Coworking spaces have become a central component of new work environments, with large international chains. The purpose of this study is to investigate whether user preferences for the physical workspace design are consistent across countries, which the uniformity of such chains seems to suggest. A comparison between the user preferences of coworking spaces between the Netherlands (n = 219), Germany (n = 98) and the Czech Republic (n = 79) is performed using a mixed multinomial logic model for each country. Besides statistical utility of attributes, also motivations for working in coworking spaces are analysed. The findings show that there are some consistencies in preferences across countries. Typical real estate characteristics like accessibility and contract options came forward to be the most important attributes in choosing which coworking space to work at in all three countries. However, significant differences in the desired quality levels of specifically these attributes were found between the countries as well, and only the less important attributes showed similar preferences internationally. This suggests that identical world-wide implementations of the same concept, might serve multi-nationals but possibly will not attract local users. The identified differences in preferences can help to position more specific, dedicated coworking spaces within local markets. For the last two decades, work practices have changed due to the collaborative economy and new forms of collaboration  was performed to identify preferences for hypothetical coworking spaces described by their most relevant characteristics. Multinomial logit modelling was used to analyse the preferences for coworking space characteristics and to see whether the countries show differences in user preferences regarding accessibility, atmosphere, layout, diversity of spaces and of tenants, services and contracts. The research has practical implications for corporates, individual user and operators and enhances theoretical understanding of coworking spaces as institutions in an entrepreneurial ecosystem.Every operational activity requires not only a distribution of tasks to the personnel resources, but always also a spatial organisation of the task completion in workplaces, rooms, buildings and locations Krüger . WhereasNowadays, the users of coworking spaces include a wide range of actors as predicted by Sargent et al. . Larger By bundling all real estate services into a space-as-a-service package, coworking providers offer external service provision. In the entrepreneurship literature, this space-as-a-service concept is considered as one component of an entrepreneurial ecosystem. Coworking spaces as the spatial entity of an entrepreneurial ecosystem can add value by knowledge management and networking opportunities. Bouncken et al.  term cowH1: Novelty based motivations to work at a coworking space are more important than those based on efficiency.Not all end-users have the same motivations for choosing a coworking space though. Spinuzzi  found thH2: The ranking of motivations to work at a coworking space is consistent across countries.As there is no previous research comparing such motivations across countries, the apparently successful ‘uniformity approach’ by large international coworking chains is used to pose the next hypothesis:To study preferences of users, it is important to identify the most important attributes of coworking spaces that can satisfy or frustrate certain user preferences. Although many hybrids between coworking spaces and other business centre concepts are increasingly present, Waters-Lynch et al.  point ouFirst, like many offices, most coworking spaces are positioned in highly accessible locations, but more ‘remote’ spaces exist as well . They also had to answer personal questions  and indicate work-related characteristics . The second part of the questionnaire was setup as a stated-choice experiment  6561 possible alternatives. Therefore, an orthogonal fraction of this design was constructed . Throughout Germany and the Netherlands, the samples were collected in many different regions/cities and coworking spaces to provide a spatial heterogeneity of the participants. In Berlin, for example, there is a very mature coworking space market, which attracts many users from the IT sector, while in Frankfurt many users come from the financial sector. Furthermore, the survey was conducted in small and medium-sized cities, so that a cross-section of coworking spaces was achieved.In Czech Republic, data was collected only in the main capital Prague. The city of Prague has been selected due to its geographic position and current situation of the coworking industry. Centrally positioned and with a midsized urban area, Prague has seen a swift growth of coworking spaces and users in recent years , first the rankings were studied themselves, after which the top 3 choices were added for each possible motivation per country to test H2 with a Chi-square analysis.i for coworking space j on choice occasion t is:To study user preferences (H3) and their consistency across countries (H4), a mixed multinomial logit model (MMNL) for each country was estimated. A MMNL is a very efficient and flexible discrete choice model  to be estimated. The error term, εijt, represents unobserved heterogeneity. This equation assumes that all the estimated parameters are equal for all users. Since the parameters relating to different levels of the same attribute are conceptually interrelated, a random parameter is estimated for only one of the parameters related to the same attribute to capture possible heterogeneity related to that attribute. As a somewhat arbitrary choice, the random parameters in the model relate to the significant, first levels of the attributes. A normal distribution is assumed for each random parameter. The non-random parameters in the model represent the second levels, plus non-significant first levels of the attributes. To estimate the parameters of the model, 1000 Halton draws were used . In Germany, many respondents (33%) work for a company with more than 11 employees and over half is a regular employee, while in the Netherlands 42% is owner/board member of their organisation. This could also be related to the fact that the hours spent in a coworking space is the highest in Germany . With regard to sector, in the sample of the Netherlands coworkers are more frequently working in the consultancy sector compared to coworkers from Germany or the Czech Republic.Figure p ≤ 0.000), so H2 (The ranking of motivations to work at a coworking space is consistent across countries) must be rejected. Both some novelty-focused motivations and some efficiency-focused motivations show particularly large differences between the observed and expected values. Regarding efficiency-focused motivations, respondents from the Netherlands more often chose flexibility  in their top 3 motivations than in Germany and the Czech Republic. German coworkers particularly seemed to select professional appearance for the company and affordable accommodation more often, while in the Czech Republic the supportive services of a professional supportive work environment were selected relatively more often. Regarding novelty-focused motivations, the coworkers from the Netherlands relatively less often put social interaction with coworkers in their top 3, while Germans selected this relatively more often. Those from the Czech Republic relatively more often mentioned the opportunity to network with coworkers towards possible new projects.As can be seen in Fig. Table Figure Not only the importance of different attributes but also the preferred ‘quality’ level for 3 of the 8 attributes differs between the countries. As 2 of these 3 attributes are at the top of the importance ranking when choosing a specific coworking space, H4 (User preferences for coworking space attributes are consistent across different countries) must be rejected. Concerning accessibility, the MMNL models showed that the part-worth utility of the level accessibility by car and public transport is the highest for the Netherlands and Germany, while in the Czech Republic the probability that coworkers choose a coworking space that is accessible only by public transport is higher. Also, coworkers in the Netherlands and the Czech Republic prefer a homey atmosphere and interior, while coworkers in Germany prefer a more modern interior. An industrial interior is the least preferred by coworkers from the Netherlands and Germany, while a modern interior is the least preferred by coworkers from the Czech Republic. And last, regarding the lease contract, differences were found between the countries as well. Coworkers from the Netherlands and the Czech Republic prefer no lease contract, while coworkers from Germany prefer a short-term lease contract.No differences were found in preferences related to the other 5 attributes: layout of the space, diversity in supply of spaces, reception and hospitality, events, and diversity of tenants. Coworkers from all the three countries prefer a coworking space with a half-open layout, which consists of open workspaces in combination with spaces for concentration and for formal meetings. Also, no differences were found with regard to the diversity in supply of spaces. Coworkers from all countries prefer a standard coworking space that offers office space with informal meeting areas and event spaces. Moreover, coworkers from all three countries mostly prefer a reception but no host, only sometimes an event (not too often) and a moderate diversity of tenants.The standard deviations of the random parameters from Table The analyses have shown several significant differences in preferences and motivations of coworking space users in the three countries that were included in this study. However, regarding motivations, the main attractiveness of coworking spaces in all countries appears to be the vibrant atmosphere and separating work from private life. A bit surprisingly, the novelty-focused motivation of a vibrant, creative atmosphere was followed by typical efficiency-focused real estate arguments of affordability and flexibility, instead of other novelty-focused motivations such as being part of a community, networking and interacting. Regarding efficiency, the Dutch seem to care more about flexibility, the Germans about affordability and the professional appearance for the company, and the Czech about the supportive services of a professional supportive work environment. As Cabral and van Winden  showed wThe second contribution is that the significant differences found in preferences for specific coworking space attributes seem to imply that international chains with uniform coworking space offerings might not satisfy local preferences to the full extent. This suggestion is particularly supported by the fact that especially those attributes coming forward as most important when choosing between different coworking space alternatives, showed significant differences in preferences between countries, namely accessibility and the type of lease contract. The main consistency across countries that came forward is that these two generally important (corporate) real estate choice attributes are indeed the most important ones in all three countries, followed by layout and the diversity of tenants in the coworking space. Only when these aspects are to one’s liking, the more typical coworking space characteristics  potentially determine the preference for one alternative over the other. Interestingly, for these more typical coworking space attributes, coworkers from all three countries did show similar preferences (atmosphere excluded). They generally seem to prefer a coworking space with a half-open layout, which consists of open workspaces in combination with spaces for concentration and formal meetings. A standard space that offers such spaces, plus a reception, some informal meeting areas and some event space appears to be preferred over a more premium alternative with a host and many events. Also, the diversity of tenants is appreciated, but only at the ‘moderate diversity’ level. Apparently, there is also something as too much diversity in tenants. So, regarding the design/service offer it seems that coworking spaces could be uniform across countries, while for accessibility, contracts and the atmosphere/aesthetics it would be wise to follow more local ‘habits’. This sample suggests that the Czech Republic coworkers care less about accessibility by car and the German coworkers prefer a more modern work environment than the ‘homey’ alternative preferred by the other two countries’ samples. It could however be, that the higher share of students in the Czech sample caused the first difference and the higher share of users from large enterprise in Germany the second one. The sample size did not allow to control for such interaction effects.The overall consistent preference for the ‘average/standard’ quality levels of these typical coworking space attributes might be explained by the fact that this sample often chose ‘to find affordable accommodation’ as a motivation for using coworking spaces in general. Additional offerings in the higher quality levels could be expected to (and most likely will) be more expensive. Repeating the study with larger samples needs to check whether this is the case for all coworking users or that this sample was specific in that. It does become clear that regarding the top ranked motivation for using coworking spaces—their vibrant and creative atmosphere—the physical support of such an atmosphere  might show different preferences across the countries but is not influencing the choice for a specific coworking space much see Fig. . So, intIn all three countries the coworking spaces seem to attract young, highly educated users, with a balance between male and female. Coworking spaces do not seem to be a place for full-time work, as the average hours spend there during the week in each country was about 20 h, which is consistent with other research Kopplin . This saOverall, the main contribution of this paper is that its findings imply that coworking space user preferences are not that different from those using other office types as assumed, with accessibility and contract being the most important attributes to choose between coworking spaces. It seems that they go through similar decision-making processes.This study did not aim to draw representative samples from coworking spaces in each country and was not able to do so (due to time limits and a limited willingness to participate among coworking providers). Rather, this paper wanted to analyse whether assumptions about internationally consistent user preferences are true, even when looking at a relatively small sample of coworking spaces in only a few countries. Future research should identify significant differences between more countries and based on representative samples, to identify successful coworking space alternatives for more ‘local’ situations. It would be interesting to look at differences between and within continents, as this paper only contains data from within Europe. Larger samples also allow for using more than 8 attributes, because one could randomly show a selection of attributes to respondents instead of showing all respondents all quality levels of all attributes. Additionally, larger samples would allow testing interaction variables in an overall model for all involved countries. Last, large samples would make it possible to do additional analyses in the form of latent class modelling, where groups of respondents with highly similar preferences are identified, and then tested on their individual demographics and other user characteristics to describe more clear target groups for certain coworking space business models.Future research should also identify why the identified differences exist and whether they are based on nationality, culture or other individual or group characteristics. The standard deviations for the standard utility parameters show that there is still more unobserved heterogeneity within the groups and thus that socio-demographic and other characteristics of individuals need to be included to help explain the choice decisions in more detail. Although some of these were included in this dataset already, the sample size did not allow for testing their prediction of certain preferences.It would be interesting to include rental prices in future research and do willingness to pay analyses for all attributes, including those that were not that high in the ranking of importance (e.g. a host). This gives providers insights in potential cost savings, that could be invested in more important attributes to attract their target group. In that sense, the clear interest in coworking spaces amongst Czech students suggests to study whether/how students can be an attractive population for coworking operators in other countries, given their limited budget to pay a fee. Besides costs, future research could study the decision-making processes of different types and sizes of companies/entrepreneurs in more detail as well, to provide more insight in sought benefits and compromises that are made in seeking goals.From a macro-organizational view, Bouncken et al.  argue thFinally, the empirical part of this study was conducted before the beginning of the current Covid 19 pandemic. It cannot be ruled out that user preferences in the countries studied have changed permanently due to the pandemic. However, this question can only be answered by further empirical analyses after the current situation has ended.This study has shed light on preferences for coworking spaces in different countries. The gathered user preferences can be used as precedents for value propositions of coworking business models that can lead to a higher loyalty of coworking users (Clauss et al. Coworking spaces are one of the unique work arrangements that can be used by users all over the world. Large operators are increasingly offering coworking space with a generic standard in their offices worldwide, which however might not serve users from all countries to a similar extent because of the findings here. They provide conformity in standards worldwide for large corporates using these coworking spaces but might not attract local users in all countries this way. Coworking operators can use the identified preferences as guidance on their path from a product-centered to a user-centric environment and help them to find their competitive advantage. Irrespective of country and user type, operators benefit from a good accessibility and can determine the success of their spaces through variable contract periods and a design with half-open layouts. The early, smaller coworking space providers are perhaps still serving a more locally and culturally determined need, which caused the rise of these spaces. The future will tell which users they will continue to attract versus the large global serviced office providers and corporates opening up coworking spaces inside their buildings as well. All will have to meet user preferences in order to compete in this fast-growing, competitive market. The decision to focus on a specific target group can be guided by the results of the study.


