likely to β-actin, with the one exception of C28/I2 chondrocytes treated with rhTNF-α (20 ng/mL) for 30 min (Figure 2A,  lane 7) where a statistically significant reduction in U-STAT3 was found (χ 2 =26.0; p<0.05). However, rhTNF-α had no effect on the ratio of p-STAT3 to β-actin (χ 2 =0.85; p=0.36) ( Figure 2B, lane 7), nor did the combination of rhTNF-α p-STAT1C (arrow) (Figure 3B and D) were detected. Two forms of p-STAT5 corresponding to p-STAT5A (arrow) and p-STAT5B (arrow) were identified at 30 min ( Figure 3F), but only p-STAT5A was detected at 60 min ( Figure 3H). We assigned the detectable p-STAT5 band at this time point to be p-STAT5A because this STAT5A/5B antibody-reactive band reflected the intensity of the anti-p-STAT5A/B antibody band migrating at ~90 kDa in all of the Western blots, whereas the p-STAT5B band was expected to migrate to ~70 kDa. However, at 60 min, p-STAT5A was detected in only 1 of the DMSO control groups ( Figure 3H, lane 3) but more importantly in the rhIL-6 treatment group ( Figure 3H, lane 4).   Ruxolitinib did not alter total U-STAT1 or inhibit phosphorylation of STAT1 (p-STAT1) in response to rhIL-6 or rhTNF-α As shown in Figure 3, ruxolitinib employed at either 50 or 100 μM did not alter total U-STAT1A, U-STAT1B, or U-STAT5A. In addition, ruxolitinib combined with rhIL-6 or rhTNF-α also generally failed to inhibit p-STAT1A, p-STAT1B, or p-STAT1C at either 30 or 60 min ( Figure 3B, D, and F, lanes 5, 6, and 8).

