Intimate partner violence (IPV) is a public health and widespread problem, and perpetrator programmes are in a unique position to work towards the end of gender-based violence. However, in order to promote safe perpetrator work, it is crucial to focus on the impact of IPV on the victims and survivors. In this context, little research has triangulated data by including both, victim’s perspectives on the impact that IPV has on them and also men’s level of awareness of the impact of their violent behaviour. In this paper, results from the “Impact Outcome Monitoring Toolkit (Impact Toolkit)” from one perpetrator treatment programme in the UK are presented. Participants were 98 in total; 49 men that were following treatment in a perpetrator program and their (ex-) partners. The differences in their perceptions of the IPV, but also on the impact of this abusive behavior on the victims, is described. Finally, recommendations for research and practice are discussed. Intimate Partner Violence (IPV) is defined by the World Health Organisation (WHO) as “any behaviour within an intimate relationship that causes physical, psychological or sexual harm to those in the relationship”  p. 1). . 1], andIn order to promote safe IPV perpetrator work, it is crucial to consider not just the presence and frequency of the abusive behavior but also the impact of this behavior on the victims and survivors. Research on the impact of violence has found that even if the short-term psychological consequences might be similar for partner and non-partner physical and sexual violence, the long-term psychological consequences are greater when the violence comes from a partner than when it is from a non-partner, emphasizing the consequences of a pattern of repeat victimization in IPV . PreviouIPV is understood and lived very differently from the perpetrators’ and victims’ side. Thus, their accounts might differ much when understanding or explaining the IPV situation that has happened. Even in terms of the attributions given for an abusive behavior some differences appear and victims give different explanations of why perpetrators exerted a given behavior than the ones’ given by the perpetrators’ themselves . In the Few studies have analyzed the perpetrators and their (ex-) partners accounts on the prevalence and/or impact of the violence behavior. One of the first researches that compared abusive men accounts with the ones of their (ex-) partners found that for physical abusive behavior men generally reported less violence  and that this difference was even more pronounced when comparing the reports on the frequency of those violent acts. As for the consequences of violence, women consistently reported receiving more injuries and more frequently than the ones reported by men. For the controlling behavior there was more concordance on the prevalence but not on the frequencies, being the women who reported more frequency of these behaviors . Sadly, In another study, Gondolf conducted a four-year follow-up evaluation of a perpetrator IPV programme in the US and found that the main predictor of re-assault was the women’s perception of their safety and concern of being re-assaulted . Thus, cAll in all, in this paper we aimed at answering the following questions: (1) are the men in treatment and their (ex-) partners views on the violent/abusive behavior similar or different at the beginning of the treatment and on which specific aspects do they converge or differ ? and (b) is the impact of the violent/abusive behavior perceived differently when comparing the men in treatment accounts with the ones’ of their (ex-) partners) at the beginning of the treatment? The tool used, the Impact Outcome Monitoring Toolkit (Impact Toolkit in what follows), allows the gathering of the same information from both parties, and thus to make comparisons within groups and within couples. Participants in this study were 49 male perpetrators of IPV enrolled in the Abusive Behavioural Change (ABC) Project for male and female perpetrators of domestic abuse, and their (ex-) partners. This programme was run by the Elm Foundation in Chesterfield, in the UK. The programme is part of a multi-agency approach against domestic violence, and regularly collaborates with police, and different social services; thus, participants are usually referred by social and child protection services or are self-referred. The 49 (ex-)couples were all the couples that joined the ABC programme between January 2017 and July 2019, and in which both the men and their (ex-)partner completed the Impact Toolkit at the beginning of the programme. They were from different cohorts of the ABC project, but the main characteristics of the programme and the requirements and profile of eligible participants have remained fairly stable. To participate in the programme, men were required to demonstrate at least some motivation prior to participation in the programme, and have to agree not to misuse alcohol and use drugs during the program. Participants were from a wide range of ages see , with thn = 18) or together (n = 11) and four were unsure about the status of their relationship. Most of them had children (n = 40). They were typically employed full-time (n = 32), while some were unemployed or unable to work (n = 12) and the rest were either employed part-time, studying or a combination of the former (n = 5). Reflecting the range of employment activity, participants were also diverse regarding their socioeconomical status , which was the most common referral route to the programme, involving more than half of the men who had children. Other channels were self-referral following publicity (n = 5), referral via mental health services (n = 5), police (n = 3), civil court mandation (n = 3), helpline (n = 3), (ex)partner (n = 2), health services (n = 2), relationship counselling service (n = 2), family and friends (n = 1), probation (n = 1), and solicitor (n = 1).Men were referred via a variety of channels. A high number of men were being pressured to attend by child protection and the social services regarding access to children (The instrument used in this study was the “Client and (ex)partner Impact Questionnaire”. This questionnaire is part of the Impact Outcome Monitoring Toolkit of the “European Network for the Work with Perpetrators of Domestic Violence (WWP EN)”. The toolkit comprises eight versions of the questionnaire, slightly adapted in relation to the phase  and in relation to the respondent (two versions: perpetrator and -ex-partner).In line with this paper’s aim to compare men’s and (ex-) partners’ perceptions of IPV at the beginning of a training programme, in this study we focus on the responses to the questionnaire for perpetrators and (ex-) partners at T1 (beginning of the programme), and on two scales of the questionnaire: violent/abusive behaviour and impact of violence/abuse. All the items of the two scales are equivalent across the men’s and (ex-) partners’ questionnaires. The first scale contains 29 items divided into three sub-scales regarding three types of IPV: emotional (10), physical (12) and sexual behaviour (7). These sub-scales can be evaluated at three different levels of frequency . The second scale, Impact of violence, comprises 16 items. It contains items about physical and emotional impacts on the (ex-) partner. Finally, the questionnaire also included three questions regarding the impact of IPV on children, related to whether the respondent thought children were affected by the abuse and whether they thought children were angry with the men and with the partner.Responses from the men and (ex-) partners were collected at the beginning of each round of the programme. The procedure used to collect the answers was different for each group. Men responded to the questionnaire on-site and on paper. They did it alone, but a facilitator was present in the room to assist with any questions or clarifications they might have. Partners and ex-partners are contacted at the beginning of the programme to inform them about the programme, its content and methods, the support services in case they needed them, and to learn about their experience of violence and their assessment of the effectivity of the programme. Thus (ex-) partners responded to the questionnaire as part of the protocol of (ex-) partners’ contact with the women’s support service. Responses were collected either over the phone or face-to-face depending on the involvement and availability of each person.Frequency of IPV. Six variables: three variables for each group for each type of abusive behaviour: emotional, physical, and sexual abusive behaviours, for each group of participants. Means were calculated for each sub-scale of the violent/abusive behaviour scale. Number of IPV behaviours. Six variables: three variables for each group for each type of abusive behaviour: emotional, physical, and sexual abusive behaviours, for each group of participants. Each item of the three sub-scales of the violent/abusive behaviour scale was recoded into a new binary item identifying presence (1) or absence (0) of each behaviour. The total number of present behaviours was calculated for each of the three sub-scales.Presence of IPV. Six variables: three variables for each group for each type of abusive behaviour: emotional, physical, and sexual abusive behaviours, for each group of participants. These three variables were binary variables that identified the presence or absence of abusive behaviours of each type. The number of IPV behaviours for each sub-scale (see above) was recoded into presence (1) or absence (0) of each type of behaviour.Impact of IPV on (ex-) partners. The number of impacts reported in the impact of violent behaviour scale was only counted by individual. The same analyses were conducted for (ex-) partners’ answers. Impact of IPV on children. Three variables: direct responses to the questions regarding impact of IPV on children were included.The present study compared men and (ex-) partners perceptions regarding IPV in relation to the following groups of variables. Each of these variables was calculated for each individual in both groups following the same procedure:n = 49) contained the answers of the man paired with those of his (ex-)partner.As we were interested in any differences between men and (ex-) partners within each couple, not in the differences between the two groups in general, responses from men were paired with data from their (ex-) partners. Thus, data were introduced into the database by couple and not by individual. Each case of the database (Descriptive statistics (mean and frequencies) for each variable and group were calculated. All the items in the two scales were given the same relative importance. We assigned a ‘type of violence profile’ to each man and (ex-) partner using the Presence of IPV variables . By combining these three variables, eight possible profiles emerge . Each individual was assigned to one of these profiles based on the type(s) of violence they reported. Frequencies of each type of profile were calculated and graphically displayed in Men and (ex-) partners’ perceptions about the frequency and number of emotional, physical and sexual abusive behaviours were compared using Wilcoxon signed-rank test, the non-parametric test used to compare matched samples. The Wilcoxon test was also used to assess differences in the impacts of violent behaviour on (ex-) partners both for the total number of impacts and for each item of the scale, and to compare men and (ex-) partners in regard to the impact of IPV on their children. Bivariate correlations among the measures of frequency of IPV behaviours  partners) and among measures of number of IPV behaviours  partners) were calculated using Pearson’s correlation coefficient. At the beginning of the programme, participants are required to sign a commitment