Zebrafish is an elegant vertebrate employed to model the pathological etiologies of human maladies such as cardiac diseases. Persistent physiological stresses can induce abnormalities in heart functions such as cardiac hypertrophy (CH), which can lead to morbidity and mortality. In the present study, using zebrafish as a study model, efficacy of the traditional Indian Ayurveda medicine “Yogendra Ras” (YDR) was validated in ameliorating drug-induced cardiac hypertrophy. YDR was prepared using traditionally described methods and composed of nano- and micron-sized metal particles. Elemental composition analysis of YDR showed the presence of mainly Au, Sn, and Hg. Cardiac hypertrophy was induced in the zebrafish following a pretreatment with erythromycin (ERY), and the onset and reconciliation of disease by YDR were determined using a treadmill electrocardiogram, heart anatomy analysis, C-reactive protein release, and platelet aggregation time-analysis. YDR treatment of CH-induced zebrafish showed comparable results with the Standard-of-care drug, verapamil, tested in parallel. Under in-vitro conditions, treatment of isoproterenol (ISP)-stimulated murine cardiomyocytes (H9C2) with YDR resulted in the suppression of drug-stimulated biomarkers of oxidative stress: COX-2, NOX-2, NOX-4, ANF, troponin-I, -T, and cardiolipin. Taken together, zebrafish showed a strong disposition as a model for studying the efficacy of Ayurvedic medicines towards drug-induced cardiopathies. YDR provided strong evidence for its capability in modulating drug-induced CH through the restoration of redox homeostasis and exhibited potential as a viable complementary therapy. Danio rerio) has emerged as a useful tool in the study of human diseases, recapitulating some or all of the observed pathologies. Through whole-genome mapping, D. rerio has been replacing small animal models in understanding disease modalities and their conserved molecular pathways. D. rerio has also been used as a model for studying cardiac stress and diseases using electrocardiograms (ECGs) and biochemical parameters, since they closely resemble those of humans [The zebrafish  is a macrolide antibiotic generally associated with gastrointestinal distress. ERY is also a motilin receptor agonist that reduces the G-coupled receptor-stimulated contractions in the smooth muscle. Studies have shown that oral treatment of patients with ERY followed by its metabolism by cytochrome P450 3A (CYP3A) leads to an increase of the drug concentration in the blood plasma, consequently leading to the development of cardiac arrhythmia and torsade de pointes (TdP) by blocking human-ether-a-go-go gene (hERG) and prolonging QTc intervals . In D. r and TdP ,9. Isoprhenotype ,11. Cardhenotype ,13. Therhenotype .Aloe vera leaves juice as the binding agent. This Ayurvedic formulation is prepared as per the procedures described in the several century-old ancient Indian medicinal texts of Bhaiṣajya Ratnāvalī and Vatavyadhi Chikitsa, for healing neuropathological, cardiovascular and diabetes diseases [Bhaiṣajya Ratnāvalī , the sloka (in sanskrit) mentions “Viśuddham rasasindūram taddwardham śuddhahāṭakam. Tatsamam kāntalauhñca tatsamañcābhrameva ca. Viśuddham mauktikañcaiva vaṅgañca tatsamam matam. Kumarikārasairbhāvayam dhānyarāśau dinatrayam” , gold (Hāṭaka) ash (Bhasma), iron  ash, mica (Abhra) ash, pearl (Mauktika) ash, tin (Vaṅga) ash are to be mixed in Aloe vera juice (Kumārī rasa) and triturated (bhaāvanā) in a vessel . It is then mixed under pressure (Mardāna) in Kumārī rasa; made into pellets and dried. The pellets are to be wrapped with Ricinus communis (Eraṇḍa) leaves and kept in a heap of dry Oryza sativa (Dhānya) seeds for 3 days, before use). The prescribed human dosage of the YDR according to Bhaishajya Ratnavali is 125 mg twice a day [Recently, there has been a growing interest in the treatment of diseases with traditional Ayurvedic medicines ,15,16,17diseases ,19. In Bce a day . Though ce a day ,21.D. rerio CH model, measured cardiac electrical activities, and tested the efficacy of YDR in protecting against drug (ERY)-induced CH. Parameters measured were modulation of electrocardiogram PQRST waves, change in heart size, production of C-reactive protein (CRP), and platelet aggregation. The mode of action for the YDR was studied under in-vitro conditions in the ISP-stimulated murine cardiomyocytes (H9C2) by measuring parameters such as the intonation of oxidative stress, mitochondrial dysfunction, and clinical and non-clinical cell signaling biomarkers for cardiac function.In the current study, we set up a Yogendra Ras (YDR) (batch number A-YGR011) was procured from Divya Pharmacy, Haridwar, India, sold under its classical name. Based on sensory characterizations, the YDR formulation was observed to be a dry, free-flowing powder and rust-brown in color, packed under inert environment. The YDR powder in dried condition was odorless, tasteless, and insoluble in water and cell culture media due to its metal-based origin under normal physiological pH and temperature. Reagents for cell culture studies such as Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), antibiotics, trypsin-EDTA, isoproterenol, 2′,7′–dichlorofluorescein diacetate (DCFDA), MitoTracker red dye, TaqMan primers and universal RT-PCR master mix for quantitative real-time PCR analysis were purchased from Thermo Fisher Scientific Inc., USA. Purified catalase enzyme was purchased from Merck India Pvt Ltd. Purified superoxide dismutase standards were purchased from Cayman Chemicals, USA. Trichloroacetic acid and Giemsa stain were purchased from Hi-media Laboratories, India. Verapamil was purchased from Sigma-Aldrich, India, and erythromycin was purchased from TCI Chemicals, India.−1.The morphological and elemental attributes of the dry YDR powder were measured using a scanning electron microscope  with an attached electron dispersive X-ray analysis system . Gold sputtering of the YDR sample to improve the electron micrograph quality was done at an accelerated voltage of 10 kV. Powder X-ray diffraction (XRD) analysis was performed using a Rigaku D-Max 2200 X-ray diffractometer applying Cu-Kα radiation at 40 kV/40 mA. Scanning was performed throughout the experiment at a step width of 0.02° over an angular range of 5° to 80° and a scanning rate of 0.5° minFor particle size distribution analysis, the YDR sample was suspended individually at a concentration of 100 mg/mL in double-distilled water and in the DMEM cell culture media containing 2% FBS. Measurements were done in triplicates using the Malvern Zetasizer Nano ZS . Inductively coupled mass spectroscopy (ICP-MS) analysis of the YDR samples was performed at the Eurofins Analytical Services India Private Limited, Bengaluru, India.D. rerio obtained from the in-house breeding facility at Pentagrit Research, Chennai, India were used in the current study. A total of 216 adult male D. rerio with a bodyweight of 0.5 g and length of 25-30 mm were selected for this study. The fish were divided randomly into two groups and acclimatized as 12 fish per polycarbonate tank containing 2 L of water, prior to the effective and therapeutic dose studies. Throughout the study duration, a period of light (14 h) and dark (10 h) cycle and a constant water temperature of 27 ± 1 °C were maintained. The fish were fed TetraMin® flakes obtained from Tetra, VA. Fish were divided into 9 groups containing 24 fish per group. All the experiments were performed following the protocol approved by the Institutional Animal Ethics Committee (IAEC) in accordance with the Committee for the Purpose of Control and Supervision of Experiments (CPCSEA), Government of India , and were in general compliance with the ARRIVE guidelines [D. rerio. All the erythromycin-exposed fish were maintained for a period of 16 days in case of an effective dose study and for 23 days in case of a therapeutic dose study  was added to 4 L of the housing water of se study . In the D. rerio were optimized at 1000× less than the relative human doses (125 mg/day BID) by body weights [D. rerio were 0.6, 4, and 18 µg/kg (®) and was extruded into uniform pellets. For feeding individual fish, a rectangular fish tank was separated into 6 independent units, and individual fish were separated from the respective study groups. Each fish was fed individually on a 24 h cycle with an estimated number of pellets, under isolated condition. Control fish were fed with unmodified fish feed under conditions similar to the exposed groups.YDR doses for  weights ,24. Henc18 µg/kg . StandarD. rerio heart is comprised of a single ventricle and a single atrium. The cardiac functions are cyclic with every systole where the ventricle pumps blood into the bulbus arteriosus; this acts as a reservoir from which blood empties into the ventral aorta [D. rerio, the heart is anatomically located right in the midline of the ventral side, immediately below the gills. The bulbus arteriosus is present on the dorso-cranial side of the ventricle. The ventricle apex is directed to the trunk. To measure the treadmill ECG, the study fish were transferred to experimental treadmill tanks as described by Depasquale et al. [Unlike the human heart, al aorta . In D. re et al. . The treThree-channel ECGs were used to measure the electrocardiograph cardiac signatures . The chaFish were sedated in cold water set at 14 °C. A small slit was made injuring the major blood vessel in the tip caudal region of the fish using a sharp scalpel. Care was taken to keep the fish alive until the clotting time was recorded. The cut was approximately 0.25 mm deep passing through the scales and the skeletal muscle. Blood was collected and immediately spotted on a glass slide. Whole blood was observed under a light microscope at 4×, and the platelet aggregation was measured as a parameter for the blood clotting time.After measurement of the platelet aggregation, all the study fish were euthanized immediately using ice-cold water set at 4 °C. Blood was collected in a glass tube containing 5 µL of 0.5% EDTA by making an incision in the region of the dorsal aorta and inferior vena cava, just posterior to the dorsal fin near the caudal region. EDTA-treated whole blood was then centrifuged at 10,000 RPM for 10 min to separate the plasma. Collected plasma was then stored at −80 °C until further use, and CRP levels were measured using the Randox CRP analysis kit and auto-analyzer system .Heart was dissected out from the euthanized fish. The isolated heart was immersed and stored in 30% formalin solution for 72 h. Post-fixative treatment of the heart was dissected in the cross-sectional direction into 3 parts. The middle section was used to measure the septal wall thickness. Microscopic images of the heart were taken at 4× magnification and the thickness was measured using Image J software.6 cells/cm2 and grown in a humidified incubator at 37 °C with 5% CO2. For the experiments, cells were plated in 96-well plates at the density of 10,000 