Discussion
The principal objective of this study was to determine the extent to which rhIL-6 activated the JAK/STAT pathway in the T/C28a2 and C28/I2 human immortalized chondrocyte cell lines. Although the IL-6/IL-6Rα/gp130 complex is the principal JAK/STAT signaling mechanism activated by IL-6, 1 the findings in the present study also indicated that rhIL-6 in combination with sIL-6R activated STAT3. This suggested that the "noncanonical" IL-6 trans-signaling pathway was likely to be active in the C28/I2 chondrocyte response to rhIL-6, as we previously reported for rhIL-6-mediated phosphorylation of STAT1 in the T/C28a2 chondrocyte line. 18 Thus, our initial experiments showed that STAT3 was constitutively phosphorylated in the T/C28a2 line of immortalized human chondrocytes. This preliminary finding prompted us to employ the C28/I2 chondrocyte line for most of the remainder of these studies, which examined the extent to which rhIL-6 or rhIL-6 plus sIL-6R activated STAT1, STAT3, and STAT5 proteins. It may be a critical issue going forward to more precisely determine the extent to which alternative IL-6 pathways, ie, induction by either IL-6 trans-signaling or mIL-6R, absent gp130, are differentially activated by rhIL-6. Although tocilizumab is a potent inhibitor of IL-6-mediated JAK/STAT activation, 14 it is unlikely that tocilizumab would be a suitable inhibitor for this purpose since it was reportedly unable to selectively inhibit the IL-6/IL-6Rα/gp130 pathway. 19 To summarize the results of these Western blots, we showed that rhIL-6 or rhIL-6 plus sIL-6R activated STAT3, which was inhibited by the JAK3-selective SMI, Janex-1. However, the results of the quantitative Western blot analyses of C28/I2 chondrocytes from the various treatment groups treated with the JAK1-/JAK2-SMI, ruxolitinib, were less consistent. Thus, based on these results, only a weak case can be made at this juncture for the involvement of JAK1 and/or JAK2 in rhIL-6-mediated changes in STAT activation in these human chondrocyte lines. Additionally, we have also come to recognize that the concentrations of ruxolitinib which were employed in these studies were higher than the reported IC 50 for ruxolitinib, with these IC 50 values having been calculated from an analysis of the ruxolitinib-inhibitory effect on JAK1/ JAK2 in other cell types. 16 What was most intriguing based on the Western blot analyses shown in Figure 3 was that the anti-STAT5A and anti-p-STAT5B antibody-reactive bands in the rhIL-6 plus ruxolitinib-treatment group as well as in the rhTNFα-treatment group were undetectable at 60 min, despite ample evidence of equal gel loading based on the results in the anti-U-STAT5 antibody and anti-β-actin-antibody lanes. However, we did detect an anti-p-STAT5A antibodyreactive band in one of the DMSO control groups as well as an even stronger anti-p-STAT5A antibody-reactive band in the rhIL-6-treatment group at 60 min. Although the overall significance of these findings remains to be completely elucidated, it appears for the present that STAT5A continued to be phosphorylated after 60 min in the DMSO control group, but also most impressively in the presence of rhIL-6.
It should be pointed out that the results of another study indicated that DMSO employed at a concentration in DMEM-F12 (1:1) plus 0.5% FBS used in the present study increased the frequency of 4,6-diamidine-2-phenylindole dihydrochloride/Terminal deoxynucleotidyl transferase dUTP nick end labeling (DAPI/TUNEL)-positive C28/I2 chondrocytes, as a measure of apoptosis, compared to C28/ I2 chondrocytes maintained in either DMEM-F12 (1:1) plus 0.5% FBS or DMEM-F12 (1:1) plus 10% FBS. 20 However, no difference was found in the frequency of DAPI/TUNELpositive chondrocytes when medium containing 0.5% FBS was compared to medium containing 10% FBS. Thus, it must be considered that DMSO has a weak cytotoxic effect on C28/ I2 chondrocytes, but this does not help to explain the effect of DMSO on phosphorylation of STAT5 at 60 min ( Figure 3H).
Finally, these human chondrocyte cell lines, especially, C28/I2, which were employed in this study, appear to be very useful for determining chondrocyte responsiveness to rhIL-6 and rhTNF-α, as has been previously shown to be the case for The Journal of Inflammation Research is an international, peer-reviewed open access journal that welcomes laboratory and clinical findings on the molecular basis, cell biology and pharmacology of inflammation including original research, reviews, symposium reports, hypothesis formation and commentaries on: acute/chronic inflammation; mediators of inflammation; cellular processes; molecular mechanisms; pharmacology and novel anti-inflammatory drugs; clinical conditions involving inflammation. The manuscript management system is completely online and includes a very quick and fair peer-review system. Visit http://www.dove press.com/testimonials.php to read real quotes from published authors.

Meszaros and Malemud
IL-1β. 7 This is particularly germane for studying human chondrocyte homeostasis in health and disease because these human chondrocyte lines can be continuously maintained in highdensity culture without losing their chondrogenic phenotype.


s2fieldsofstudy: Biology Medicine

text:
Effect of the p53–tristetraprolin–stathmin-1 pathway on trophoblasts at maternal–fetal interface

Problem To reveal the effect of p53–tristetraprolin–stathmin-1 signaling on trophoblasts and recurrent spontaneous abortion (RSA). Method of study Stathmin-1 (STMN1), p53, and tristetraprolin (TTP) expression in paraffin-embedded villus tissue was determined using immunohistochemistry. HTR-8/SVneo cells were treated with doxorubicin to activate p53; STMN1 and TTP levels were detected by quantitative reverse transcription–PCR and western blotting. Western blotting and immunofluorescence were used to investigate STMN1 expression after TTP overexpression or knockdown in HTR-8 cells. Results STMN1 was downregulated and p53 was upregulated in the villus tissue from patients with RSA. Doxorubicin decreased STMN1 expression but enhanced TTP expression in HTR-8 cells. In vitro, TTP overexpression inhibited STMN1 production; TTP knockdown promoted it. TTP downregulated STMN1 expression in trophoblasts by directly binding its 3ʹ untranslated region. Conclusions TTP modulates trophoblast function and interacts with STMN1 and p53, and is related to pregnancy outcomes.