contract, which also includes a data protection agreement informing them about the use and treatment of their personal information. All the participants agreed that their data would be used only for therapeutic and research purposes and that any use of this data for research purposes would ensure, at any time, the confidentiality and complete anonymity of data. Men and (ex-) partners were assigned an identification number which was used to answer the questionnaire. Programme staff were responsible for individuals’ personal information and the identification number assigned to each person. The authors of this paper did not have access to participants’ personal information at any time of the research.p < 0.01).At the beginning of the programme 23 men reported some kind of emotionally abusive behavior, 29 admitted at least one physical abusive behaviour and only 7 reported at least one sexual abusive behavior. In contrast, 34 (ex-) partners reported suffering some kind of abusive behavior, 33 reported at least one physical abusive behavior and only 4 reported suffering sexual abusive behavior. In the within pairs analysis, these differences were only statistically significant for presence of emotional abusive behaviour  coincided in the profile they reported: six reported no violent behavior of any type, eight reported emotional and physical but no sexual violent behaviors; and in one (ex-) couple both parties reported the three types of violent behavior. In the other 34 (ex-) couples, man and (ex-) partner reported different violent behavior profiles. The most prevalent combinations among them were partners who reported both emotional and physical violent behaviors with men who reported only physical behavior (n = 6), only emotional behavior (n = 5), all three types of behavior (n = 4) or no behaviors at all (n = 3).When comparing the profile report by men and their (ex-) partners within each couple, results show that less than a third of them , especially for sexual behaviour, with no differences between emotional and physical behaviours in terms of frequency see . Regardip < 0.01) and for frequency and number of physical behaviour (p < 0.05). Men and (ex-) partners were not different in regards to both measures of sexual behaviour.(Ex-) Partners considered violent behaviours to be slightly more frequent than men reported, and they reported experiencing a bigger number of violent emotional and physical behaviours than men did. The Wilcoxon test for paired samples showed these differences to be significant for frequency and number of emotional behaviours (n = 15); “Extreme jealousy or possessiveness” (n = 15); and “Threats to hurt partner/ex” (n = 12). As for the physical behaviours, the most frequent behaviours, according to men, were: “Slapped / pushed / shoved” (n = 23); “Restrained/held down/tied up” (n = 15); and “Physically threatened” (n = 13). Finally, the most frequently mentioned sexual behaviours were: “Disrespected boundaries or safe words” (n = 4); “Touched in way which caused her fear/alarm/distress” (n = 4).Within the group of men, the most frequently mentioned emotional behaviours were “Told partner what to do/not do, where to go/not go, who to see/not see” (n = 28); “Told what to do/not do, where to go/not go, who to see/not see” (n = 22); and “Threats to hurt you” (n = 21). As for the physical behaviours, the most frequent behaviours, according to (ex-) partners, were: “Slapped / pushed / shoved” (n = 26); “Stalked/followed/harassed you” (n = 18); and “Restrained/held down/tied up” (n = 17). Finally, the most frequently mentioned sexual behaviours was: “Touched in way which caused fear/alarm/distress” (n = 4).According to (ex-) partners, the most frequent emotional behaviours were “Extreme jealousy or possessiveness”  partners yielded statistically significant correlations among all eight measures of emotional and physical behaviours (frequency and number according to men and (ex-) partners) see . This shp = 0.094).Looking at the individual items, Within pairs, Wilcoxon test for matched samples showed statistically significant differences between men and (ex-) partners in regards to three items: “Injuries such as bruises/ scratches/ minor cuts”; “Depression or sleeping problems”; and “Partner having to be careful of what she said or did”. (Ex-) partners reported all these items more frequently than men.Finally, as for the impact of men’s behaviour on children, among the 40 men who had children, most of them thought the children were affected by the abuse and only 3 thought they were not. Although this number is higher according to (ex-) partners (n = 8), the differences are not statistically significant. The number of men and (ex-) partners who thought the children was upset with the men or the partner were very similar. This is the first study in which men and (ex-) partners accounts around the presence, frequency and impact of the violent/abusive behaviour were compared using the same tool for both, and some interesting results have been obtained.In regard to the presence and frequency of abusive behaviours several issues have emerged. First, comparing the most common emotional, physical, and sexual abusive behaviours stated by the men and their (ex-) partners, they seemed to be quite similar. The main difference was with regard to the “stalking” that (ex-) partners mentioned as one of the main abusive behaviours, whereas the men did not recognize it. Second, despite having quite similar views on the most common abusive behaviours, there were differences between the men and their (ex-) partners regarding the number and frequency of the emotional abusive behaviours. This was also visible in the profiles of violence per individual: while many men reported only physical behaviour, this profile was very rare among (ex-) partners who mostly reported physical connected to emotional behaviours. This seems to indicate that some men struggle to recognise or admit emotional abuse, which resonates with previous studies which found that the higher presence of emotional abuse, the greater possibility that victims also experience physical abuse . MoreoveThird, despite those differences, correlations among these two types of behaviours are still significant, which indicates that, although men report fewer emotional behaviours, the amount and frequency of these behaviours they report is similar to their (ex-) partners’ reports; and that the more emotional behaviours they report, the more likely they are to report more physical behaviours (both men in treatment and their (ex-) partners). This study has shown that the reports by men in treatment and their (ex-) partners are correlated, and so they seem to be able to identify an increase and/or changes in the abusive behaviour in a similar way. Despite this, the total amount and frequency of abusive behaviours detected by both is still quite different, and so, in order to assess risk and safety, it is crucial to always count on (ex-) partners’ accounts. As already pointed out by Gondolf , the  partners with regard to the frequency and number of sexual behaviours reported, when the profiles of each individual are analysed, surprisingly, more men report exerting sexual abusive behaviour. Moreover, sexual abusive behaviour did not correlate with the other violent behaviours, and there was no correlation between men’s and (ex-) partners’ perceptions of this type of abusive behaviour either. The low number of reported behaviours of this type could partially explain these results. Another plausible explanation would be that there is a general reluctance by both perpetrators and victims to disclose sexual violence; as showed in several reports, disclosure of adult sexual violence is quite low ,20. IndeFinally, a significant number of men, but also (ex-) partners, did not acknowledge that the men used any emotional, physical and/or sexual abusive behaviours. This could be the result of a series of factors: first of all, possible denial of violence from the perpetrators side , and also some minimization of violence from the victims side  as a resRegarding the impact of men’s violent behaviour, the main results obtained show, first, that both groups reported a similar number of impacts, but the most reported impacts were different; therefore, it seems that men recognize a similar number of impacts than their (ex-) partners, but those impacts are slightly different than the ones recognized by the (ex-) partners. More specifically, men had more difficulties identifying their (ex-) partners’ short-term physical impacts (such as bruises/ scratches/ minor cuts), and long-term psychological impacts such as: depression or sleeping problem; and the fact that the partner had to be careful of what she said or did because of the long-term coercive control exerted. Finally, regarding the impact of the abusive behaviour on the children, most of men are aware of the impact that the abusive behaviour had on them, which might be influenced by the fact that many of them men in this study had been told by social services/child protection to attend the programme. In what refers to the limitations of this study first of all it must be considered that the findings of this study are specific of a particular context, time, and group of individuals, and might be not representative of men and their (ex-) partners in other regions, with different backgrounds and cultures. Particularly, too, comparisons within couples with higher levels of violence might also yield different but very interesting and necessary insights. Moreover, it must be bear in mind that it was not possible to obtain an explanation for the reason of the differences observed among men in treatment and their (ex-) partners within this study as we did not include any open questions that would have allowed for a qualitative analysis of these reasons. For future studies, we aim at including qualitative data in order to explore further explanations. Moreover, in future research it would also be interesting to include information on the changes that men did during the course of the treatment, and on the evolution of these couples during the treatment. Finally, as recommendations for future research, it could be interesting to undertake an in-depth analysis of the profiles of violence according to men and their (ex-) partners including information of history of abuse or exposure to IPV in childhood for both participants. Comparing the accounts of men in treatment and their (ex-) partners has shown interesting results and has given some indications that are crucial for perpetrator programmes. It has become clear that in order to ensure safety and to assess the risk of perpetrator programmes it is crucial to include the (ex-) partners’ accounts as their accounts on the frequency and number of violent/abusive behaviors differ from the ones of their partners . Moreover, the impact of this violent/abusive behavior is also seen differently from both members of the couple, therefore, having the (ex-) partners’ accounts might help on increasing awareness (or accountability) of these impacts on the men’s side. All in all, the results obtained in this study have clear implications on perpetrator programme practice, and support the survivor contact element of IPV perpetrator programmes. As suggested by previous studies  the surv