cells/well. The cells were pre-incubated before exposure to the stimulant and the drugs.The H9C2 (rat embryonic cardiac) cells were purchased from the ATCC licensed cell repository National Centre for Cell Science (NCCS), Pune, India. The H9C2 cells were cultured in DMEM media supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics. Cells were cultured at the density of 2 × 1020) and 50% (IC50).H9C2 cells were treated with ISP or YDR mixed in fresh cell culture media in semi-log doses between 0 and 1000 µg/mL for 24 h. At the end of the exposure time, the old medium was removed, and cells were washed with 100 μL PBS. A total of 100 μL of 0.5 mg/mL 3--2,5-diphenyltetrazolium bromide (MTT) was added to each well, and the plates were incubated for 3 h at 37 °C. At the end of the exposure period, 75 μL of MTT dye was removed. Fifty microliters of DMSO was added, and the plates were placed on a shaker for 10 min. The absorbance of each well was read using the PerkinElmer Envision microplate reader at 595 nm wavelength, and cell viability percentage was calculated along with an inhibitory concentration of 20% (ICH9C2 cells were exposed to ISP (50 µM) and YDR (30 µg/mL) individually or in combination for 24 h. At the end of the exposure period, cells were washed with lukewarm PBS, and fresh media containing 10 µg/mL of 2′,7′-dichlorofluorescein diacetate (DCFH-DA) dye was added to all the wells. The plates were incubated in the dark for 45 min at 37 °C. Fluorescence intensity was measured using the PerkinElmer Envision microplate reader at the excitation and emission wavelengths of 490 and 520 nm, respectively.H9C2 cells were exposed to ISP (50 µM) and YDR (30 µg/mL) individually or in combination for 24 h. At the end of the exposure period, the cells were washed with lukewarm PBS and stained with 1 mM of MitoTracker Red probes for 45 min. Cells were washed with 1× HBSS buffer after removing staining media, and fluorescence intensity was measured on the PerkinElmer Envision microplate reader with excitation and emission at 584 and 606 nm, respectively.2. Cells were counterstained with 2% safranin for 10 min. Stained cells were observed under an inverted microscope at 10× for the formation of blue-purple formazan crystals. The formazan crystals were solubilized with 2 M KOH and 0.5 N HCl to calculate the percentage of NBT reduction [H9C2 cardiomyocytes were seeded in the 6-well culture plate, and after 60–70% confluence, cells were treated with ISP and YDR each for 24 h. After the completion of the exposure, 0.3% NBT was added to each well and incubated for 1 h in an incubator at 37 °C with 5% COeduction . Elutionv/v), Tris-HCl , EDTA (1 mM), PMSF, and protease inhibitor cocktail and incubated for 1 h on ice with intermittent tapping. The lysate was then centrifuged at 13,000 rpm for 10 min at 4 °C. Protein concentration was quantified in the collected supernatant using a BCA kit  as per the manufacturer instructions. Unused supernatant was stored at −80 °C for further analysis.H9C2 cells were exposed to ISP (50 µM) and YDR (30 µg/mL) individually or in combination for 24 h. At the end of the exposure period, the cells were washed with lukewarm PBS, trypsinized, and lysed in RIPA buffer NaCl (150 mM), NP-40 (10% 2O2 (30% v/v) in 100 mL of potassium phosphate buffer. Then, 125 µg/mL of extracted protein was added to 0.1M potassium phosphate buffer (pH 7.0), and the reaction was initiated by adding 30 mM H2O2. Absorbance was recorded spectrophotometrically for every 30 s at a wavelength of 240 nm for 3 min in a quartz cuvette. Purified catalase was used to plot the standard curve. The enzyme activity was expressed as millimolar H2O2 consumed per min per mg of protein.Catalase activity was measured in isolated proteins as per the method mentioned by Weydert et al. . InitialSOD enzyme activity was measured based on the method of Beauchamp and Fridovich using Cayman’s superoxide dismutase assay kit as per the manufacturer instructions . The rea−ΔΔCt was calculated, and data were analyzed for fold change in mRNA expression.Gene expression studies using quantitative real-time PCR (qRT-PCR) were performed to evaluate the mRNA expression level of clinical and non-clinical cardiac biomarkers in H9C2 cells. The cells were exposed to ISP (50 µM) and YDR (30 µg/mL) individually or in combination for 24 h. At the end of the exposure period, the cells were washed with lukewarm PBS and lysed using TRIZOL reagent , and total RNA was isolated. RNA was purified and quantified by taking absorbance at 260 and 280 nm. Each RNA sample was processed to cDNA using a one-step Verso cDNA synthesis kit . For qRT-PCR, TaqMan chemistry-based FAM single tube primer assays of NOX-2 and -4, COX-2, ANF, CLRS-1, TNN-T and TNN-I, along with TaqMan universal master mix were used. Beta-actin (ACTB) housekeeping gene was used as an internal control to normalize the expression of other genes. Real-time PCR assay was run on a Biometra TProfessional RT-PCR machine , and cycling parameters included initial denaturation at 95 °C for 10 min and primer extension at 95 °C for 15 s and 60 °C for one min with 40 cycles. Ct values were obtained, relative expression 2p-value was ≤ 0.05.All the experiments were performed in technical triplicates as well as in biological triplicates. Data from all the experiments are presented as the mean ± SEM. An unpaired t-test was used for comparison between two groups. For groups of three or more, the data were subjected to one-way analysis of variance (ANOVA) followed by Dunnett’s post-hoc test. Differences were considered statistically significant if the Evaluation of the scanning electron microscope (SEM) images of YDR showed them to be heterogeneous in shape and size A. YDR wa2O3), arsenic trioxide (As4O6), and AsFe were identified in the YDR samples (Inductively coupled plasma mass spectroscopy (ICP-MS) analysis of YDR samples showed the presence of 9.8% mercury (Hg) and 7.9% gold (Au). X-ray diffraction (XRD) crystallography analysis of the YDR sample confirmed the presence of Hg in the form of Cinnabar and Metacinnabar (HgS) A. Using  samples A. Severa samples B. HoweveChange in the size distribution of the YDR particles in the presence of the cell culture media biomolecules was further supported by the polydispersity index (PdI) values indicating that the YDR particles formed a more stable colloidal suspension in the DMEM cell culture media supplemented with 2% FBS (PdI = 0.174 ± 0.06) as compared to the particles suspended in pure water (PdI = 0.406 ± 0.080) B.D. rerio heart showed structurally intact bulbus arteriosus, atrium, and ventricle  time-dependent septum wall thickness increase in the ERY-stimulated D. rerio in comparison to the normal control fish  for both the 7-day (effective dose screening) and 14-day (therapeutic dose testing) exposure studies showed a widening in the angle of QRS complex compared to the normal control fish , medium (4 µg/kg), and high (18 µg/kg)  Biii–v. A8 µg/kg) Biii. The8 µg/kg) Biv. The 8 µg/kg) Bv. Septu8 µg/kg) A. All th8 µg/kg) Biii–v. T8 µg/kg) Biii–v. A8 µg/kg) Biii–v.D. rerio showed a complete recovery of the ERY-stimulated ECG parameters related to the onset of CH, as compared to the disease control fish  levels present in the blood serum of the mulation B. Elevatmulation C. This rf action E.D. rerio led to a decrease in the platelet aggregation time as compared to the normal control fish (D. rerio with different doses of YDR (0.6–18 µg/kg) significantly  recovered the platelet aggregation time back to normal as compared to the disease control fish. Therapeutic treatment of the ERY-stimulated fish with YDR (18 µg/kg) and the standard drug verapamil also showed a significant recovery of the platelet aggregation compared to the disease control  and 50% (IC50) for ISP were determined to be 50 and 122.4 µM, respectively. Similarly, IC20 and IC50 doses for the YDR were determined to be at the concentrations of 30 and 205 µg/mL, respectively. On the basis of this dose screening, 50 µM of ISP and 30 μg/mL of YDR were selected for the subsequent cell biological experiments.Cell viability analysis showed a dose-dependent change in the H9C2 cells following exposure to ISP or YDR A. Based p < 0.05; 3 µg/mL p< 0.05; 10 µg/mL p < 0.01; 30 µg/mL p < 0.01) reduced the intracellular generation of ROS in a dose-dependent manner (The stimulation of H9C2 cells with ISP (50 µM) for 24 h produced a significant  1.3-fold increase in the intracellular ROS generation B. Elevatt manner B.p-value < 0.01) escalation in the mitochondrial membrane potential (MMP)  reduced the elevated level of MMP in a dose-dependent manner (Mitochondria play an important role in the production and release of intracellular ROS. Stimulation of the H9C2 cells with ISP (50 µM) led to significant  C. This ial (MMP) B,C. Co-tt manner C.−2) were detected intracellularly using a nitroblue tetrazolium assay. We observed a significant  rise in the generation of intracellular O−2 in the H9C2 cells when treated with ISP (50 µM) alone  reduced the stimulated release of O−2  alone D. Concure of O−2 D. These e of O−2 B,C. Hencp-value < 0.01) reduction in the levels of the anti-oxidants i.e., superoxide dismutase (SOD) and catalase, indicating onset of oxidative stress  recovered through the co-treatment of the ISP-stimulated H9C2 cells with YDR showing a recovery in the levels of both SOD and catalase. The SOD and catalase enzymatic assay result further correlated well with the previously observed high induction of O−2 and ROS in the H9C2 cells when treated with ISP alone and its amelioration following a co-treatment with YDR (Stimulation of the H9C2 cells with ISP (50 µM) led to a significant  fold increase following the induction of H9C2 cells with ISP (50 µM), while the elevated levels of NOX-2 were not statistically significant. YDR alone did not induce any stimulated increase in the inflammatory mediators. In the present study, concurrent treatment of ISP-stimulated H9C2 cells with YDR (30 µg/mL) led to a (p <0.05) reduction in the expression of COX-2 and NOX-4 (ROS generation is known to induce the expression of Cyclooxygenase (COX) and the subclass of NADPH oxidases (NOX), which further act as inflammatory mediators during the induction of cardiotoxicity. Therefore, we studied the oxidative stress-stimulated up-regulation of the mRNA expression profiles of pro-inflammatory mediators COX-2, NOX-2, and NOX-4 A–C. Bothnd NOX-4 A,C. The nd NOX-4 B. Thus, p-value <0.01) overexpression of fetal cardiac gene atrial natriuretic factor (ANF) was observed , gold (Au), and tin (Sn). Generally, YDR is used as a neurostimulator and acts as a catalyst with other Ayurvedic formulations to increase their therapeutic efficacy . AccordiBhaisajya Ratnāvalī [4O6). Mercury in the form of Cinnabar has been extensively used in traditional Indian and Chinese medicines in treating various diseases such as neuropathy and cardiac diseases [2) and demonstrates 1000× lower toxicity as