Introduction
Miscarriage, the most common complication of pregnancy, is defined as the spontaneous loss of pregnancy before the fetus has reached viability [1]. Recurrent spontaneous abortion (RSA) is defined as the occurrence of two or more consecutive pregnancy losses before 24 weeks of gestation [2]. In recent years, many molecules (including eukaryotic translation initiation factor 5A [eIF5A] [3] and peroxiredoxin 2 [4]) and immune cells such as dendritic cells, natural killer (NK) cells [5,6], invariant NK T (iNKT) cells [7,8], T helper (Th)1, and Th17 cells [9] have been linked with RSA.
Stathmin-1 (STMN1), also referred to as OP18, is a ubiquitous cytosolic phosphoprotein, proposed to be a small regulatory protein and a relay integrating diverse intracellular signaling pathways involved in controlling cell proliferation, differentiation, and activities [10]. In rodent uteri, STMN1 expression is upregulated at the embryo implantation site and may play a role in the early stages of pregnancy [11]. Recent reports have shown differential STMN1 expression in human endometrial and placental cells and that it may participate in decidualization. We found that STMN1 was downregulated in the chorionic villi from patients with RSA. It is a trophoblast proliferation-and invasion-associated microtubule regulatory protein that participates in the pathogenesis of RSA [12].
Many studies have suggested that STMN1 is a target of p53. Alli and colleagues found that silencing STMN1 induced a tumor suppressor function in breast cancer cell lines harboring mutant p53 (p53 MUT ) [13]. Another study showed that p53 inhibits STMN1 expression and that it is associated with G2/M block [14]. STMN1 is also believed to be important in ovarian cancer by increasing p53 MUT protein stability and transcriptional activity [15]. The p53 gene, first described in 1979, was the first tumor suppressor gene identified [16]. The p53 protein regulates the course of transcription of many genes in response to a wide variety of stress signals. Following DNA damage, p53 regulates key processes, including DNA repair, cell cycle arrest, senescence, and apoptosis, thereby suppressing cancer [17].
p53 is a potential mediator of pregnancy via estrogen and progesterone activation [18]. Studies on the p53 signaling pathway have suggested an association between polymorphisms and infertility, implantation failure after in vitro fertilization, and RSA. It is believed that p53 controls the apoptosis pathways (p53-cyclin-dependent kinase inhibitor [CDKN]1A and p53-Bax) and that it may be involved in the pathogenesis of RSA [19,20].
The regulation of mRNA turnover is critical for controlling gene expression. The 3 0 untranslated regions (3 0 UTRs) of the mRNAs of transiently expressed genes frequently contain an AU-rich sequence. The AU-rich elements (AREs) are the recognition signal for an mRNA processing pathway that specifically degrades the mRNAs of certain lymphokines and cytokines, including the T cell-specific cytokine interleukin (IL)-2 [21]. A set of RNA-binding proteins termed AU-binding proteins (AUBPs) specifically bind to AREs, either stabilizing mRNAs or promoting their destruction [22].
Different signaling pathways may participate in mammalian cell mRNA decay, including ARE-mediated mRNA decay [23]. Tristetraprolin (TTP/Zfp36) is a phosphorylating protein that recruits one or more degradative enzymes and initiates mRNA decay. TTP binds to AREcontaining mRNAs, marking them for delivery to processing bodies (P-bodies), where transcripts are degraded by mRNA decay enzymes [24]. TTP mRNA and protein levels are increased in the reproductive tract of mice during the estrous cycle [25]. In addition, the TTP family member TIS11D is crucial to female fertility and embryonic development, as disrupted TIS11D resulted in infertility in mice [26].
Previously, we found that STMN1 plays a key role in regulating trophoblast invasion and that impaired STMN1 expression may lead to RSA [12]. Little is known about the effects of p53 and STMN1 on trophoblast migration during pregnancy. We aimed to reveal the effects of p53-TTP-STMN1 signaling on trophoblast function and pregnancy outcomes.
The cells were seeded onto 6-well plates and incubated overnight at 37˚C with 5% CO 2 . The cells reached approximately 60-70% confluence after 24 h. Then, the cells were changed to new DMEM/F12 supplemented with 10% FBS and 0.1, 0.25, 0.5, 1.0, 1.5, or 2.5 μmol/mL doxorubicin (Sigma-Aldrich, St. Louis, MO, USA). The cells were harvested at 6 h, 12 h, and 24 h after treatment. mRNA was analyzed by quantitative real-time PCR (qRT-PCR); proteins were analyzed by western blotting and immunofluorescence. We also recruited 18 women aged 22-34 years with normal early pregnancies as the healthy controls. These women had all had previous pregnancies without any history of RSA, preterm labor, or pre-eclampsia. Induced abortion was performed by suction apparatus to terminate their unwanted pregnancy at 8-12 weeks of gestation, and samples of villous tissue were collected and stored in liquid nitrogen.