Methods. In this study, 3T3-L1 cells were induced adipogenic differentiation and then treated with CGA. We investigate the effect of CGA in inhibiting adipogenesis and evaluate its role in modulating Wnt10b (wingless integration1 10b), β-catenin, glycogen synthase kinase-3β (GSK-3β), and peroxisome proliferator-activated receptor γ (PPAR-γ) involved in the Wnt (wingless integration1)/β-catenin signaling pathway. Results. The result showed that after CGA treatment, lipid accumulation and TG level decreased significantly in 3T3-L1 cells, indicating that CGA could inhibit adipogenesis. In addition, CGA repressed the induction of adipocyte differentiation biomarkers as PPAR-γ, adipocyte protein 2 (aP2), fatty acid synthase (FAS), and lipoprotein lipase (LPL), and the secretion of GSK-3β in a dose-dependent manner upregulated the expression of β-catenin and Wnt10b both in gene and protein levels. Moreover, CGA induced phosphorylation of GSK-3β and promoted the accumulation of free cytosolic β-catenin in 3T3-L1 adipocytes. Conclusion. Overall, these findings gave us the implications that CGA inhibits adipogenesis via the canonical Wnt signaling pathway.In our previous in vitro study, we found that chlorogenic acid (CGA) inhibited adipocyte differentiation and triglyceride (TG) accumulation, but the underlying mechanism is still unclear. Accumulative genetic evidence supports that canonical Wnt signaling is a key modulator on adipogenesis.  Obesity is becoming a worldwide health concern for its close association with cardiovascular disease, type 2 diabetes, hypertension, metabolic syndrome, osteoarthritis, and certain types of cancer . Accordiγ and CCAAT-enhancer-binding proteins (C/EBPs), along with the regulation of several signaling pathways, including Wnt, mitogen-activated protein kinase (MAPK), Hedgehog, and Insulin-Like Growth Factor 1 (IGF-1) [β-catenin pathway. The importance of peculiar Wnt/β-catenin signaling has directed considerable attention in the future production of therapeutic approaches in obesity [Adipogenesis is triggered by a cascade of transcriptional networks, mainly PPAR- (IGF-1) . One of  obesity .There are many options for treating obesity on the market that are prevented from being widely used in a clinical environment because of their side effects such as hypertension, cardiovascular problems, damage to liver function, and psychiatric disorders . Thus, sCGA , a phenoThe 3T3-L1 cell line has been used extensively and reliably in in vitro models for studying the conversion of preadipocytes into adipocytes . Severalγ, aP2, FAS, LPL, and GSK3β were obtained from cell signaling .Dulbecco's modified eagle medium , penicillin-streptomycin , Cell Counting Kit-8 (CCK-8) solution , dimethyl sulfoxide , Phosphate-Buffered Saline , 3-isobutyl-1-methylxanthine , Dexamethasone , Insulin , fetal bovine serum , CGA , lithium chloride , and Oil Red O  were used. Polyclonal antibodies against PPAR-4 cell/cm2 in 24-well plates. Following a 48 h culture after cells reached 90–95% confluence (Day 0), the cells were treated with adipogenic medium containing DMEM with 10% FBS, 0.25 mM 3-isobutyl-1-methylxanthine (IBMX), 1 µM dexamethasone, and 500 nM insulin for 2 days (Day 2). And then, cells were maintained in 10% FBS/DMEM with 500 nM insulin for an additional 4 days (Day 6). Thereafter, the cells were maintained in postdifferentiation medium containing 10 g/mL insulin in 10% FBS/DMEM. Differentiation was measured by the appearance of lipid droplets with the use of the dye ORO. Cells were maintained at 37°C in a humidified 5% CO2 and differentiated for a total of 12 days with medium changed every 2 days.The 3T3-L1 cells were obtained from the Sojubio . This cell line has been used to study physiopathological mechanisms of adipogenesis . PrimaryµM previously reported by researchers were evaluated for CGA in the present study [µg/mL of CGA were added to the media on Day 0. The cell viability of the extracts was determined at 12, 24, and 48 h. CCK-8 solution at 10% was added to each well, and the plates were incubated for 4 h at 37°C. After incubation, absorbance was read at 450 nm as optical density (OD) value by Multiskan Microplate Photometer (FC) enzyme-labeled instrument Multiscan MK3 , and the surviving cell fraction was calculated. The cell viability by OD values and the percentage of viable cells following treatment with the CGA compared to the vehicle control (0.1% DMSO) is expressed as follows.The proliferation rate = average OD value of certain time point/average OD value of Day 0 × 100%The inhibition rate =  × 100%The nontoxic concentrations ranging from 1 to 500 nt study . The inf3T3-L1 cells were washed with PBS three times and fixed for 10 min in 4% paraformaldehyde. Fixed cells were stained with the ORO-isopropanol at 3 mg/mL in 60% (v/v) isopropyl alcohol for 1 h at 25°C, and the excess stain was washed by 70% ethanol and water. Cells were visualized by using an IX-73 inverted biomicroscope  to evaluate stained lipid droplets. The diameters and areas of lipid droplets were analyzed by the Image pro plus 6.0 .Adipogenesis was assessed in the well-characterized mature 3T3-L1 cell models by measuring the accumulation of TG as described previously using Triglyceride Assay Kit  according to the manufacturer's instruction.β-catenin antibody  overnight. After washing three times with PBS, cells were incubated with Rhodamine (TRITC)-conjugated goat anti-rabbit IgG (H + L)  at 1:200 for 1 h at room temperature in the dark and then stained with 4′, 6-diamidino-2-phenylindole  for 10 min. Slides were then washed three times and mounted. Samples were viewed with a Leica confocal microscope .Cells were fixed in 4% paraformaldehyde for 30 min and permeabilized in 0.2% Triton X-100 for 5 min. After blocking with goat serum for 30 min, the cells were incubated overnight at 4°C with diluted monoclonal mouse anti-TM, Carlsbad, CA, USA), according to the manufacturer's instructions. The sequence and GenBank accession number for the forward and reverse primers used to quantify mRNA are listed in µL containing 0.5 mM of all the primers. Briefly, first-strand complementary deoxyribonucleic acid (cDNA) was synthesized from 0.2 µg of total RNA using M-MLV Reverse Transcriptase . The total reaction volumes of RT-PCR reactions were 20 µL. The system consisted of 2 µL RNA, 1 µL Oligonucleotide 18 primer, 9 µL RNase-free water, 4 µL 5× reaction buffer, 1 µL Riblock Rnase Inhibitor, 2 µL 10 mM deoxyribonucleoside triphosphate mixture (dNTP MIX), and 1 µL Moloney Murine Leukemia Virus reverse transcriptase (MMulV). The reaction products were stored at −80°C for long-term preservation. The cDNA was amplified in QuantiFast SYBR Green RT-PCR Kit  in a Bio-Rad iCycler iQ Real-Time PCR System . The sequence and GenBank accession number for the forward and reverse primers used to quantify mRNA are listed in µL containing 0.5 mM of all the primers. The mixture was subjected to the following program of amplification: the first segment of 10 min at 95°C for preincubation of the enzyme, the second segment of amplification consisting of 35 or 40 cycles with each cycle consisting of a thermal ramp to 20°C/s, an alignment segment to 61°C with 7 s thermal ramp to 20°C/s, and an amplification segment at 72°C for 10 s with a thermal ramp of 20°C/s. The program consisted of an initial denaturation at 95°C with a thermal ramp of 20°C/s, realignment at 65°C for 15 s with a thermal ramp of 20°C/s, and finally, a slow denaturation to 95°C at a thermal ramp of 0.1°C/s, with continuous measurement of the fluorescent signal integrity of PCR products that were verified by electrophoresis in 2% agarose gel stained with ethidium bromide .The total Ribonucleic Acid (RNA) was extracted from cells using TRIzol reagent (Invitrogen∆∆Ct) method was used to analyze the data. Each experiment was repeated independently three times. Results are reported as a fold change of the value obtained for the vehicle control.The statistical analysis of the qRT-PCR was obtained by using the comparative threshold-cycle (CT) method, which calculates the relative changes in gene expression levels of the target gene normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The relative quantification , 2 mM phenylmethylsulfonyl fluoride, 80 µg/mL leupeptin, 3 mM NaF and 1 mM dithiothreitol (DTT), and a protease inhibitor cocktail to obtain the total cell lysates. The total cell lysates were centrifuged at 12,000 rpm for 20 min at 4°C to remove insoluble materials. Protein extract samples (20 μg) were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes at 150 mA for 1 h. The membranes were then blocked for 1 h at room temperature with phosphate-buffered saline (PBS) containing 5% skim milk and 0.1% Tween 20. The membranes were then incubated with 1:1000 dilution of primary antibodies overnight at 4°C and subsequently with a horseradish peroxidase-conjugated anti-rabbit secondary antibody for 1 h at room temperature. The protein bands were detected with an enhanced chemiluminescence (ECL) detection system  according to the manufacturer's instructions and visualized with the G:BOX EF imaging system and the Gene Snap program . The Western blotting results were confirmed twice by independent experiments.After 12 days of differentiation, cells were removed from the plates, and the protein expression of the key molecules was evaluated by Western blotting. Cells were washed in ice-cold PBS and then were resuspended in lysis buffer containing 50 mM Tris-HCl (pH 8.0), 0.4% Nonidet P-40, 120 mM NaCl, 1.5 mM MgClt-test and one-way analysis of variance (ANOVA) followed by the post hoc Bonferroni test were used. A P value of less than <0.05 was considered statistically significant.All experiments were repeated independently at least 3 times. All quantitative data are presented as means ± SEM. Statistical significance for comparisons between treated samples and corresponding untreated samples was performed using Statistical Product and Service Solutions (SPSS) 22.0 . Student's μg/mL. 240 μg/mL CGA slightly decreased the proliferation rate and increased the inhibition rate of 3T3-L1 cells from Day 1 to Day 2  value by Cell Counting Kit-8 (CCK-8) assay. 3T3-L1 cells incubated for 12, 24, and 48 h with CGA do not cause significant loss of cell viability at concentrations ranging from 50 to 240 to Day 2 .μg/mL) and lithium chloride (LiCl) of 20 mM separately to the dimethyl sulfoxide (DMSO) and MDI  induction medium of cells on Day 0 of differentiation. The final concentration of DMSO was kept at 0.1% throughout the differentiation period. Oil Red O (ORO) staining was applied in adipocytes-induced differentiation for 7 days (μg/mL CGA significantly reduced the areas and diameters of lipid droplets (∗P < 0.05); however, compared with the MDI group, 50 μg/mL CGA did not change the areas and diameters of lipid droplets (∗∗P > 0.05). These data indicate that 50 μg/mL CGA had almost no impact on them. Based on these, our in vitro study was focused on the concentration range of 100 and 200 μg/mL CGA.We further investigated the effect of CGA on lipid accumulation by adding CGA  and 20 mM LiCl to the DMSO with MDI induction media of cells. As the undifferentiated group, the vehicle control was cultured in the 0.1% DMSO. By inducing differentiation for 12 days, 95% 3T3-L1 cells were characterized with increasing numbers and sizes of lipid droplets as the mature adipocyte phenotype. On Day 12, the intracellular lipid accumulation and lipid droplets are greater in the MDI group as compared to those of vehicle control (0.1% DMSO). 200 μg/mL CGA resulted in a greater reduction in intracellular lipid content formation compared to that treated with 100 μg/mL CGA . CGA downregulated the TG contents in a dose-dependent manner, 200 μg/mL CGA had a more notable effect on the reduction of TG content as compared with 100 μg/mL CGA (P < 0.01).On Day 12, the total cellular TG content of adipocytes was evaluated. Consistent with ORO staining, Triglyceride Assay showed a very low level of TG was present in the medium of vehicle control and increased approximately 20 times in the medium of MDI group  in a dose-dependent manner by comparison with the MDI group  has approved several drugs such as Qsymia, Belviq, Contrave, and Saxenda for the treatment of obesity, the use of these drugs has been associated with side effects, including the development of cardiovascular disease and adverse reactions of the gastrointestinal tract . Researcβ inhibitor) can inhibit adipocyte differentiation [β-catenin signaling, LiCl can significantly inhibit adipogenesis by acting on the Wnt signaling pathway [Suppression of lipogenesis and adipogenesis is a feasible approach against obesity, and lipid accumulation reflected the process of differentiation of preadipocytes into adipocytes . Consistntiation . In this pathway . 