compared to methyl mercury [Based on the physicochemical characterization, YDR was found to be heterogeneous in shape and size, containing large quantities of Hg, Sn, and O. Complementing these findings, ICP-MS analysis of the YDR particles showed the presence of Hg (9.8%) and Au (7.9%). Interestingly, the absence of Au peak in the EDX and in the ICP-MS analysis might be due to the masking effect induced by the gold-palladium coating process of the samples. The detected elemental constituents of the YDR were close to those mentioned in the ancient Indian text of atnāvalī . Hence, diseases ,38. Cinn mercury . Other cD. rerio as earlier studies have shown that this can induce CH [D. rerio contains a two-chambered heart, directing the hypertrophy toward the left ventricle is not possible. In the present study, based on the ECG reading development of CH was observed through an increase in the amplitudes of the QRS wave. Furthermore, analysis of the Cornell product, Cornell voltage, Sokolow–Lyon, and Romhilt–Estes point criteria obtained from the ECG readout clearly showed the induction of CH [D. rerio with YDR significantly reduced the stimulated release of CRP and platelet aggregation, indicating a reduction in the level of CH. Our results confirmed the earlier ECG and oxidative stress-related findings and showed that the YDR has equal potential in ameliorating drug-induced CH. Therefore, this is the first study to report the efficacy of metal-based ancient Ayurvedic medicine in modulating CH.In the present study, we applied erythromycin as an inducer of CH in the nduce CH . Since Don of CH . Induction of CH ,40. Elevon of CH ,41. Trea20) concentration of ISP in the H9C2 cells, we further studied the efficacy of YDR. ISP induced loss of cell viability, redox imbalance, and up-regulation in the mRNA expression of non-clinical and clinical biomarkers of CH. YDR given to the H9C2 cells in combination with the ISP ameliorated these drug-induced CH biomarkers.Based on the in-vivo study results, we expected YDR to play a major role in cardioprotection, by reducing stress signaling, and cardiac remodeling. In our in-vitro studies, we applied isoproterenol (ISP) for the induction of hypertrophy. ISP helps in normalizing the diminished heartbeat. However, prolonged use of ISP could also lead to change in the left and right ventricular wall function and physiology inducing cardiomyopathy . Therefo−2 generation. Potential growth in the ROS generation leading to a reduction in the cardiac mitochondrial cytochrome c and ubiquinone levels further modulating the mitochondrial membrane potential has been well-documented [−2. Hence, YDR acted as an anti-oxidant, or as a stimulator for cellular anti-oxidant  levels. Both these antioxidants were significantly reduced through the treatment of H9C2 cells with ISP alone and enhanced through a co-treatment with YDR.Oxidative stress-induced redox imbalance in the cardiomyocytes is one of the primary reasons for the development of myocyte hypertrophy . Mitochocumented ,46. H9C2−2 in cardiomyocytes [Cyclooxygenases (COX) and nicotinamide adenine dinucleotide phosphate oxidases (NADPH oxidases/NOX) represent the primary group of enzymes involved in the generation of ROS. Overexpression of COX-2 has been associated with angiotensin-II induced oxidative stress signaling pathways and CH . In the myocytes . NOX plamyocytes . A steadmyocytes . In our Oxidative stress activates the cell-death-associated cascades and contributes to maladaptive myocardial remodeling. The induction of hypertrophy in the adult myocardium is associated with the partial recapitulation of the embryonic program observed during cardiac development. Atrial natriuretic factor (ANF) is associated with remodeling during the induction of CH. It is a very sensitive marker, and overexpression of ANF in adult heart can lead to the development of defects in the chamber myocardium of the atria, ventricles, atrioventricular canal, and pacemaker tissue patients . In our D. rerio CH model, YDR acted as a therapeutic medicine and helped in the normalization of cardiac parameters, represented by ECG, heart morphology, and septum wall thickness. YDR also ameliorated ERY-stimulated release of pro-inflammatory C-reactive protein and normalized platelet aggregation time. In the in-vitro model of ISP-stimulated H9C2 cells, YDR was found to act as an anti-oxidant agent, helping in the reduction of excessive intracellular reactive oxygen species and superoxide anions generation and an associated increase in mitochondrial membrane potential. YDR also reduced the ISP-stimulated changes in the levels of antioxidant enzymes and, at molecular levels, clinical and non-clinical biomarkers associated with CH. Taken together, the results suggest a role for YDR as an alternative therapeutic medicine for the treatment of oxidative stress and inflammation-associated cardiac ailments such as cardiac hypertrophy.In conclusion, zebrafish represent an effective model for the study of cardiac hypertrophy and its amelioration by traditional Indian medicine Yogendra Ras (YDR). YDR was found to be formed mainly of cinnabar, gold, and tin. In the ERY-stimulated


Odontoblast cells generated from human dental pulp stem/progenitor cells (hDPSCs) secrete reparative dentin in responds to an injury. Endogenous Wnt signaling is also activated during this process, and these Wnt-activated cells are responsible for the following repair response. R-spondin 2 (Rspo2) is a potent stem cell growth factor, which strongly potentiates Wnt/β-catenin signaling and plays a vital role in cell differentiation and regeneration. However, the role of Rspo2 during odontoblast differentiation in hDPSCs has not yet been completely understood. This study investigated the effects of Rspo2 on hDPSCs to provide therapeutic insight into dentin regeneration and reparative dentin formation. HDPSCs were extracted from human molars or premolars. Immunofluorescence staining and flow cytometric analysis were used to detect the mesenchymal stem cell markers in hDPSCs. EdU assay and Cell Counting Kit-8 (CCK-8) were performed to explore cell proliferation. The odontogenic differentiation levels were determined by measuring the mRNA and protein expression of DSPP, DMP-1, ALP, and BSP. Immunofluorescence staining was performed to detect the localization of β-catenin. The biological effects of Rspo2 on hDPSCs was investigated using the Lentivirus-based Rspo2 shRNA and recombined human Rspo2 (rhRspo2). Recombined human DKK-1 (rhDKK-1) and recombined human Wnt3a (rhWnt3a) were used for further investigation. The cells generated from human dental pulp expressed mesenchymal stem cell markers Vimentin, Stro-1, Nestin, C-kit, CD90, and CD73, while were negative for CD3, CD31, and CD34. The mRNA expression levels of the odontogenic-related genes DSPP, DMP-1, ALP, and BSP were upregulated in the rhRspo2 treated cells. Silencing Rspo2 suppressed the proliferation and differentiation of the hDPSCs. Blockade of Wnt signaling with DKK-1 inhibited Rspo2-induced activation of Wnt/β-catenin signaling and cell differentiation. The combined use of rhWnt3a and rhRspo2 created a synergistic effect to improve the activation of Wnt/β-catenin signaling. Rspo2 promoted the proliferation and odontogenic differentiation of hDPSCs by regulating the Wnt/β-catenin signaling pathway. Several conditions can lead to exposure of the pulp of a tooth, such as dental caries, trauma, or mechanical responses. The consequences of pulp exposure may be severe, requiring a root canal treatment or extraction. An alternative therapy is direct pulp capping, by placing a material directly over the exposed pulp tissue, in an effort to retain pulpal vitality and health. A successful direct pulp capping can avoid the loss of a tooth or the need for replacement. Calcium hydroxide and trioxide mineral aggregates (MTA) are common pulp capping materials. Calcium hydroxide has consistently been considered the “gold standard” of the available direct pulp capping materials. HoweverRecombinant growth factors have been successfully used in clinical applications. For example, BMP-2 and BMP-7 have been approved by the FDA in the United States for spine fusion surgery . CanonicFtl mice (deficient in Rspo2) showed reduced osteogenesis compared with BMSCs from wild-type litter mates  are potent stem cell growth factors which strongly potentiate Wnt/β-catenin signaling . Severaler mates . Rspo2 ilization . Howeverlization . Therefoin vitro. We also investigated the pathway involved. We hypothesized that Rspo2 should be an agonist of hDPSCs to promote odontogenic differentiation through the Wnt/β-catenin signaling pathway.In the present study, we examined the effects of Rspo2 in the proliferation and odontoblast differentiation of hDPSCs 2, the medium was changed every 3 days.Human third molars or premolars were obtained from patients (18–25 years old) at The First Affiliated Hospital of Zhengzhou University. All of the donors signed an informed consent form. HDPSCs were isolated and cultivated, as described previously . Cells fDifferent concentrations, either 0, 50, 100, or 200 ng/mL, of recombinant human Rspo2 (rhRspo2) from PeproTech (United States) were added to the medium for use in subsequent experiments. In addition, 100 ng/mL recombinant human Wnt3a (rhWnt3a) from R&D Systems (United States) or 800 ng/mL recombinant human Dickkopf-related protein 1  from PeproTech (United States) were used in this study.For odontogenic differentiation, hDPSCs were cultured with odontogenic inductive medium containing 10% FBS, 10 mM β-glycerophosphate, 50 mg/ml ascorbic acid, and 100 nM dexamethasone  for 10 days, the medium was changed every 3 days. Differentiated cells then were collected for the quantitative real-time PCR (qRT-PCR), immunofluorescence staining, and western blot analysis.6 cells were trypsinized and incubated with anti-human CD90, CD73, CD3, CD33, and CD34 antibodies, respectively. All of the antibodies were purchased from BD Biosciences (United States). Flow cytometric analysis was conducted on a FACSAria II flow cytometer .Flow cytometric analysis was used to detect the surface marker of hDPSCs. A total of 1 × 104 cells were seeded on coverslips in each well of 24-well plates. At 80% confluence, cells were fixed with 4% formaldehyde, followed by permeabilized treatment with 0.5% Triton X-100. Subsequently, the cells were blocked with 5% bovine serum albumin (BSA) and then incubated at 4°C overnight with the following primary antibodies: anti-Stro-1 , anti-Vimentin , anti-Nestin , anti-C-kit , anti-dentin sialophosphoprotein (DSPP) , anti-osteopontin (OPN) , anti-dentin matrix acidic phosphoprotein 1 (DMP-1) , anti- alkaline phosphatase (ALP) , anti-osteocalcin (OCN) , anti-bone sialoprotein (BSP)  and anti-active β-catenin . The cells were washed and incubated with fluorescent-labeled secondary antibodies Dylight 488 goat anti-mouse IgG or Dylight 594 goat anti-rabbit IgG  for 1 h at room