Human sample characteristics
The Medical Ethics Committee of International Peace Maternity & Child Health Hospital of the China Welfare Institute, Shanghai Jiao Tong University School of Medicine approved this study. Written informed consent was obtained from all patients who participated in the study.

TTP and p53 overexpression or knockdown
Lentiviruses carrying short hairpin RNA (shRNA) and overexpressing lentiviral vectors targeting human TTP were purchased from GeneChem (Shanghai, China) and transduced into the HTR-8 cells according to the manufacturer's instructions. Control siRNA and on-target individual siRNAs against p53 were synthesized by Shanghai GenePharma (Shanghai, China) and transfected into the cells at a final concentration of 100 nmol/L using Oligofectamine reagent (Life Technologies).

Immunofluorescence
HTR-8 cells were cultured on poly-L-lysine-coated coverslips in 24-well plates, treated with control vector (vector) or lentiviruses carrying short hairpin RNA (shTTP), cultured for 48 h, washed three times with phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde-PBS for 10 minutes, and stained with primary antibodies [rabbit anti-STMN1 monoclonal antibody (1:150; Abcam) and mouse anti-TTP monoclonal antibody (1:100; Santa Cruz Biotechnology)] overnight at 4˚C. The next day, secondary fluorescent Alexa Fluor 488 donkey anti-rabbit IgG (H+L) and Alexa Fluor 594 donkey anti-mouse IgG (H+L) antibodies (Life Technologies) were used after the plates had been washed three times with PBS. Nuclei were counterstained using 4,6-diamidino-2-phenylindole (DAPI; Abcam). Coverslips were mounted on the glass slides and the slides were visualized using a Leica microscope (Wetzlar, Germany). For the chorionic tissues, rabbit anti-p53 monoclonal antibody (1:1600; Cell Signaling Technology) was used as primary antibody.

Western blotting
Total protein from HTR-8 cells was prepared using 1× sodium dodecyl sulfate (SDS) lysis buffer (60 mmol/L Tris-HCl [pH 6.8], 10% glycerol, 2% SDS, sodium salt, 0.01% bromophenol blue, 100 mmol/L DL-dithiothreitol). Then, the mixture was heated in a water bath for 8 min at 100˚C. Equal amounts of protein samples were subjected to 12% polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories, Richmond, CA, USA). Then, 5% non-fat milk in Tris-buffered saline with Tween (TBST) was used to block the PVDF membranes for 1 h at room temperature. The membranes were incubated with antibodies against STMN1

Luciferase assay
To generate the STMN1 3 0 UTR-luciferase reporter constructs, a~1.5-kb DNA fragment containing TTP binding sites was amplified and cloned into a pGL3 luciferase vector (Promega, Madison, WI, USA). STMN1 3' UTR constructs carrying mutations in the putative TTP binding sites were generated using a QuikChange Lightning Multi Site-Directed mutagenesis kit (Stratagene, La Jolla, CA, USA). HTR-8 cells were transfected with the luciferase reporter plasmids and the vector or TTP overexpression plasmids, and were harvested after 24 h. Luciferase activity was measured using a dual luciferase reporter assay kit (Promega).