20 mM tγ is a ligand-activated factor and the most important transcriptional modulator of adipocyte development in all types of adipose tissue by mediating the expression of target genes as aP2, FAS, and LPL [γ in MDI-treated 3T3-L1 cells. Meanwhile, the expression of target genes aP2, FAS, and LPL was also decreased in a dose-dependent manner after CGA treatment  in the 3T3-L1 cells.PPAR- and LPL . In the reatment . Similartment (γ . Howeveripocytes . The difβ-catenin dependent) and noncanonical (β-catenin independent). Canonical Wnt signaling has been explored more thoroughly than noncanonical signaling [β-catenin signaling plays differential regulatory roles in adipogenesis by suppressing the differentiation of preadipocyte cells to mature adipocytes. It was demonstrated that the Wnt/β-catenin signaling is similar to an adipogenic switch—when it is on, adipogenesis is repressed; when it is off, adipogenesis is initiated [β-catenin is particularly crucial in that the cytoplasmic accumulation and then nuclear translocation of β-catenin (nonphosphorylated β-catenin) enhance the transcriptional activity and regulate the target genes expression [β-catenin pathway [β-catenin was mainly located in the cytoplasm rather than the nucleus in differentiated adipocytes, indicating that the Wnt/β-catenin pathway was inactive, while CGA promoted the nuclear translocation of β-catenin from the cytoplasm /CK1α (Casein Kinase1α), so that β-catenin in the cytoplasm remains at a relatively low level [β leads to the disintegration of the complex and the nondegradation of β-catenin. After the accumulation in the cytoplasm, β-catenin enters the nucleus and upregulates the transcription of genes [β, suggesting that CGA might inhibit adipocytes and lipid accumulation by activating the β-catenin signaling pathway via inducing GSK-3β phosphorylation. This is the first study showing the mechanism of CGA inhibiting adipocyte differentiation and its effects on the Wnt/β-catenin signaling pathway after adipocyte differentiation.Wnt family proteins can inhibit preadipocyte differentiation through a canonical pathway (ignaling . Considenitiated . As the pression . In the  pathway , 31. Ourytoplasm . Consist Figures , suggestow level . Therefoof genes . In the β-catenin signaling pathway. As the preliminary study, this research investigated the underlying mechanism of the inhibitory effect of CGA on adipogenesis via the Wnt/β-catenin signaling pathway in vitro. Further studies will be needed in vivo using gene knockout animal models to clarify the mechanism based on the Wnt/β-catenin signaling pathway.Our findings suggest that CGA may be a potential therapeutic agent for obesity with the mechanism involving the Wnt/


CDH1 gene, which encodes the E-cadherin protein, is the best known gene associated with these tumors. The structural and functional integrity of E-cadherin is regulated by interconnecting molecular pathways which might participate in the development of this disease. Hence, we analyzed the protein expression in key genes in E-cadherin-related pathways associated with primary SRC/Histiocytoid carcinoma of the eyelid. SRC/Histiocytoid carcinoma diagnosed in the eyelid/orbit at MD Anderson Cancer Center from 1990 to 2016 were evaluated. Clinicopathologic findings were studied to confirm the primary site of origin. Immunohistochemical studies for the expression of E-cadherin, β-catenin, c-Myc, Cyclin D1, Src, and p53 were analyzed. Next generation sequencing for the detection of somatic mutations was performed on each tumor with matched normal tissue, examining 50 cancer-related genes. Four primary SRC/Histiocytoid carcinomas of the eyelid were diagnosed in four male patients aged 40-82 years. Immunohistochemically, two tumors with loss of E-cadherin expression had weak β-catenin and low cytoplasmic staining for Src while the other two cases with intact E-cadherin showed strong β-catenin expression and high cytoplasmic expression for Src. Cyclin D1 was focally positive in three cases. Somatic mutations in CDH1, PIK3CA, and TP53 genes were detected in two cases. Our results suggest an abnormality in the convergence of E-cadherin/β-catenin pathways which may promote tumorigenesis by inducing expression of oncogenes such as Cyclin D1 and C-Myc. Mutations in CDH1, PIK3CA, and TP53 genes could induce E-cadherin dysfunction which takes part in the development and progression of this malignancy.Signet Ring Cell (SRC)/Histiocytoid carcinoma of the eyelid is a rare neoplasm that shares histological and immunohistochemical similarities with diffuse gastric cancer and breast lobular carcinoma. The  Primary Signet Ring Cell (SRC)/Histiocytoid carcinoma of the eyelid is a rare, aggressive neoplasm favored to arise from the eccrine sweat glands. To date, less than 40 cases of SRC/Histiocytoid carcinoma arising from the eyelids have been described in English literature –3. CliniCDH1, a tumor suppressor gene encoding E-cadherin protein, is one of the known pathogenic mechanisms for this disease which correlates well with loss of E-cadherin expression by immunohistochemical evaluation [β-catenin complex is required to maintain the integrity of epithelial cell-cell contact, keeps Wnt/β-catenin signals in check, and is involved actively in epithelial-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) which plays an important role in embryo development, tissue fibrosis, and cancer progression [CDH1-E box elements by the transcription factors Snail and Sip-1; and phosphorylation by tyrosine kinase such as EGFR, c-Met, FGFR, and c-Src [β-catenin promotes tumorigenesis by upregulating the expression of oncogenes such as c-Myc and Cyclin D1 [β-catenin complex.Histologically, this tumor shows diffuse involvement of the eyelid by predominantly single cells with a spectrum of histologic features encompassing atypical cells with intracytoplasmic vacuoles and eccentric nuclei, which exhibit signet ring cell appearance and/or cells with amphophilic cytoplasm and large nuclei resembling histiocytes. These histologic findings in the eyelid are comparable to signet ring cell carcinoma (SRCC) of the gastrointestinal tract and breast. In SRCC of gastric origin and breast lobular carcinoma, E-cadherin plays a central role in the pathogenesis, and hereditary loss of aluation . Physiolgression . Loss ofnd c-Src –16. The yclin D1 . As mechWe searched the pathology files at The University of Texas MD Anderson Cancer Center (MDACC) from 1990 to 2016 for carcinomas of the eyelid or orbit with diagnosis of/or description that included histiocytoid or signet ring cell features. Five cases of SRC/Histiocytoid carcinoma of the eyelid and orbit were identified at our facility. Clinical and histopathologic findings were evaluated including imaging workup to exclude metastasis to this site. Metastatic adenocarcinoma of the breast to the eyelid was found in one of the 5 cases and was excluded from the analysis. The histopathologic slides of the primary four cases with SRC/Histiocytoid carcinoma of the eyelid were retrieved and analyzed. Clinical and surgical records were evaluated for reporting in conjunction with the histopathologic and immunohistochemical findings. The study was approved by the institutional review board of MDACC. The approach to the pathologic evaluation whether during immediate intraoperative assessment or for permanent processing must account for the ill-defined nature of the disease and potential widespread infiltrative single-cell pattern of growth. As performed in two cases of orbital exenteration, circumferential mapping of the eyelids around the globe at each clock designation aided in determining the extension of the disease which was beyond the clinically apparent tumor in all the cases .β-catenin , c-Myc , Cyclin-D1 , Src , p63 , Her-2/neu protein , and p53 . Immunohistochemical studies were performed on an automated stainer . All antibodies and testing were performed in a CLIA-certified laboratory with standardized clinical methodology and positive control tissue.Formalin-fixed, paraffin-embedded 4-micra unstained tissue sections were immunohistochemically analyzed with monoclonal antibodies: cytokeratin 7 (CK7) , cytokeratin 20 (CK20) , E-cadherin , β-catenin patterns of expression were analyzed using the adjacent normal epithelium as an internal positive control. Cytoplasmic and membranous immunostaining for E-cadherin and the pattern of membranous staining for β-catenin immunoreactivity were analyzed based on prior study by Karayiannakis et al.[β-catenin (cytoplasmic or nuclear) was also considered within the category of abnormal expression. Sections with 10% or less stained tumor cells or with complete absence of staining for E-cadherin and/or β-catenin were considered negative. Src cytoplasmic and membranous staining was evaluated using the adjacent normal keratinocytes. We defined negative as no cytoplasmic immunoreactivity, weakly positive when decreased/low cellular intensity was seen in less than 25% of tumor cells, and positive/high immunoreactivity when 25%-75% of tumor cells exhibited cytoplasmic staining with the same or higher intensity as normal adjacent epidermal cells. [Evaluation of each immunohistochemical marker included the cellular distribution and percentage of tumor staining. Cytoplasmic immunoreactivity was evaluated for CK7 and CK20 while nuclear immunoreactivity for c-Myc, Cyclin-D1, p63, and p53 were assessed. We defined negative as no reactivity or less than 1% immunoreactive tumor cells, focal (+) when positivity was seen in 1% to less than 25%, moderate (++) when reactivity was seen in 25% to 50%, and strong diffuse (+++) when immunoreactivity was present in more than 50% of the tumor cells, based on immunohistochemical findings of previous studies [is et al.. When 90l cells. The interpretation of Her-2/neu status by immunohistochemistry as well as fluorescence in situ hybridization (FISH) was based upon American Society of Clinical Oncology/College of American Pathologist's criteria (2013 updated guidelines) for Her-2/Neu testing in breast cancer.Molecular profiling using next generation sequencing (NGS) on the Ion Torrent PGM (Life Technologies) was performed on each tumor with matched normal tissue. Fifty cancer-related genes were examined utilizing the AmpliSeq Cancer HotSpot v2 Panel . NGS canμm thick as described previously [Serial sections of formalin-fixed paraffin-embedded tissues were reviewed by our pathologists to circle the tumor-rich area and estimate tumor purity of more than 60%. DNA was extracted from a random number of unstained sections, each 0.4 eviously . DeparafAn 82-year-old male consulted MDACC with a right infraorbital ulcerated subcutaneous nodule and a right nontender neck mass. He had history of SRCC arising in the right orbit diagnosed 13 years prior to presentation at MDACC, followed by radiation therapy, chemotherapy, and right orbital exenteration. His family history revealed lung cancer in one brother, and one sister died of stomach cancer. A CT scan of the maxillofacial area and neck confirmed soft tissue thickening along the right infraorbital region  and metaA 65-year-old male with a right lower eyelid mass had history of adenocarcinoma of the left orbit, status post radiation therapy, followed by left orbital exenteration, 12 years prior to presentation at MDACC. An MRI of the orbit, face, and neck performed at MDACC