temperature. The coverslips were moved and mounted on glass slides with the presence of 10 mg/mL DAPI (4′6-diamidino-2-phenylindole)  for nuclei staining. Cell images were recorded using a fluorescence microscope  or a ZEISS confocal microscope (Germany).A total of 5 × 10For DSPP, OPN, DMP1, ALP, OCN, and BSP detection, cells were incubated in odontogenic differentiation medium with 0, 50, or 200 ng/mL rhRspo2 for 10 days before fixation. For β-catenin detection, cells were stimulated with or without rhRspo2 (200 ng/mL), rhDKK-1 (800 ng/mL), or rhWnt3a (100 ng/mL) for 2 h before fixation.4 hDPSCs were seeded into 96-well plates in complete medium (DMEM supplemented with 10% FBS). The next day, the medium was removed, and the cells were incubated in DMEM without FBS for 24 h. Next, the medium was changed to complete medium with different concentrations of rhRspo2 . After 24 h, the proliferation of hDPSCs was assessed using the EdU assay . Following incubation, the medium was removed, and the cells were incubated in DMEM containing 50 μM EdU for 2 h at 37°C. The cells were fixed with 4% formaldehyde, and were then incubated in the dark with Apollo dye solution for 30 min followed by a Hoechst33343 reaction solution for 30 min in room temperature. Using the fluorescence microscope , images were recorded.A total of 1 × 103 hDPSCs were seeded into 96 well plates in complete medium. The next day, different concentrations of rhRspo2  were added to the medium. Following 0, 1, 2, 3, 4, and 5 days of incubation, the proliferation of hDPSCs was assessed using the CCK-8 assay . Briefly, following incubation, the medium was removed, and the cells were incubated in DMEM containing CCK-8 for 1 h at 37°C. The optical density (OD) of each well was read at 450 nm using a microplate reader CMax Plus .A total of 5 × 105 cells were seeded into a 6-well plate. Infection was performed using the lentivirus at a MOI (multiplicity of infection) of 40 when cells were grown to a confluence of 30%. After 12–16 h, the medium was replaced with fresh complete medium. In addition, 72 h after infection, the frequency of the green fluorescent protein (GFP)-positive cells were used to assess the efficiency of infection. Thereafter, the cells were treated with 3 μg/mL puromycin for 5 days to obtain stabled-transfected cells. The knockdown efficiency of Rspo2 was verified by quantitative real-time PCR and western blotting.Lentivirus-based Rspo2 shRNA (shRspo2) and non-targeted negative control shRNA (shNC) were constructed, respectively . Approximately 0.6–1 × 10TMRT reagent Kit . Thereafter, the cDNA sample was analyzed using TB GreenTMPremix Ex TaqTMII  on Quantstudio 5 Real-Time PCR system . Cycle conditions were as follows: 95°C for 30 s, followed by 45 cycles of 95°C for 5 s, 55°C for 30 s, and 72°C for 30 s. Primers are listed in Total RNA was extracted from the cells with RNAiso Plus  according to the manufacturer’s procedure. A 1 μg sample of total RNA was reverse-transcribed to cDNA utilizing the PrimeScriptCells were washed and lysed with RIPA lysis buffer  supplemented with 1 mM protease inhibitor and incubated on ice for 30 min. Subsequently, the lysate was centrifuged at 12,000 × g for 5 min at 4°C, and then the supernatant was collected. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes . The membranes were blocked with 5% BSA in TBST and then incubated overnight at 4°C with the following primary antibodies: DSPP , DMP-1 , ALP , BSP , Non-phospho (Active) β-catenin , β-catenin  and GAPDH . HRP-conjugated goat anti-rabbit and goat anti-mouse secondary antibodies  were used and then incubated with ECL plus  and visualized using an Amersham Imager 600 Super Sensitive Multifunctional Imaging Instrument . The bands intensity were analyzed using Image J software.t-test. A p < 0.05 was considered statistically significant.All experiments were performed in triplicate and repeated three times. The data were analyzed using Prism6 software , and statistical analyses were performed with the Student’s To evaluate hDPSCs, we examined these cells for the presence of mesenchymal stem cell markers by immunofluorescence and flow cytometry. As demonstrated by immunofluorescence, hDPSCs were positive for the mesenchymal stem cell markers, which include Vimentin, Stro-1, Nestin, and C-kit . ResultsEdU and CCK-8 assays were performed to assess whether different concentrations of rhRspo2 could affect the proliferation of hDPSCs. As shown in In order to investigate the effect of Rspo2 on odontoblastic differentiation, cells were seeded into a 6-well plate and treated with odontogenic inductive medium with either 0, 50, and 200 ng/mL rhRspo2 for 10 days. We performed qRT-PCR to examine the gene expression of the odontogenic-related markers, which include DSPP, DMP-1, ALP, and BSP. The mRNA expression levels of these genes were up-regulated in rhRspo2-treated cells, with the highest mRNA expression levels found with the 200 ng/mL treatment group .In addition, using fluorescent microscopy to co-stain the markers DSPP, OPN, DMP-1, ALP, OCN, and BSP, we measured changes in fluorescent intensity as a readout to assess the function of odontogenic differentiation in hDPSCs. The fluorescence intensity increased significantly when the concentration of rhRspo2 was 200 ng/mL .To further explore the effect of Rspo2 on hDPSCs, we successfully constructed Rspo2 knockdown cells using Lentivirus-based Rspo2 shRNA. The knockdown efficiency of Rspo2 was verified using qRT-PCR and western blotting . Using tCells were seeded into 6-well plates and treated with an odontogenic medium to confirm whether silencing Rspo2 would influence odontogenic differentiation in hDPSCs. After 10 days, cell lysates were collected, and the mRNA and protein levels of the odontoblast markers, DSPP, DMP1, ALP, and BSP, were examined. We found that silencing Rspo2 impaired the odontogenic potential of hDPSCs significantly . We alsoWnt signaling pathway played a pivotal role in odontoblast differentiation . PreviouDickkopf-related protein 1 (DKK-1) is a ligand for the Wnt receptors LRP5 and LRP6 and a negative regulator of Wnt/β-catenin signaling . TherefoIn addition, the expression level and localization of β-catenin were assessed by western blotting and immunofluorescence, respectively. We induced the cells by treating with or without the rhRspo2 (200 ng/mL) or the rhDKK-1 (800 ng/mL) for 2 h. As depicted in Wnt3a is a Wnt protein that could activate the canonical β-catenin pathway and induces Tcf/LEF transcription factors to regulate the expression of the target genes . ZNRF3 cWe compared the mRNA expression levels of Wnt and Rspo2 related receptors between shRspo2 cells and shNC cells. As depicted in Stem cells isolated from different tissues such as bone marrow, muscle, adipose tissue, etc. all share two basic stem cell characteristics including self-renewal and the ability of multilineage differentiation. Stem cells derived from human dental pulp have been proven to have the advantages in myogenic differentiation, odontogenic/osteogenic differentiation, and chondrogenic differentiation . HDPSCs Canonical Wnt signaling plays a vital role in tissue generation, regeneration, and self-renewal . HoweverRspo2 has been reported to play a vital role in several types of differentiation, including neuroretina differentiation , chondroWe found that inhibition of Wnt/β-catenin signaling by DKK-1 significantly suppressed the enhanced odontoblast differentiation seen with Rspo2 treatment alone. Further, we found that silencing Rspo2 significantly inhibited the expression of active β-catenin. These data suggest that Rspo2 produced a positive effect on the odontogenic differentiation in hDPSCs via modulating Wnt/β-catenin signaling. These data correspond to findings regarding the osteoblast induction ability of Rspo2.The mechanism of activation for the Wnt/β-catenin signaling pathway by the R-spondin family is not fully understood. R-spondin functions via forming a ternary complex that prevents Wnt receptor degradation, which is mediated by RNF43 or ZNRF3 by binding to the R-spondin cognate receptors LGR4-6 and the E3 ubiquitin ligases RNF43 and ZNRF3 . It seemWe also compared the mRNA expression levels of Wnt and Rspo2 related receptors between shRspo2 cells and shNC cells. Rspo2 may regulate Wnt/β-catenin signaling pathway through binding to LGR4 receptor. The lower expression levels of LRP5 and LRP6 in shRspo2 cells may lead to the downregulation of the Wnt/β-catenin signaling pathway activated through Wnt ligands. However, in order to clarify the real condition, further investigations are needed to determine whether these membrane receptors are involved in the remodeling events of Rspo2 during odontoblast differentiation.Several studies have attempted to use growth factors or molecules for reparative dentin formation, such as Semaphorin 3A , Wnt3a , lithiumin vivo experiments to investigate the clinical uses of R-spondins and Wnt ligands, as well as their potential application in dentin formation.In summary, our findings suggested that Rspo2 played a vital role in the proliferation and odontogenic differentiation in hDPSCs, and knockdown of Rspo2 suppressed this activity. Inhibiting Wnt/β-catenin signaling by DKK-1 reduced the odontogenic effect of Rspo2. Wnt3a in combination with Rspo2 produced a synergistic effect during the activation of the Wnt/β-catenin signaling pathway. Our results demonstrated the feasibility of Rspo2 as a pulp capping agent to treat pulp exposure by activating endogenous Wnt signaling. Future work will focus on All datasets generated for this study are included in the article/supplementary material.The studies involving human participants were reviewed and approved by the local ethical committee of Zhengzhou University. The patients provided their written informed consent to participate in this study.YG, SY, and JS conceptualized and planned the experiments. YG performed all the experiments and wrote the manuscript. SY and JS contributed toward editing and proofreading the manuscript. YW and SL performed the experiments of immunofluorescence staining. WD and RG performed the experiments of quantitative polymerase chain reaction. RL was responsible for the overall project design and manuscript organization, revision, and finalization. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The uncharacterized protein FAM47E interacts with and stabilizes the protein arginine methyltransferase PRMT5, regulating its epigenetic functions thereby modulating target gene expression. Protein arginine methyltransferase 5 (PRMT5) symmetrically dimethylates arginine residues in various proteins affecting diverse cellular processes such as transcriptional regulation, splicing, DNA repair, differentiation, and cell cycle. Elevated levels of PRMT5 are observed in several types of cancers and are associated with poor clinical outcomes, making PRMT5 an important diagnostic marker and/or therapeutic target for cancers. Here, using yeast two-hybrid screening, followed by immunoprecipitation and pull-down assays, we identify a previously uncharacterized protein, FAM47E, as an interaction partner of PRMT5. We report that FAM47E regulates steady-state levels of PRMT5 by affecting its stability through inhibition of its proteasomal degradation. Importantly, FAM47E enhances the chromatin association and histone methylation activity of PRMT5. The PRMT5–FAM47E interaction affects the regulation of PRMT5 target genes expression and colony-forming capacity of the cells. Taken together, we identify FAM47E as a protein regulator of PRMT5, which promotes the functions of this versatile enzyme. These findings imply that disruption of PRMT5–FAM47E interaction by small molecules might be an alternative strategy to attenuate the oncogenic function(s) of PRMT5. Arginine methylation is a widely prevalent, important posttranslational modification affecting various cellular processes . ProteinPRMT5 plays a critical role in the differentiation of primordial germ cells, nerve cells, myocytes, and keratinocytes . NotablyThe enzymatic activity, substrate specificity, subcellular localization, and functions of PRMT5 is often regulated by its interaction partners . For insGiven the versatile functions of PRMT5 in the cell and its multiple interaction partners and substrates, the identification and characterization of new interaction partners is very important to obtain a comprehensive understanding of the diverse roles of PRMT5 in the cell. To address this, we performed yeast two-hybrid (Y2H) screening to identify new interaction partners of PRMT5 and identified a novel interaction partner FAM47E , an hitherto uncharacterized protein. In addition to identifying the new interaction partner of PRMT5; here, we have characterized the functions of FAM47E. We report that FAM47E regulates the stability, chromatin association and methyltransferase activity of PRMT5, with potential implications in normal physiology and in diseases.To identify new interaction partners of PRMT5, we performed Y2H screening of PRMT5 using universal normalized human cDNA library. Three positive clones were obtained in the initial screening with low stringency selection medium which scores for the expression of two reporter genes. Of these three clones, one of them failed in high stringency selection medium which assesses the expression of four reporter genes and hence it was not considered for further study. Sequencing of the other two positive clones revealed that they code for full length COP9 signaling complex subunit 5 (COPS5 protein) and the C-terminal region of FAM47E isoform 2 (43 amino acid to 295 amino acid). The COPS5 protein is known to interact with GAL4 DNA-binding domain directly and produce false positive results in GAL4-based Y2H screenings  and hencTo confirm the PRMT5-FAM47E interaction in vivo, we performed the co-immunoprecipitation (Co-IP) experiments by co-expressing Myc-tagged PRMT5 with GFP or GFP-tagged FAM47E in HEK293 cells. Similarly, we co-expressed HA-tagged FAM47E with GFP or GFP-tagged PRMT5 for reverse Co-IP experiments. We found that GFP-tagged FAM47E efficiently co-precipitated the Myc-tagged PRMT5 in forward Co-IP  and GFP-We next assessed whether the ectopically expressed GFP-tagged FAM47E could interact with endogenous PRMT5. For this, we overexpressed GFP or GFP-tagged FAM47E in HEK293 cells and performed the Co-IP experiments. We found that GFP-FAM47E efficiently co-precipitated the endogenous PRMT5 suggesting that FAM47E could interact with endogenous levels of PRMT5 . To asseBecause the WD40 repeat protein, MEP50, interacts and forms a stable hetero-octameric complex with PRMT5 , we inveBecause PRMT5 interacts with FAM47E, we investigated if PRMT5 methylates FAM47E. For this, we overexpressed GFP-FAM47E and the known PRMT5 substrate, GFP-SmD3, as a positive control, in HEK293 cells in the presence and absence of PRMT5 inhibitor, EPZ015666 . GFP-FAMProtein–protein interaction may result in alteration of (i) protein stability of the interacting proteins (ii) functional outcomes or (iii) both. We first tested if PRMT5-FAM47E interaction alters the stability of PRMT5 and/or FAM47E. To investigate this, we overexpressed GFP-tagged FAM47E and Myc-tagged PRMT5 individually or in combination. Whereas the overexpression of PRMT5 did not alter the levels of FAM47E, the overexpression FAM47E increased the PRMT5 protein levels . We nextFAM47E might regulate the levels of PRMT5 by (i) increasing the synthesis of PRMT5, (ii) stabilizing PRMT5 protein by binding to it and preventing its degradation or (iii) both. To gain mechanistic understanding on how FAM47E regulates PRMT5 protein levels, we first quantified the levels of PRMT5 transcripts in HEK293 cells upon perturbation of FAM47E by using qRT-PCR. We observed that the knockdown of FAM47E did not alter the levels of PRMT5 mRNA levels significantly . This suBecause the E3 ubiquitin ligase CHIP interacts with PRMT5 and promotes its proteasomal degradation through ubiquitination , we inveWe next investigated whether FAM47E binding to PRMT5 affects the functionality of PRMT5. One of the well-established functions of PRMT5 is the epigenetic regulation of gene expression through histone arginine methylation. In this regard, we first tested if FAM47E binding affects the association of PRMT5 to chromatin. For this, we perturbed the FAM47E levels in HEK293 cells  and isolWe next investigated if the increase in chromatin association of PRMT5 upon the overexpression of FAM47E translates into increased methylation of histones by PRMT5. To test this, we quantified the histone modifications that could be introduced by PRMT5 viz. (i) symmetric dimethylation of arginine 2 of histone 3 (H3R2me2s), (ii) symmetric dimethylation of arginine 8 of histone 3 (H3R8me2s), and (iii) symmetric dimethylation of arginine 3 of histone 4 (H4R3me2s) in histones isolated from HEK293 cells, upon overexpression or knockdown of FAM47E. We observed that the overexpression of FAM47E significantly increases the levels of H3R2me2s, H3R8me2s, and H4R3me2s modifications  and cyclin D1 (CCND1) genes (DCLK1), pentraxin-related protein (PTX3), and tumor necrosis factor α–induced protein 3 (TNFAIP3) genes (CAD and CCND1) that are negatively regulated by PRMT5 and reduced expression of genes  that are known to be positively regulated by PRMT5, suggesting that FAM47E facilitates PRMT5-mediated epigenetic regulation of gene expression  genes , which a1) genes  and (ii)3) genes , which a3) genes . These r3) genes  and S8B.3) genes . When FApression .Because FAM47E regulates the PRMT5-mediated epigenetic regulation, we investigated the binding of FAM47E and PRMT5 at the promoters of the tested PRMT5 target genes by using chromatin immunoprecipitation (ChIP). The details about the promoter regions of these genes for ChIP analyses were obtained from previous studies . We founPRMT5 levels are elevated in several cancer types and are associated with poor clinical outcomes . PRMT5 oBased on our above observations, we hypothesized that increased cell proliferation and colony forming capacity upon FAM47E overexpression could be mediated through increase in PRMT5 levels/activity. To test if the effect of FAM47E overexpression on cell proliferation and clonogenic capacity is mediated by PRMT5, we overexpressed the FAM47E in PRMT5-depleted HeLa cells and investigated the cell proliferation and clonogenic capacity of the cells. The knockdown of PRMT5 in these cells was confirmed by qRT-PCR and immunoblotting  and S11BGenome wide association studies indicated that the FAM47E is associated with chronic kidney disease and Parkinson’s disease . HoweverPRMT5 is a versatile protein which is involved in (i) epigenetic regulation via chromatin modifications and (ii) regulation of various other cellular processes through methylation and interaction of non-histone proteins. We found that the FAM47E increases PRMT5-mediated chromatin modifications by enhancing its association to the chromatin strongly and by decreasing the levels of PRMT5 in soluble fractions. This suggests that FAM47E promotes the epigenetic functions of PRMT5 and mitigates its non-epigenetic functions.We also report that elevated levels of FAM47E could contribute the oncogenic properties of the cells as it increases the cell proliferation and colony forming capacity of the cells and demonstrate that the oncogenic functions of FAM47E is mediated by PRMT5. PRMT5 levels are up-regulated in several cancers and the depletion of PRMT5 reduces the carcinogenic properties of cells which makes the PRMT5 enzyme as an important therapeutic target for cancer therapy. This led to the discovery of selective inhibitors for PRMT5 enzyme and some of them entered clinical trials . HoweverNM_001242936.1, Isoform 2) and PRMT5 (NM_006109.4) sequences were PCR-amplified and cloned in different vectors. FAM47E was cloned in pGADT7 vector (Clontech) using EcoRI and BamHI sites, pGEX-6P2 vector  using BamHI and XhoI sites, and pCDNA3-EGFP vector (Invitrogen) using BamHI and EcoRI sites to generate pGADT7-FAM47E, pGEX-FAM47E, and pCDNA-GFP-FAM47E constructs, respectively. The oligo encoding the HA tag was introduced in to pCDNA4/myc-HisA vector (Invitrogen) using XhoI and ApaI sites to generate pCDNA4-HA vector. FAM47E was also sub-cloned in pCDNA4-HA using BamHI and EcoRI sites to generate pCDNA4-HA-FAM47E construct. Similarly, full-length PRMT5 was cloned in pGBKT7 vector (Clontech) using EcoRI and BamHI sites, pCDN4/myc-HisA (Invitrogen) using EcoRI and XhoI sites, pET28a (Novagen) using BamHI and XhoI sites and pEGFP-C1 vector (Clontech) using XhoI and BamHI sites to generate pGBKT7-PRMT5, pCDNA4-Myc-PRMT5, pET28-PRMT5, and pEGFP-PRMT5 constructs, respectively. The sequence encoding the full length MEP50 (NM_024102.4) and the E3 ubiquitin ligase CHIP (NM_005861.4) was cloned in pCDN4/Myc-HisA vector using BamHI and XhoI sites to generate pCDNA4-Myc-MEP50 and pCDNA4-Myc-CHIP constructs, respectively. The sequence encoding the full-length SmD3 (NM_004175.5) was cloned in pEGFP-C1 vector using EcoRI and BamHI sites to generate pEGFP-SmD3 construct. The bacterial expression and purification of GST-tagged FAM47E and His-tagged PRMT5 was performed as described previously  screening was performed using Matchmaker Gold Yeast two Hybrid system (Clontech) as per the manufacturer’s instructions. To screen the interaction partners for PRMT5, pGBKT7-PRMT5 construct was used as a bait and human normalized cDNA library  were used as prey. The normalized human cDNA library lacks the abundantly expressed transcripts, resulting in improved representation of low abundance cDNA. The positive interactions in Y2H screening were identified by profiling the expression of reporter genes. The screening identified FAM47E as putative interaction partner for PRMT5.AUR1-C and MEL1. Contrarily, SD−Trp/−Leu/−His/−Ade/+Aba/+X-α-Gal medium selects the co-transformants that express the four