Statistical analyses
Statistical analysis was performed using GraphPad Prism version 6.0 (GraphPad, La Jolla, CA, USA). Experiments were performed in technical duplicates of at least three biological replicates. All results are reported as the mean ± SD [28]. All P-values are two-sided. Student's ttest was used to determine significant differences. P < 0.05 was considered significant.

STMN1 is downregulated and p53 is upregulated in patients with RSA
We investigated the STMN1 and p53 expression patterns at the maternal-fetal interface in patients with RSA. Immunohistochemistry and immunofluorescence analysis showed that STMN1 levels were significantly decreased in the patients with RSA as compared to the healthy controls (Fig 1a, 1c, 1e and 1f). On the contrary, p53 levels were increased in the patients with RSA (Fig 1a, 1b, 1g and 1h). The western blotting studies confirmed these findings. p53 was upregulated and STMN1 was downregulated in the villus tissue from patients with RSA (Fig 1d).

Doxorubicin activation of p53 reduces STMN1 expression in trophoblasts
Doxorubicin intercalates into DNA and inhibits the production of topoisomerase II (an enzyme that unwinds DNA), which halts DNA replication and induces the accumulation and transcriptional activity of p53 [29]. To determine the relationship between p53 and STMN1, we used the HTR-8 cell line, a first trimester human extravillous cytotrophoblast-derived cell line, to construct a cell model in which doxorubicin activates p53 in vitro. First, we added doxorubicin to the HTR-8 medium and harvested the cells at 6 h, 12 h, and 24 h to perform qRT-PCR and western blotting. qRT-PCR showed that STMN1 expression was obviously decreased at the optimal concentration of 0.5-1.5 μmol/mL doxorubicin and at the optimal time point of 24 h (Fig 2a and 2b). The western blotting results conformed to that of the qRT-PCR and doxorubicin treatment had no impact to expression of p53 but increased the expression of phosphor p53 (p-p53) and p21 (Fig 2c and 2d). Transfection with p53 siRNA (sip53) or control siRNA (sictrl) revealed that STMN1 was upregulated after knockdown of the p53 gene (Fig 2e-2g). The results suggest that excessive p53 production may inhibit STMN1 in trophoblasts.

TTP is upregulated in patients with RSA and activates p53 via doxorubicin in vitro
To investigate the relationship between p53 and STMN1, we searched the literature and found a key molecule: TTP. Prediction of the human STMN1 region using bioinformatics software p53-TTP-STMN1 effects on trophoblasts  p53-TTP-STMN1 effects on trophoblasts TRANSFAC (Transcription factor database) showed that STMN1 3'UTR region has two TTP binding sites (Fig 3a). It may be the key of linking p53 with STMN1. To confirm our hypothesis, we subjected paraffin-embedded tissue to immunohistochemistry and immunofluorescence assays. TTP levels were significantly higher in the patients with RSA than in the healthy controls (Fig 4a-4d). qRT-PCR and western blotting were performed to explore TTP expression after doxorubicin treatment in HTR-8 cells. TTP was significantly increased in a timeand concentration-dependent manner (Figs 2c, 2d, 4e and 4f). Interestingly, the change in TTP expression was the opposite of that of STMN1 and was in keep with p53 levels. As previously reported, TTP is an ARE-mediated regulator of mRNA decay [23]. Our findings indicate that TTP regulates STMN1 and that STMN1 expression in trophoblasts correlates inversely with TTP levels in patients with RSA.