demonstrated a thickened enhancing lesion adjacent to the right nasolacrimal duct measuring 4.1 × 1.0 cm  which exFour years of documented follow-up  after the first surgery done for the lower eyelid mass, he was still alive. Currently, the patient is being seen at an outside facility with his only eye globe intact. He has had slowly progressive distant lymph node metastatic disease for which standard treatments have not been offered, and he is not keen to enroll in clinical trials.A 51-year-old male presented to MDACC for evaluation of a left upper eyelid mass. On review of the outside needle core biopsy sections, scant histiocytoid cells (CK7 positive) were noted percolating among normal lacrimal gland tissues. The microscopic assessment was a poorly differentiated adenocarcinoma with indeterminate origin due to the limited biopsy sample. An MRI of orbit and face showed an infiltrative lesion involving pre- and postseptal regions of the left upper and lower eyelids, the lacrimal gland, and the focal intraconal extension . The patA 40-year-old male consulted MDACC with a history of thickening of the left medial lower eyelid and enlargement of a caruncle. A CT scan of orbits and head and neck soft tissue demonstrated an enhancing and infiltrative mass along the inferomedial aspect of the left anterior orbit  and no eImmunohistochemical studies are summarized in β-catenin and low cytoplasmic expression of Src  was observed as well as focal positive nuclear staining for c-Myc in case 4. Additional immunohistochemical studies were performed in three of our cases  which were received for consultation from outside facilities. The three cases were positive for gross cystic disease fluid protein 15 (GCDFP-15) stain while negative for Her2Neu. Androgen receptor (AR) was positive in cases 2 and 4.Cyclin-D1 was positive in three of our cases, two of them demonstrating preserved E-cadherin/TSC2  and FGFR4 . The polymorphism phenotyping detected on FGFR4 was probably deleterious.No mutations or copy number changes were identified. Germline polymorphisms were noted on PIK3CA (E545K and N1044K) and one CDH1 mutation was found in exon 13 (frameshift). Copy number variations were not identified  and MLH1 . The polymorphism phenotyping was considered benign in both genes.No mutation or copy number changes were detected. Germline polymorphisms were noted on PIK3CA (H1047R) and TP53 (D259Y). Benign germline polymorphism was noted on ERBB2 . No copy number changes were noted  [% 6–9, 22. PrimarReview of the literature indicates that primary SRC/Histiocytoid carcinoma of the eyelid shows strong and diffuse reactivity for CK7, CAM 5.2, high molecular weight cytokeratin (HMWCK), keratin cocktail AE1/AE3 and MNF116, carcinoembryonic antigen (CEA), epithelial membrane antigen (EMA), gross cystic disease fluid protein-15 (GCDFP-15), p63, mucin-1 (MUC-1), and BerEP4; moderate positivity for -smooth muscle actin (ASMA), tissue-specific transcription factor 1, MUC-2, podoplanin, N-cadherin, and E-cadherin; and weak positivity for epidermal growth factor receptor (EGFR) [β-catenin complex expression has not been described previously. Underexpression of the E-cadherin protein complex is found in gastric and other cancers such as colon, lobular carcinoma of the breast, lung, prostate, and in plasmacytoid-variant bladder carcinoma [Immunoreactivity for E-cadherin, in SRC/Histiocytoid carcinoma of the eyelid, has been studied in prior case reports –3, 7; hoarcinoma , 24–26.β-Catenin is a multifunctional cytoplasmic protein and has been found to be an important member in the Wnt signaling pathway, which plays important roles in cellular development, proliferation, and differentiation. Conventionally, Wnt signaling causes β-catenin accumulation in a complex with T cell factor/lymphoid enhancer factor (TCF/LEF), which regulates target gene expression. Reduction and loss of β-catenin or other molecules might disrupt stability and integrity of the E-cadherin-catenin complex and disturb cellular adhesive junction. Additionally, in the absence of Wnt signals, β-catenin is sequestered in a complex with the adenomatous polyposis coli tumor suppressor (APC), AXIN, and a serine threonine glycogen synthetase kinase-3β (GSK-3β), enabling phosphorylation and degradation of free β-catenin by the ubiquitin-proteasome system, processes that have been associated with a wide variety of human malignancies [α,β-catenins significantly correlated with differentiation, and lymph node and liver metastases of pancreatic cancer.It has been proposed that the loss of E-cadherin-mediated cell adhesion is a prerequisite for tumor cell invasion and metastasis formation , 27. E-cgnancies . Nelson gnancies  have desβ-catenin or E-cadherin results in increased stabilization of the cadherin-catenin complex while phosphorylation by nonreceptor tyrosine kinases such as Src or epidermal growth factor receptor (EGFR) disrupts binding of β-catenin to cadherin [Src is an integral part of a cadherin-activated cell signaling pathway that can positively regulate adhesion and cell contact integrity as mentioned by McLachlan et al. [Src has a bimodal impact on cadherin function, exerting a positive supportive role at lower signal strengths, but inhibiting function at high signal strengths which suggests that quantitative changes in signal strength can lead to distinctly different functional outcomes. One potential pathway for Src to influence cadherin function is through PI3-kinase which is capable of supporting cell-cell adhesion when it is active at cadherin cell-cell contacts. Moreover, Src might participate as an upstream element in signaling pathways that activate PI3-kinase [The structural and functional integrity of the cadherin-catenin complex is regulated by phosphorylation. Serine/threonine phosphorylation of cadherin . Src is n et al. . However3-kinase .Cyclin D1 and c-Myc genes are important cell cycle regulators playing important roles in the control of cell growth, differentiation, apoptosis, and neoplastic transformation. The correlation between abnormal expression of β-catenin and overexpression of target genes, Cyclin D1 and c-Myc, in pancreatic cancer and colorectal carcinoma have been previously evaluated and correlated with loss of tumor differentiation, metastasis and prognosis [APC gene as well as alteration in the Wnt–β-catenin signaling pathway.rognosis , 30. Theβ-Catenin can no longer interact with E-cadherin, and it is either degraded or protected from degradation [Destabilization of epithelial adherens junctions is present during EMT. E-cadherin is cleaved at the plasma membrane and subsequently degraded. radation . EMT proHerein, we present four male patients with histopathologic diagnosis of SRC/Histiocytoid carcinoma of the eyelid arising in the eccrine glands with ages between 40 and 69 years at the initial diagnosis. Metastatic tumors from the breast, gastrointestinal tract, and urothelial lining have been excluded. Although, histopathologically, the tumors  disclose predominantly an infiltrative, diffuse growth pattern of histiocytoid cells, more cohesive neoplastic cells with signet ring cells are noted in case 4.β-catenin and the decreased cytoplasmic expression of Src in cases 1 and 2 is observed while an intact/strong expression of E-cadherin/β-catenin is seen along with a high cytoplasmic expression of Src in cases 3 and 4.An association between the absence/abnormal expression of E-cadherin/β-catenin with a heterogeneous pattern of staining in ductal carcinoma in situ of the breast (54%) with high concordance between β-catenin expression patterns in in situ and invasive components (81%). We observe a similar heterogeneous pattern of staining in cases 1 and 2, which might indicate that changes in β-catenin expression may occur early during the development of these tumors. The retained expression of E-cadherin/β-catenin with positive/high cytoplasmic expression for Src, seen in cases 3 and 4, could be associated with an abnormality in different stages of the convergence of the β-catenin and cadherin pathways which could also be supported by the positive expression of p53, c-Myc, and Cyclin-D1 as noted in case 4.Karayiannakis et al. describeβ-catenin (case 1) associated with positive Cyclin-D1 expression, cases 3 and 4 demonstrate normal β-catenin expression with also positive Cyclin-D1 expression. Guo et al. [β-catenin and positive expression of Cyclin-D1 in patients with breast cancer, and they also report that breast cancer patients with normal expression of β-catenin and positive expression of Cyclin-D1 exhibit longer tumor-free survival time.Although there is an abnormal expression of o et al.  indicateCDH1 gene provides instruction for making E-cadherin protein and is the best known gene associated with the hereditary diffuse gastric cancer syndrome (HDGC). The literature review demonstrates that mutation in the CDH1 gene predisposes primarily to diffuse gastric cancer (DGC). Multiple DGC cases in a family, DGC at a young age in an individual, or the combination of DGC and lobular breast cancer (LBC) in an individual or a family defines the hereditary DGC syndrome. Testing for germline CDH1 mutations is warranted in HDGC [CDH1 [CDH1 somatic mutations and the frequencies of CDH1 somatic mutations, previously reported, can vary from 3% to greater than 50%. In the same work, PIK3CA and TP53 are found the most frequently mutated genes in DGC and intestinal gastric cancer. PIK3CA is an oncogene whose mutated form exhibits increased kinase activity, causing cancer cell proliferation and the relative high mutation of this gene in DGC may reflect the specificity of mutations in this gene to DGC.The  in HDGC . ApproxiGC [CDH1 . HoweverGC [CDH1  report tCDH1 (case 2), PIK3CA (cases 2 and 4), and TP53 genes, detected in the present study, indicate abnormalities in the E-cadherin structure as well as in the calcium signaling pathway which is essential for the function of E-cadherin as has been previously reported by Lee et al. [The somatic mutations on the e et al. . We can e et al. .PIK3CA mutation in the hotspot E545K is detected in case 2 which is a well-known mutation in DGC and other malignancies [CDKN1B and NTRK3 genes have been recently found associated with this tumor [gnancies . In addiβ-catenin complex and associated molecular pathways. The spectrum of histological, immunohistochemical, and molecular findings in this malignancy might represent abnormalities in the different stages of the convergence of E-cadherin/β-catenin pathways which affect the clinical outcomes seen in these patients. All four patients received adjuvant chemotherapy and/or radiotherapy with three developing recurrent disease. One of those patients (case 1) died five years after his final treatment .Although our molecular studies reveal no somatic mutations in cases 1 and 3, further research studies are mandatory to determine and understand the different interconnecting molecular pathways participating in the pathogenesis of this tumor. Up to date, there are just few investigations targeting cancer-related genes associated with this malignant neoplasm. We believe that this eyelid tumor is biologically similar to primary SRC/Histiocytoid carcinoma described in the stomach and breast, among others with alterations in expression of the E-cadherin/While E-cadherin abnormalities cannot be specifically targeted, clinical research trials aiming at associated pathways are being evaluated for therapeutic consideration in similar tumors in other sites. The molecular heterogeneity identified in this study emphasizes the need to consider genetic testing in patients with SRC/Histiocytoid carcinoma of the eyelid to gain further insight into this rare disease. Moreover, with improved understanding of altered biologic pathways, patients may ultimately benefit from targeted personalized therapy.