reporter genes viz. HIS3, ADE2, AUR1-C, and MEL1. The expression of AUR1-C confers resistance to the toxic drug, Aureobasidin A, and the MEL1 codes for an enzyme, α-galactosidase which acts on the chromogenic substrate X-α-Gal. The positive interactions were confirmed by assessing the expression of reporter genes.The authenticity of the PRMT5–FAM47E interaction was investigated in Y2H assay by co-transforming pGBKT7-PRMT5 with pGADT7 vector or pGADT7-FAM47E construct in MM Gold yeast strain (Clontech). Similarly, co-transformation was also carried out using pGADT7-FAM47E construct and pGBKT7 vector. The transformants were plated in a (i) synthetically defined medium which lacks tryptophan and leucine (SD−Trp/−Leu), (ii) synthetically defined medium which lacks tryptophan and leucine but contains Aureobasidin A and X-α-Gal  and (iii) synthetically defined medium which lacks tryptophan, leucine, histidine, and adenine but contains Aureobasidin A and X-α-Gal  (Clontech). SD−Trp/−Leu medium allows the growth of all the co-transformants, whereas SD−Trp/−Leu/+Aba/+X-α-Gal media allows the growth of co-transformants which express the two reporter genes viz. 2. The plasmid constructs were transfected using standard calcium phosphate precipitation method. The siRNAs, siControl , siFAM47E , and siPRMT5 (5′-CUU UGA GAC UGU GCU UUA U 3′) were transfected using Lipofectamine 2000 Transfection Reagent (Thermo Fisher Scientific).HEK293 and HeLa cells were purchased from National Centre for Cell Science, India and grown in DMEM (HiMedia) supplemented with 5% fetal bovine serum (HiMedia) and glutamine–penicillin–streptomycin solution (HiMedia). The cells were grown in incubator supplied with 5% COCo-Immunoprecipitation (Co-IP) experiments were performed to investigate the interaction of PRMT5 and FAM47E. For forward Co-IP, pCDNA4-Myc-PRMT5 construct was co-transfected with either pCDNA-GFP vector or pCDNA-GFP-FAM47E construct in HEK293 cells. For reverse Co-IP, pCDNA4-HA-FAM47E construct was co-transfected with either pEGFP vector or pEGFP-PRMT5 construct in HEK293 cells. After 48 h of transfection, the cells were harvested and lysed in RIPA buffer . For co-immunoprecipitation, the cell lysates were incubated with GFP-Trap A beads (ChromoTek) for 8 h at 4°C. After the incubation, the beads were washed extensively with RIPA buffer.To investigate the interaction of FAM47E and MEP50, pCDNA-Myc-MEP50 construct was co-transfected with either pCDNA-GFP vector or pCDNA-GFP-FAM47E construct in HEK293 cells. To study the influence of FAM47E on PRMT5-MEP50 interaction, pCDNA-Myc-MEP50 construct was co-transfected with the combinations of pEGFP vector and pCDNA-HA vector or pEGFP-PRMT5 construct and pCDNA-HA vector or pEGFP-PRMT5 construct and pCDNA-HA-FAM47E construct in HEK293 cells. To study the influence of FAM47E on PRMT5-CHIP interaction, pCDNA-Myc-CHIP construct was co-transfected with the combinations of pEGFP vector and pCDNA-HA vector or pEGFP-PRMT5 construct and pCDNA-HA vector or pEGFP-PRMT5 construct and pCDNA-HA-FAM47E construct in HEK293 cells. After 48 h of transfection, the cells were harvested and lysed in lysis buffer . For co-immunoprecipitation, the cell lysates were incubated with GFP-Trap A beads (ChromoTek) for 8 h at 4°C. After the incubation, the beads were washed extensively with lysis buffer or wash buffer .The bound proteins were eluted by boiling the beads at 100°C for 5 min, and the eluted proteins were separated in 12% SDS–PAGE and transferred to PVDF membrane . The membrane was blocked overnight with blocking agent , washed with TTBS buffer  and probed with anti-Myc antibody  or anti-HA antibody . The whole cell extract which was used in co-immunoprecipitation were immunoblotted and probed with anti-Myc antibody or anti-HA antibody or anti-GFP antibody . The blots were developed using Super signal West Pico chemiluminescent substrate (Thermo Fisher Scientific) as per the manufacturer’s instructions and the images were captured in X-ray sheets in darkroom.To investigate the interaction of GFP-tagged FAM47E with endogenous PRMT5, HEK293 cells were transfected with either pCDNA-GFP vector or pCDNA-GFP-FAM47E construct. After 48 h of transfection the cells were lysed in RIPA buffer and immunporeciptation was carried out using GFP-Trap A beads (ChromoTek) as described above. The bound fractions were immunoblotted and probed with anti-PRMT5 antibody .To investigate the interaction PRMT5 and FAM47E at their endogenous levels, HEK293 cells were lysed in a lysis buffer . The lysates were incubated with rabbit IgG antibody  or anti-FAM47E antibody  or anti-PRMT5 antibody for 1 h at 4°C. After the incubation, 30 μl of Protein A Dynabeads (Invitrogen) were added to the lysates and incubated in rotator for 10 h at 4°C. The beads were washed thrice with wash buffer . The bound proteins were eluted by boiling the beads at 100°C for 5 min. The eluted proteins were immunoblotted and probed with anti-FAM47E primary antibody or anti-PRMT5 antibody. The whole cell extract which was used in immunoprecipitation was also immunoblotted and probed with anti-FAM47E primary antibody or anti-PRMT5 antibody.Glutathione sepharose 4B  beads were coupled with either 50 μg of GST protein or GST-tagged FAM47E protein in ice cold interaction buffer containing 20 mM Hepes (pH 7.5), 150 mM KCl, 0.2 mM DTT, 1 mM EDTA, and 10% glycerol. Then the beads were blocked with interaction buffer containing 5% bovine serum albumin for 1 h at 4°C. The blocked beads were incubated with 25 μg of His-tagged PRMT5 protein in binding buffer  for 3 h at 4°C. After incubation the beads were washed thrice with wash buffer . The bound proteins were eluted by boiling the beads with 2× LAP at 100°C for 5 min. The eluted proteins were immunoblotted and probed with anti-His antibody .To investigate the stability of Myc-PRMT5 protein upon GFP-FAM47E overexpression or vice versa, HEK293 cells were transfected with pCDNA-GFP-FAM47E construct or pCDNA4-Myc-PRMT5 construct individually or in combination. The cells were collected 48 h of post-transfection and lysed in RIPA buffer. The lysates were immunoblotted and probed with anti-GFP antibody or anti-Myc antibody or anti-GAPDH antibody .To study the effect of FAM47E perturbation on endogenous PRMT5 levels, HEK293 cells were transfected with control siRNA or FAM47E siRNA or pCDNA-GFP vector or pCDNA-GFP-FAM47E construct. After 40 h of transfection, the cells were treated with or without 50 μM of MG-132 and incubated for 8 h and lysed in RIPA buffer. The lysates were immunoblotted and probed with anti-PRMT5 antibody or anti-FAM47E antibody or anti-GFP antibody or anti-β actin antibody . The band intensities were quantified using the ImageJ software.g for 10 min at 4°C. The supernatant thus collected was labeled as soluble fraction and the pellet was resuspended in digestion buffer  and incubated in ice for 45 min. The benzonase digestion was stopped by adding 2 mM EDTA, reaction mixtures were centrifuged at 21,100g for 20 min at 4°C and the supernatants were collected and labeled as chromatin fractions. The soluble fractions were immunoblotted and probed with anti-PRMT5 antibody or anti-β actin antibody and the chromatin fractions were immunoblotted and probed with anti-PRMT5 antibody or anti-histone 3 antibody . The band intensities were quantified using the ImageJ software.The levels of PRMT5 association with the chromatin was investigated as described previously  with fewTo investigate the effect of ectopic expression of FAM47E on histone arginine methylation modifications, HEK293 cells were transfected with pCDNA-GFP vector or pCDNA-GFP-FAM47E construct or control siRNA or FAM47E siRNA. After 48 h of transfection, the cells were harvested and histones were isolated from these cells by standard acid extraction method as detailed previously . The isoHEK293 cells were transfected with pCDNA-GFP vector or pCDNA-GFP-FAM47E construct or control siRNA or FAM47E siRNA or co-transfected with pCDNA-GFP-FAM47E construct and control siRNA or PRMT5 siRNA. After 48 h of post-transfection, the cells were harvested, total RNAs were extracted using Trizol reagent (Invitrogen) according to manufacturer’s protocol and the cDNAs were prepared using Maxima H Minus Reverse Transcriptase (Thermo Fisher Scientific). The qRT-PCR analyses were performed using SYBR green Master mix (Roche) as per manufacturer’s protocol. The qRT-PCR reactions were performed in triplicates and each assay was repeated at least three times. The expression of target genes was normalized to the expression of GAPDH. To quantify the expression of different FAM47E isoforms, the total RNA was isolated from HEK293 cells and qRT-PCR analyses were performed as described above using isoform specific primers. The primers used in the qRT-PCR are listed in Table S1.List of primers used in the quantitative RT-PCR for various investigations.Table S1 The chromatin was prepared from HEK293 cells as described previously . For theHEK293 cells were transfected with pEGFP-SmD3 construct or pCDNA-GFP-FAM47E construct and treated with or without PRMT5 inhibitor, EPZ015666 (Sigma-Aldrich) to investigate the methylation of FAM47E by PRMT5. The pEGFP-SmD3 construct was co-transfected with pCDNA-HA vector or pCDNA-HA-FAM47E construct in HEK293 cells to study the influence of FAM47E on the methylation of SmD3 by PRMT5. After 48 h of transfection, the cells were lysed in the lysis buffer  and incubated with GFP-Trap A beads (ChromoTek) for 2 h at 4°C. After the incubation, the beads were washed extensively with lysis buffer and bound fractions were eluted by boiling the beads at 100°C for 5 min and the eluted proteins were separated in 12% SDS–PAGE, transferred to PVDF membrane and probed using or anti symmetric dimethyl arginine antibody, SYM10 , or anti-GFP antibody .For immunofluorescence, HEK293 cells were grown on the coverslip up to 80% confluency. Then the cells were washed with PBS; fixed by using 4% formaldehyde and permeabilized with PBS containing 0.25% Triton X-100. Then the cells were incubated with anti-FAM47E antibody  for overnight at 4°C and probed with anti-rabbit IgG Dylight 633 antibody . Then the cells were stained with DAPI and embedded using the Mowiol mounting medium, and the images were taken using the confocal microscope (LSM 510 Meta instrument).HeLa cells were transfected with pCDNA-GFP vector or pCDNA-GFP-FAM47E construct or control siRNA or FAM47E siRNA or co-transfected with pCDNA-GFP-FAM47E construct and control siRNA or PRMT5 siRNA. After 36, 48, 72 and 96 h of transfection, the cells were harvested, stained with trypan blue, and counted using a hemocytometer. MTT assays were performed as described previously .HeLa cells were transfected with pCDNA-GFP vector or pCDNA-GFP-FAM47E construct or control siRNA or FAM47E siRNA or co-transfected with pCDNA-GFP-FAM47E construct and control siRNA or PRMT5 siRNA. After 24 h of transfection, the clonogenic assay was carried out as described earlier .