TTP regulates STMN1 expression in vitro
To gain further insight into the relationship between TTP and STMN1, HTR-8 cells were transduced with shTTP or TTP-overexpressing lentiviral vector, and incubated for 48-72 h. TTP expression was upregulated after transfection of the TTP-overexpressing vector and was decreased after shTTP transduction (Fig 5a, 5b, 5c and 5e). Western blotting showed that TTP overexpression in HTR-8 cells resulted in decreased STMN1 expression as compared with the   control cells, but did not affect p53 expression (Fig 5a). Western blotting and bicolor immunofluorescence assay showed that TTP knockdown in HTR-8 cells increased STMN1 expression as compared with the control cells, but did not affect p53 expression (Fig 5b-5e). Obviously, TTP did not directly regulate p53 expression. Furthermore, three luciferase reporters were constructed with a control of either the wild-type human STMN1 3'UTR (WT reporter) or two mutants in which two putative TTP binding sites had been deleted (MUT1 and MUT2 reporter, respectively) (Fig 3a and 3b). As expected, STMN1 expression was increased in HTR-8 cells which were transfected with MUT1 or MUT2 reporter as compared with transfection in WT reporter. Moreover, the simultaneous addition of MUT1 and MUT2 had an obvious effect on STMN1. In a rescue experiment using TTP overexpression, MUT1, MUT2, and MUT1 +MUT2 transfection significantly decreased STMN1 expression as compared with transfection in the vector control cells (Fig 3c). Our findings strongly imply that STMN1 is an authentic target of TTP and that TTP may regulate trophoblast function and may be involved in the pathogenesis of RSA.

Discussion
Successful embryo implantation depends on embryo hatching, trophoblast development, and proper maternal-fetal cross-talk and immune regulation. The invasive potential of trophoblasts is essential for embryo implantation [30]. In our previous study, gain-and loss-of-function analyses showed that STMN1 regulates trophoblast proliferation and migration in vitro. The present study demonstrates that TTP affects the changes in STMN1 expression at the maternal-fetal interface in patients with RSA.
TTP is now known to bind to so-called class II AREs within the mRNAs that encode tumor necrosis factor (TNF)-α and granulocyte/macrophage colony-stimulating factor (GM-CSF). TTP protein is localized in both the nuclear and cytosolic compartments in fibroblasts, and its translocation from the nucleus to the cytosol can be accomplished rapidly, i.e., within 5 min, in response to stimuli such as polypeptide growth factors and phorbol esters, but not by agents that elevate cyclic adenosine monophosphate (cAMP) levels [31]. Cytokines are a prime target of TTP. A well-characterized example is TNF-α, whose expression is efficiently controlled by TTP as a part of a negative feedback loop [32]. TTP modulates various other inflammatory mediators, such as macrophage inflammatory protein-2 (MIP-2) [33], GM-CSF [34], interferon-γ [35], IL-22 [36], IL-10 [37], IL-6 [38], and post-transcriptional gene regulation is frequently organized in functional units. TTP is believed to play a key role in tumorigenesis, as its mRNA encodes the LATS2 tumor suppressor and the E6-AP ligase that directs p53 destruction [39]. Interestingly, a recent report on the TTP family and their selected targets during porcine pregnancy found that the shift in TTP expression and TIS11D points to a potential role of these genes in regulating spontaneous fetal loss [40]. However, little is known about its function in human trophoblasts at the maternal-fetal interface.
p53 is required for TTP induction in cancer cells [41], and doxorubicin enhances p53 activity [14], suggesting that doxorubicin may induce TTP expression in trophoblasts. Accordingly, we designed in vitro cell culture experiments to verify this premise. Our results showed that, in HTR-8 cells, doxorubicin increased both mRNA and protein levels of TTP in a dose-and time-dependent manner. Previously, we had found that STMN1 expression was significantly decreased in uterine NK cells from CBA/J × DBA/2J mouse matings as compared with CBA/ J × BALB/c mouse matings [42] and in patients with RSA [12], implying a link between STMN1 and p53/TTP signals.
In conclusion, we identified TTP as a downstream target of p53 and as a mediator of the anti-proliferative effects of STMN1 in trophoblasts. Specifically, excessive p53 reduced STMN1 expression by upregulating TTP to suppress trophoblast migration and invasion at the early stage of placentation. Regrettably, because RSA samples are difficult to collect, number of samples is not enough. Further we will continue to collect more RSA samples to validate our discovery. Although, the relation between p53, STMN1 and TTP is complicated, many other elements that could affect them, including mechanism of TTP increasing. But our study provides an important foundation for future studies investigating mRNA destabilizing factors at the maternal-fetal interface. Trophoblast proliferation, migration, invasion, and endocrine secretion play important roles in a successful pregnancy. Along the way, we will also learn much about possible therapeutic strategies for preventing and/or treating pregnancy complications.