Functionally, C20 treatment also prevented the reduction in left ventricle fractional shortening, which was typically observed in the vehicle-treated ex mdx mice. Based on these findings, we propose that PKCθ pharmacological inhibition could be an attractive therapeutic approach to treating dystrophic cardiomyopathyChronic cardiac muscle inflammation and subsequent fibrotic tissue deposition are key features in Duchenne Muscular Dystrophy (DMD). The treatment of choice for delaying DMD progression both in skeletal and cardiac muscle are corticosteroids, supporting the notion that chronic inflammation in the heart plays a pivotal role in fibrosis deposition and subsequent cardiac dysfunction. Nevertheless, considering the adverse effects associated with long-term corticosteroid treatments, there is a need for novel anti-inflammatory therapies. In this study, we used our recently described  DMD gene, encoding for the dystrophin protein [Duchenne Muscular Dystrophy (DMD) is a severe X-linked genetic uncurable disease that leads to progressive skeletal and cardiac muscle wasting, affecting roughly 1:3000–5000 of males. It is caused by mutations in the  protein ,3,4, a l protein ,5,6,7. B protein ,4,5,6,7. protein ,9,10. DM protein ,12,13.mdx mouse [To date, no definitive cure exists for DMD. Several therapeutic approaches are being tested for their ability to restore dystrophin synthesis: viral vectors carrying micro-dystrophin isoforms, exon skipping compound or stop codon readthrough agents, as well as gene editing with the novel CRISPR/Cas technology ,16,17,18dx mouse ,27,28,29mdx mice, together with an increase in muscle performance and preservation of the muscle stem cell (MuSCs) pool [mdx mouse model presents a much milder cardiac pathology that develops late in the mice lifespan [exercised mdx” (ex mdx) mouse model, which is characterised by accelerated heart pathology, with clear signs of cardiomyopathy, such as diffuse ventricular fibrosis, appearing as early as 12 weeks of age [Previously we demonstrated the potential of using C20, a PKCθ inhibitor, to ameliorate skeletal muscle and diaphragm pathology by targeting early T cell recruitment ,31. PKCθCs) pool ,42. We, lifespan ,44,45, ws of age .ex mdx treated with C20 showed a strong reduction in cardiomyocyte necrotic cell death, as well as a sharp decrease of the ventricular fibrotic area. Heart function was also improved by the treatment, with C20-treated mice showing increased left ventricle fractional shortening. Overall, these results suggested that PKCθ inhibition may be considered as an attractive novel therapeutic approach to ameliorate the phenotype and function of dystrophic cardiac muscle. We showed that C20 treatment reduced the number of all the immune cell components infiltrating the dystrophic heart, suggesting a critical role of immune cells in DMD-related cardiac inflammation. The hearts of mdx model (ex-mdx) we previously described and characterised [mdx mice were divided into three groups at 3 weeks of age. One group received C20 treatment, while another one received vehicle. A third group of non-exercised, non-treated, age-matched mdx were used as control. Vehicle and C20-treated groups were exercised as previously described [+ and CD11b+ immune cells. After two weeks of treatment, we found that twice-weekly administration was as effective as daily administration . As shown in To examine heart muscle organisation in C20 and vehicle-treated ex mdx by immune-staining of the ventricle cryosections with anti-mouse IgG conjugated to a TRITC fluorochrome. As shown in mdx. Taken together, these results demonstrated that C20 treatment ameliorates dystrophic heart phenotype in the ex mdx model by reducing immune cell infiltration and cardiomyocyte necrosis.Since immune cell infiltration might be triggered by necrotic cell death of cardiomyocytes ,48, we aex mdx mice, cryosections of the heart ventricles were stained with Sirius red, and the extent of collagen deposition was analysed using ImageJ software. As shown in ex mdx was similar to the level observed in unexercised age-matched mdx. Accordingly, Collagen1α mRNA was significantly decreased in C20-treated hearts compared with vehicle-treated controls, as shown by qRT-PCR . The mice were housed in the Histology Department-accredited animal facility at the University of Sapienza. All the procedures were approved by the Italian Ministry for Health and were conducted according to the EU regulations and the Italian Law on Animal Research. Only males were used.C57BL/10ScSn-The exercise was carried out as previously described  using a mdx mice were intraperitoneally injected twice a week with C20, at the dose of 5 mg/kg, or with vehicle, in a total volume of around 0.1 mL, using 500 μL syringes with 27 G needles. The treatment was maintained throughout the exercise protocol, which started one week later at the age of 4 weeks, for the following 8 weeks. The bi-weekly C20 or vehicle injection preceded the bi-weekly exercise session of about 24 h. Mice were sacrificed 48 h after the last exercise session.C20 was prepared as a mother solution at the concentration of 100 ug/uL in DMSO and then diluted 1:100 in 0.9% NaCl saline solution. The vehicle was prepared diluting DMSO 1:100 in the same saline solution. Three-week-old Heart ventricles were embedded in tissue-freezing medium after dissection  and snap-frozen in liquid nitrogen-cooled isopentane. Frozen heart ventricles were cut into 8 μm sections (four sections per heart) and stored at −20 °C until use. Histochemistry and immunofluorescence analyses were performed as previously described ,31,42. https://imagej.nih.gov/ij/, accessed on 31 December 2021). Quantification of collagen deposition was determined using the Color Deconvolution plugin  of ImageJ software. Briefly, for histological analysis, the sections were stained with haematoxylin/eosin or with Sirius red/picric acid . The sections were photographed in a Zeiss Axioskop 2 Plusfluorescence microscope, using a 10× objective and a 10× eyepiece. The whole ventricular surface was acquired for each section. Quantification of cell infiltration patches size and number, as well as total ventricular area, was performed using built-in functions in ImageJ open-source software  was performed on cryosections after fixation. After three PBS washes, sections were blocked in 5% goat serum and incubated with rabbit anti-laminin primary antibody  for 1 h at room temperature, at the dilution of 1:400 in 1% PBS-BSA. After three washes in 1% PBS-BSA, sections were incubated with goat anti-mouse IgG antibody coupled to TRITC and goat anti-rabbit IgG coupled to Alexa Fluor 488 . Nuclei were counterstained with Hoechst 33,342 . The sections were photographed in a Zeiss Axioskop 2 Plusfluorescence microscope, using a 10× objective and a 10× eyepiece. All the ventricular necrotic areas were acquired for each section. The images were then analysed using ImageJ software. Cytofluorimetric analysis was performed as previously described ,30. BrieTotal RNA from ventricle tissue was extracted using the TRIsure solution  and converted in cDNA using the SensiFast cDNA Synthesis kit from Bioline, according to the supplier’s instructions. PCR amplification was performed using the SensiMix SYBR Lo-Rox Mix, from Bioline, following the manufacturer’s protocol. All PCR reactions were carried out in duplicate. All qPCR results are expressed as relative ratios of the target cDNA transcripts to GAPDH and normalised to those of the reference condition. The reaction was carried out using a 7500 Real-Time PCR System, and the analysis was carried out with the 7500-software, both from Applied Biosystem, Waltham, MA, USA. To amplify the genes of interest, we used the following primers pairs: GAPDH (for) 5′-ACCCAGAAGACTGTGGATGG-3′ (rev) 5′-CACATTGGGGGTAGGAACAC-3′: Collagen1α1 (for) 5′-ACCCAGAAGACTGTGGATGG-3′ (rev) 5′-CAGATTGGGGGTAGGAACAC-3′, IL-6(for) 5′-CCCGAAGCGGACTACTATGC-3 (rev) 5′-CATAGATGGCGTTGTTGCGG-3′.Echocardiographic analyses were performed as previously described ,81. Brie1H-NMR  δ 9.40 . C20 was synthesised at the Dept. of Drug Chemistry and Technologies, La Sapienza, Roma, according to Cywin et al., 2007 . Compouno-trifluoromethoxybenzylamine (0.46 mmol) was added followed by the dropwise addition of triethylamine (0.92 mmol). The resulting precipitate was filtered off after 16 h of stirring at room temperature (TLC: EtOAc:Hex 2:1) providing the desired intermediate III (5-nitro-4-thiocyanato-N-(2-(trifluoromethoxy)benzyl)pyrimidin-2-amine), which was used without further purification in the next step. m.p.: 190–192 °C; yield: 51%; 1H-NMR  δ 4.79 , 7.36–7.54 , 9.51 , 9.75 .Compound II (0.46 mmol) was suspended in abs EtOH, then 1H-NMR  δ 0.50–1.50 , 2.86 , 3.21 , 4.63 , 7.34–7.41 , 8.69 , 8.71 , 8.89 ; MS (M + H) = 455.01.Compound III (0.39 mmol) was suspended in dry DCM and 1,4-Cyclohexanebis(methylamine) (1.56 mmol) and the reaction was left stirring for 16 h at rt. The crude was loaded on a silica gel column using chloroforom/methanol/ammonia 12:1:0.1 as the eluent system, yielding pure C20 (N4-((4-(aminomethyl)cyclohexyl)methyl)-5-nitro-N2-(2-(trifluoromethoxy) benzyl)pyrimidine-2,4-diamine). m.p.: 105–107 °C; yield: 52%; t-test with Welch’s correction was used for statistical comparison between two groups, and one-way ANOVA (with Bonferroni’s correction for multiple comparisons) was used for comparisons between multiple groups. A p-value of ≤0.05 was considered statistically significant.All statistical analyses were performed using Prism software, version 6 . Data are presented as mean ± SEM. Unpaired two-tailed Student’s