Patients with coexpression of fascinhigh and FAKhigh or high β-catenin downstream targets showed the worst survival outcome, whereas in fascinlow, patient coexpression of FAKhigh or high β-catenin targets had less significant effect on the survival. Altogether, our data demonstrated the critical role of fascin-mediated β-catenin activation and its dependence on intact FAK signaling to promote breast CSC function. These findings suggest that targeting of fascin–FAK-β-catenin axis may provide a novel therapeutic approach for eradication of breast cancer from the root.Cancer stem cells (CSCs), a rare population of tumor cells with high self-renewability potential, have gained increasing attention due to their contribution to chemoresistance and metastasis. We have previously demonstrated a critical role for the actin-bundling protein (fascin) in mediating breast cancer chemoresistance through activation of focal adhesion kinase (FAK). The latter is known to trigger the β-catenin signaling pathway. Whether fascin activation of FAK would ultimately trigger β-catenin signaling pathway has not been elucidated. Here, we assessed the effect of fascin manipulation in breast cancer cells on triggering β-catenin downstream targets and its dependence on FAK. Gain and loss of fascin expression showed its direct effect on the constitutive expression of β-catenin downstream targets and enhancement of self-renewability. In addition, fascin was essential for glycogen synthase kinase 3β inhibitor–mediated inducible expression and function of the β-catenin downstream targets. Importantly, fascin-mediated constitutive and inducible expression of β-catenin downstream targets, as well as its subsequent effect on CSC function, was at least partially FAK dependent. To assess the clinical relevance of the  Recent years have witnessed increased overall survival of breast cancer patients that could be attributed to early detection and the substantial progress in developing more effective therapies. Nonetheless, tumor-related mortality remained high largely due to the presence of residual cancer cells, also called cancer stem cells (CSCs), which are chemoresistant and metastatic , 2. Fascβ-Catenin signaling cascade is initiated when cytoplasmic β-catenin is stabilized and translocated to the nucleus where it interacts with members of the T-cell factor (TCF)/Lef transcription factors and subsequently activates expression of the target genes. Mounting evidences have shown a critical role for β-catenin signaling in enhancing fascin expression and increasing cancer cell migration in multiple types of cancer. Indeed, β-catenin/TCF was reported to bind fascin promoter and increase its expression in colon cancer , 11, whihigh was coexpressed with high FAK or high β-catenin downstream targets, demonstrating a clinical relevance of our in vitro data. Altogether, the data presented in this study demonstrated that fascin–FAK-β-catenin axis plays a critical role in promoting breast CSC function, and the interference with this axis may thus provide an attractive approach for therapeutic targeting of breast cancer from the root.Here, we assessed the impact of fascin gain and loss of function on β-catenin expression and its subsequent effect on breast CSC function. Our results demonstrated that fascin directly enhances the constitutive and inducible expression of β-catenin downstream targets in a FAK-dependent manner. Furthermore, fascin-mediated activation of β-catenin signaling via FAK was critical for promoting self-renewability of CSCs. Importantly, gene expression analysis in breast cancer patients demonstrated shorter survival when fascinThe MDA-MB-231 (HTB-26) and T-47D (HTB-133) breast cancer cell lines were purchased from ATCC  and were maintained in culture as previously described (−) and control (fascin+) were established in the naturally fascin-positive MDA-MB-231 cells using fascin and scrambled ShRNA, respectively, as previously described (−) MDA-MB-231 cells using fascin ORF (FORF) or control ORF as previously described . The anti–cyclin D1 antibody was from Abcam .The focal adhesion kinase inhibitor (FAKi) and the glycogen synthase kinase 3β inhibitor (GSK-3βi) were obtained from Sigma , and the stock solution was diluted to desired concentrations in culture medium. Whenever applicable, cells were pretreated for 24 h with either 2 μM of FAKi or 5 μM of GSK-3βi or both, harvested, and added to the respective assay.−ΔΔCT equation as previously described (First-strand complementary DNA (cDNA) was synthesized from ~2 μg of extracted total RNA using High Capacity RNA-to-cDNA Kit . TaqMan fluorogenic probes for the specific gene were from Applied Biosystems  and all quantitative reverse transcriptase–PCR reactions were performed using Applied Biosystems 7500 Fast detection system. The relative mRNA expression was normalized against the housekeeping gene (GAPDH) and analyzed using 2escribed . QuantitCells lysates were prepared, and proteins were quantified as previously described . An optiThe tumorsphere assay was performed as previously described . Cells wThe colony-forming assay was performed in 6-well plates (Corning) as previously described  was added to the lower chamber, and cells (100 μL) were added to the upper chamber. For invasion, 25 μL of Matrigel was used to coat the upper chamber for 4 h in the 37°C incubator prior to the addition of cells. The cells were allowed to settle for 30 min at room temperature before loading the plate into the instrument, and measurements were taken at 15-min intervals for 24 h.Migration and invasion assays were performed using CIM-plate 16 on xCELLigence Real Time Cell Analysis instrument, from ACEA Biosciences Inc. , which allows quantitative kinetic measurement of cell movement from the upper chamber to the lower chamber. Cells were cultured for 24 h until they reach 60–80% confluence followed by 6-h serum starvation [1% fetal bovine serum (FBS)] before they were harvested and adjusted at 30 × 10Data acquisition was stopped after 24 h of measurement, and the average migration or invasion of all the replicates was calculated using the xCELLigence software. Migration or invasion signal was considered positive if it has a mean Cell Index of ≥0.1. Rate of cell migration and invasion was assessed by increases in the slope (1/h) of the curve during 24 h, and a graph displaying change in the slope between the different groups was generated.https://kmplot.com/analysis/)  . The datt-test between control and treated samples. Data were presented in mean or replicates ± SD. Any p-value of <0.05 was considered statistically significant.The analysis was performed using two-tailed paired Student −) in basal-like triple-negative MDA-MB-231 breast cancer cells (+) (p < 0.05) reduced the protein expression of β-catenin, as well as TCF3 and cyclin D1, known β-catenin targets (+ MDA-MB-231 cells at significantly (p < 0.05) higher levels as compared to their fascin− counterparts (− MDA-MB-231 as compared to their fascin+ counterparts (p < 0.05) increased the inducible expression of CTNNB, TCF3, CCND1, and c-Myc in fascin+ MDA-MB-231 cells more than in their fascin− counterparts  , signifi targets . We thenterparts . To valiterparts . Consistterparts . We haveterparts , which hterparts . Thus, wterparts , demonst− MDA-MB-231 cells (− MDA-MB-231 that was transfected with scrambled ShRNA (NORF), fascin-rescued cells (FORF) showed significantly increased expression of TCF3, CCND1, and c-Myc, which were comparable to that of the fascin+ (NORF) group  in fascin− MDA-MB-231 cells was diminished when treated with FAKi. To further validate the dependence of β-catenin signaling on fascin expression, FORF was used to express fascin in T-47D  group . Fascin in T-47D , hormonein T-47D . Importa+ MDA-MB-231 cells formed more primary and secondary tumorspheres as compared to their fascin− counterparts  counterparts. Both tumorsphere  with FAKi suppressed GSK-3βi–mediated induction of tumorsphere and colony formation. The dependence of GSK-3βi–mediated induction of tumorsphere and colony formation on fascin expression was also validated in the naturally fascin-negative T-47D breast cancer cells (NORF) and compared with their fascin-expressing counterparts (FORF). Glycogen synthase kinase 3β inhibitor failed to induce tumorsphere  on the function of fascinorsphere  and coloorsphere  formatioorsphere  or colonorsphere  formatioin vitro results above showed that fascin-mediated β-catenin signaling was FAK-dependent and indicated that this process was critical for promoting breast CSC function. To understand the clinical relevance of these findings, we have used the publicly available Kaplan–Meier plotter portal . The survival analysis revealed that patients with fascinhigh/FAKhigh expression have the worst RFS as compared with fascinhigh/FAKlow, fascinlow/FAKhigh fascinlow/FAKlow-expressing patients . The power of significance  of FAKhigh effect on RFS was dramatically reduced from <0.0001 in fascinhigh to 0.0152 in fascinlow patients  effect on worse RFS in fascinhigh patients, this significance was lost (p = 0.0654) in fascinlow patients (high expression showed significant (p < 0.0001) effect on worse RFS in fascinhigh patients; this significance was lost (p = 0.1613) in fascinlow patients (high or c-Mychigh) or dramatically reduced (FAKhigh or TCF3high) in fascinlow patients strongly suggests that the observed effect of FAKhigh and high β-catenin targets on RFS in fascinhigh patients was at least partially dependent on fascin expression. The poor RFS in patients that coexpress fascinhigh with FAKhigh or high β-catenin downstream targets demonstrated a clinical relevance to our in vitro findings. Collectively, these data suggest that fascin, FAK, and β-catenin downstream targets promote progression of breast cancer, and their levels of coexpression could serve as biomarkers for assessing the prognosis of the disease.The r portal  dataset patients . Similarpatients . To detepatients . Similarpatients . While Cpatients . Similarpatients . The facin vitro results, analysis of the publicly available survival dataset showed poor RFS in patients that coexpress fascinhigh with FAKhigh or high β-catenin downstream targets, demonstrating a functional relationship between these proteins that associate with worse patient survival outcome.Growing evidence has shown that fascin is not only a target of the β-catenin signaling pathway, but it can also activate Wnt/β-catenin signaling to promote EMT , a featuin vitro and in vivo when Wnt/β-catenin signaling was inhibited with small molecule antagonist (Increased expressions of fascin, β-catenin, and FAK were independently reported in breast cancer and were found to associate with enhanced self-renewability. In particular, the Wnt/β-catenin signaling pathway controls many cellular processes, but was also reported to be deregulated in many type of cancers including breast . Indeed,tagonist , and β-ctagonist . FAK tartagonist . Chen ettagonist  demonstrhigh and high β-catenin downstream targets is in line with the data reported by Shen et al. (high with FAKhigh or high β-catenin targets, whereas high expression of FAK or β-catenin targets has less significant effect on the survival of fascinlow patients. The survival data together with our in vitro results strongly suggest the dependence of β-catenin expression and function on fascin expression and the central role of FAK in this fascin-mediated β-catenin expression. The patient data reported in this study indicated poor survival when fascinhigh was coexpressed with FAKhigh or with high β-catenin downstream targets, strongly suggesting that their coexpression presents challenge for effective treatment of cancer. The demonstrated role of fascin in regulating breast cancer chemoresistance, metastasis, and CSC function makes it recognized as an attractive therapeutic target for the treatment of cancer. Our reported results provided insight into the role of fascin in mediating β-catenin activation through FAK and thus may give a new dimension to the field of breast cancer developmental therapeutics. Given that there are only limited studies that have targeted fascin through small molecules (The poor survival that we observed in patients that express fascinn et al. , where holecules , 29 and olecules . Interesolecules . Kim et olecules  suppressThe findings reported in this study of fascin-activating β-catenin signaling through FAK and the subsequent effect of this signaling cascade on CSC function present a feasible window for therapeutic targeting of fascin-FAK-β-catenin axis in order to effectively exterminate the core of cancer.All datasets generated for this study are included in the article/RB and SA-K performed the experiments and analyzed some of the data. TB performed all patient survival analysis using kmplot website. MF obtained fund for SA-K and provided materials support. AA edited the manuscript. HG performed some data interpretation and edited the manuscript. MA-A conceived and designed the experiments, analyzed the data, and wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.