Objective: Activation of β-catenin causes podocyte injury and proteinuria, but how β-catenin signalling is regulated during podocyte injury remains elusive. Nuclear receptor interacting protein 2 (NRIP2) modulates the Wnt pathway in colorectal cancer-initiating cells, but the role of NRIP2 in podocyte injury has not yet been investigated. We aimed to examine the interaction between NRIP2 and β-catenin signalling.Materials and Methods: Knockdown or overexpression of NRIP2 and β-catenin and chemical treatments were performed in cultured human podocytes. Immunoprecipitation, immunoblotting and immunofluorescence assays were used to assess protein interactions and expression. Data from the GEO dataset and kidney tissues from patients with focal segmental glomerulosclerosis (FSGS) and surgical nephrectomy were examined. An adriamycin (ADR) nephropathy model was established in NRIP2 knockout mice.Results: NRIP2 knockdown accelerated β-catenin degradation, which was reversed by MG132; specifically, NRIP2 bound β-catenin and stabilized it to prevent its degradation through the ubiquitin proteasomal pathway. Overexpression of NRIP2 led to β-catenin activation and Snail1 induction, and these effects were attenuated by β-catenin knockdown. NRIP2 knockdown blocked ADR-stimulated β-catenin activation. In ADR mice, genetic knockout of Nrip2 ameliorated podocyte injury and loss, glomerulosclerosis, and proteinuria by inhibiting β-catenin activation. Moreover, NRIP2 was significantly upregulated in podocytes of FSGS patients and colocalized with nuclear β-catenin.Conclusion: These results established NRIP2 as a stabilizer of β-catenin activation through the ubiquitin proteasomal pathway in podocyte injury. Constitby MG132 . Existinby MG132 , DKK1  was initially described as a suppressor of the nuclear receptor (NR) by selectively binding the retinoic acid receptor (RAR) and the thyroid hormone receptor (THR) in a ligand-dependent manner in the mouse brain . RecentlRabbit anti-NRIP2 ; Mouse anti-WT1 ; Rabbit anti-WT1 ; Mouse anti-Total β-catenin ; Rabbit anti-Active β-catenin ; Rabbit anti-Ubiquitin ; Rabbit anti-Col1a1 ; Rabbit anti-α-SMA ; Goat anti-Synaptopodin ; Rabbit anti-GAPDH ; Mouse anti-β-Tubulin .Negative control, human NRIP2 siRNAs , human β-catenin siRNA (GGAUGUUCACAACCGAAUUTT) were purchased from RIBOBIO Company . NRIP2 and β-catenin (Full length and mutants) were synthesised from GenScript company and cloned into pcDNA3.1 backbone. pRK5-HA-ubiquitin was obtained from MiaoLing Plasmid Sharing Platform (P1761).in vitro DNA & siRNA transfection reagent  were used according to the manufacturer’s instructions. In brief, 2 µg plasmid was diluted into 200 µl jetPRIME buffer, mixed by vortex. After adding 4 µl jetPRIME reagent and incubating for 10 mins, the transfection mixture was added to the cells in serum containing medium. For siRNA transfection, 110 pmole siRNA was diluted into 200 µl jetPRIME buffer, mixed by vortex. After adding 3 µl jetPRIME reagent and incubating for 10 mins, the transfection mixture was added to the cells in serum containing medium. Cells were harvest at 24 h after transfection. Cells were treated with 10 µM Adriamycin  for 6 h and 5 µM MG132  for 1 h before harvested.The human podocyte cell line was provided by M. Saleem  Univers. Human pHuman podocytes were lysed in M-PER™ Mammalian Protein Extraction Reagent  with protease inhibitors, cell lysates were fractionated by SDS-PAGE  and then transferred to polyvinylidene difluoride (PVDF) membranes  for immunoblot analysis.For Immunoprecipitation, cells were lysed with Minute™ Total Protein Extraction Kit , lysates were incubated with β-catenin antibody with protease inhibitors on a rotator overnight at 4°C. The protein/β-catenin complexes were prepared by adding 15 µl protein A/G Magnetic Beads  for 3 h at 4°C. After several washes with cold PBS, the complexes were resuspended with 2 × SDS buffer and boiled for 10 min; supernatants were subjected to SDS-PAGE and immunoblot analysis.For ubiquitination assays, transfected podocytes treated with or without MG132 were lysed in SN-002 with protease inhibitors, lysates were incubated with primary antibody as indicated on a rotator overnight at 4°C, and subjected to 15 µl protein A/G Magnetic Beads incubation  for 3 h at 4°C. After several washes with cold PBS, the complexes were resuspended with 2 × SDS buffer and boiled for 10 min; supernatants were subjected to SDS-PAGE and immunoblot analysis with anti-Ubiquitin antibody.Nrip2 Knockout (KO) mice were customized in Cyagen Biosciences Inc. . KO mice were generated using CRISPR/Cas9 system (Target-1: TGGAGCTAGTCATGCACCTCTGG and Target-2: AGGTGGACTAGACCGGAGAGGGG) on C57BL/6N background. ADR  were injected at 20 mg/kg body via the tail vein in WT and Nrip2 KO mice at the age of 8-weeks-old, saline-injected mice were served as normal controls. ADR mice were sacrificed at 4 weeks after injection. Urine albumin was quantified by Albuwell M ELISA kit , Urine creatinine were examined with the same samples using Creatinine Companion . The urine albumin excretion was assessed using the ratio of albumin to creatinine. Genotyping primers used were: F1- CAAAGAAAGGAAGCCAAGCTGGTAC, R1- ACCCTGAGCCTTCTACCTGTCCTAG, and R2- CTGCTGGCTCCATCCCAAAATAG.Kidneys of mice were fixed with 4% PFA overnight at 4°C and embedded with paraffin. Paraffin-embedded kidney tissues were cut into 2-μm. Sections were stained with Periodic Acid Schiff (PAS) for histology analysis. Quantification of glomerulosclerosis was measured using ImageJ (NIH).4 with 0.1M cacodylate buffer (pH 7.4) for 24 h, dehydrated in a series of ethanol and infiltrated overnight in a 1:1 mixture of epoxy resin and propylene oxide. Specimens were embedded with Eponate 12  and polymerized at 60°C for 24 h. Ultrathin sections were stained with lead citrate and uranyl acetate and viewed on a HITACHI H-7500 microscope.Kidney tissues were fixed in 4% glutaraldehyde with 0.1M cacodylate buffer (pH 7.2) for 4 h. Samples were further rinsed in cacodylate buffer and post-fixed in 1% OsOPatients with FSGS were renal biopsy proven at National Clinical Research Center of Kidney Diseases, Jinling Hospital, Nanjing, China. The clinical characteristics of patients were detailed in Paraffin-embedded kidney sections from human and mouse were deparaffinized, blocked with 10% BSA in PBS for 30 min at room temperature (RT) and then incubated with primary antibodies at 4°C overnight. The next day, after five times washes with PBS, incubate with secondary antibodies at RT for 1 h. For IHC staining, diaminobenzidine (DAB) color reaction was kept with a fixed exposure time for all experiments among the groups. Quantitation of IHC staining in the glomeruli was measured using ImageJ (NIH). For IF staining, Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed secondary antibody, Alexa Fluor 488  and Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed secondary antibody, Alexa Fluor Plus 555  were incubated for 2 h in dark, and mounted with DAPI- Aqueous, Fluoroshield  for imaging.p < 0.05 were considered statistically significant.Data were represented as mean ± SD. Data between 2 groups were analysed using with two-tailed unpaired Student’s t test. All experiments were repeated for 3 times, representative experiments were exhibited. All protocols regarding the use of human samples were approved by the Human Subjects Committee of Jinling Hospital and written/signed consent was obtained from all patients (2013KLY-013-02). Mice were grown and maintained following standard procedure approved by the Institutional Animal Care and Use Committee at Jinling Hospital.To investigate a direct role for NRIP2 in regulating β-catenin, we first examined whether NRIP2 and β-catenin are expressed under physiological conditions. We used whole-cell extracts for an immunoprecipitation assay, and the interaction between endogenous NRIP2 and β-catenin was detected in cultured human podocytes . FurtherSubsequently, we examined whether NRIP2 regulates the abundance of β-catenin. NRIP2 knockdown with siRNA (#2 siRNA was used in the following experiments) decreased the levels of both total and active β-catenin ; conversβ-Catenin signalling plays a central role in mediating podocyte dedifferentiation and mesenchymal transition. We sought to ascertain the role of NRIP2 in regulating β-catenin signalling during podocyte injury and found that NRIP2 knockdown greatly inhibited ADR-induced expression of total and active β-catenin , transloTo investigate the protective effects of NRIP2 deletion in a murine model of experimental nephropathy, we established a model of ADR nephropathy. We generated mice with global allelic knockout of Nrip2 using the CRISPR/Cas9 approach  on a C57As expected, ADR-treated WT mice developed severe albuminuria, while ADR-treated KO mice displayed much lower proteinuria levels . Histolohttp://www.nephroseq.org)  , which sseq.org)  that gloseq.org)  that gloseq.org)  of NRIP2seq.org) . We alsoseq.org) . These rIn summary, the present work revealed a role of NRIP2 in stabilizing β-catenin in podocytes, and this role of NRIP2 is involved in podocyte injury in NS patients.β-Catenin signalling has a central role in mediating podocyte damage and proteinuria. During podocyte injury, β-catenin activation is dependent on receptor of advanced glycation end products (RAGE)-mediated NADPH oxidase induction, reactive oxygen species generation, and nuclear factor-κB activation ; β-catenStabilization of β-catenin is important for its nuclear translocation and subsequent activation . To dateHere, we describe that NRIP2 is necessary for β-catenin stabilization and activation in podocytes. Enhanced β-catenin degradation was observed in NRIP2-knockdown podocytes but was blocked by MG132 treatment, which means that β-catenin degradation is ubiquitin–proteasome-dependent. The interaction of NRIP2 and β-catenin was required for β-catenin stabilization. Additionally, reinforced expression of NRIP2 in podocytes was sufficient to activate β-catenin. Collectively, our findings indicate that NRIP2 interacts with and stabilizes β-catenin in podocytes.in vitro and in vivo data demonstrate that NRIP2 is required for β-catenin activation during podocyte injury. In vitro, β-catenin activation was dramatically activated in ADR-treated podocytes but totally blocked in NRIP2-knockdown podocytes. In vivo, NRIP2 knockout ameliorated podocyte injury and proteinuria in mice with ADR nephropathy by inhibiting β-catenin activation and the expression of its downstream target genes. In NS patients, glomerular expression of NRIP2 was found to be significantly upregulated and mainly localized in podocytes. The interaction of NRIP2 and β-catenin was observed in podocytes from FSGS patients. These data summarize the importance of NRIP2 for β-catenin activation during podocyte injury.Furthermore, our It is well known that β-catenin signalling is essential for kidney development , but no In conclusion, we describe a previously unsuspected protein–protein interaction between NRIP2 and β-catenin and show that this interaction is important for stabilizing β-catenin in podocytes.


Runx2 and/or Osterix  transcription. Precisely, how Osx activity is dictated by ß-catenin or p53 is not clarified and represents a knowledge gap that, until now, has largely been taken for granted. Using conditional lineage-tracing mice, we demonstrated that chondrocytes gave rise to a sizable fraction of MPCs, which served as progenitors of chondrocyte-derived osteoblasts (Chon-ob). Wnt/ß-catenin activity was only required at the stage of chondrocyte-derived mesenchymal progenitor (C-MPC) to Chon-ob differentiation. ß-catenin– C-MPCs lost osteogenic ability and favored adipogenesis. Mechanistically, we discovered that p53 activity was elevated in ß-catenin– MPCs including ß-catenin– C-MPCs and deleting p53 from the ß-catenin– MPCs fully restored osteogenesis. While high levels of p53 were present in the nuclei of ß-catenin– MPCs, Osx was confined to the cytoplasm, implying a mechanism that did not involve direct p53-Osx interaction. Furthermore, we found that p53’s anti-osteogenic activity was dependent on its DNA-binding ability. Our findings identify chondrocytes as an additional source for MPCs and indicate that Wnt/ß-catenin discretely regulates chondrocyte to C-MPC and the subsequent C-MPC to osteoblast developments. Most of all we unveil a previously unrecognized functional link between ß-catenin and p53, placing p53’s negative role in the context of Wnt/ß-catenin signaling-induced MPC osteogenic differentiation.The developmental origins of mesenchymal progenitor cells (MPCs) and molecular machineries regulating their fate and differentiation are far from defined owing to their complexity. Osteoblasts and adipocytes are descended from common MPCs. Their fates are collectively determined by an orchestra of pathways in response to physiological and external cues. The canonical Wnt pathway signals MPCs to commit to osteogenic differentiation at the expense of adipogenic fate. In contrast to ß-catenin, p53’s anti-osteogenic function is much less understood. Both activities are thought to be achieved through targeting  The cellular means by which a fully differentiated chondrocyte gains the plasticity to evolve into a mature osteoblast, as well as what signaling pathways govern this event, remains elusive.Endochondral bone formation occurs through a cartilage to bone conversion process, during which cartilaginous tissue serves both as a template for ossification and as an innate source of osteoblasts10. In Osx-expressing MPCS, Wnt/ß-catenin plays a switch role between osteogenic and adipogenic fates. Despite the lack of convincing evidence7, it is currently accepted that ß-catenin promotes osteogenesis through activating Runx2 and/or Osx transcription13.Canonical Wnt signaling plays diverse roles at different stages of bone development and growth14. The precise functions and regulatory mechanisms of p53’s physiological roles remain much less understood and appreciated. In limited reports, crosstalk between p53 and Wnt/ß-catenin signaling has been shown to play various roles in a context-dependent manner, such as in smooth muscle cells15 and in embryonic stem cells16.p53 is a well-established tumor suppressor. It is also a vital regulator of cell fate and differentiation18 Nonetheless, p53 downstream molecular events leading to osteogenic inhibition are not yet defined. p53 null marrow mesenchymal stem cells are more osteogenic and display no apparent difference in their adipogenic and chondrogenic capacities20. One study shows that p53 inhibits osteoblastic differentiation through microRNA-34-mediated Runx2 suppression22. To date, the physiological context of this inhibitory function remains entirely elusive.p53 exhibits osteo-inhibitory activity in various mouse modelsCol10a1) and Aggrecan (Agc1)-driven ß-catenin conditional lineage-tracing mice to delineate how Wnt/ß-catenin regulates chondrocyte to osteoblast reprogramming. We showed that chondrocytes evolved into osteoblasts through at least two steps, which were differentially regulated by Wnt/ß-catenin. Mechanistically, we discovered that the ß-catenin-deficient MPCS acquired elevated p53 activity and their defect in osteogenic capacity was fully reinstated by merely deleting p53 from them, indicating that Wnt/ß-catenin promotes osteogenesis via a p53 suppression-dependent mechanism.Here we used Collagen X  cells was observed within the marrow cavity of both Col10a1-Cre;ROSA26R-Tomato (XTomato) control and Col10a1-Cre;Ctnnb1fl/fl;ROSA26R-Tomato (X/CKOTomato) mutant animals, each of which presented a distinct pattern of distribution. At postnatal day 2 (p2) of XTomato control animal, the majority of Col10a1-Cre-induced Tm+ (X/Tm+) cells were localized at the primary spongiosa physically in contact with trabeculae (trabecula-bound) and showed mature osteoblast-like morphology  cells in the p2 X/CKOTomato mutant animal were scattered rather evenly throughout the marrow cavity. Most of them were not connected with trabeculae (non-trabecula-bound) and were morphologically distinct from mature osteoblasts , whereas fewer XCKO/Tm+ cells were found on the endostea (0.63/ko vs 6/con cells/100 µm), embedded within cortices (0/ko vs 13.4/con cells/10 mm2) or trabecular matrixes (16.6/ko vs 38.9/con cells/10 mm2) compared to the X/Tm+ cells  and Agc1-CreERT2;Ctnnb1fl/fl;ROSA26R-Tomato (iAgc/CKOTomato) mice. These mice were analyzed after 6- and 8-week chases post tamoxifen injection at p10. The iAgc/CKOTomato mice developed a low trabecular bone volume phenotype resembling that of the X/CKOTomato mice . Conversely, numbers of the trabecula-bound, endosteum, and cortex-embedded iAgcCKO/Tm+ cells were all fewer than that of the corresponding iAgc/Tm+ cells    Fig. . The invm2) Fig. . The phem2) Fig. .Col10a1-Cre;Ctnnb1EX3fl/fl;ROSA26R-Tomato (X/ΔEXTomato) mice with stabilized β-catenin showed delayed primary ossification and developed an osteopetrosis-like phenotype after birth, opposite of the X/CKOTomato mice  and bone (Col1+) matrixes and was filled with XΔEX/Tm+ cells. It appeared that many XEX/Tm+ cells, especially toward the middle, were producing both Col10 and Col1  mice and sorted for YFP-positive  cells by fluorescence-activated cell sorting (FACS) , CD140a+(90.85%), CD140b+(99.97%), CD105+(96.78%), and negative signals for hematopoietic cell marker CD45, endothelial cell marker CD31, and erythroid cell marker Ter119  and CD105 (10.9%) 23. To evaluate Osx expression in chondrocyte-derived non-trabecula-bound stromal cells, we did experiments using Col10a1-Cre;Osxfl/+ (X/Osxfl/+) mice, in which Osx-expressing cells are identified by GFP upon Cre-mediated LoxP recombination24. Taking the approach shown in Supplementary Fig. S+ cells  and X/CKOTomato (CKO) mice for flow analysis. It revealed an elevated percentage of Tm+ stromal cells from CKO mice compared to that from control mice  , X/CKOTomato (CKO), and X/ΔEXTomato (ΔEX) mice were collected and grown in culture. The attached cells were analyzed by FACS. As shown in the fresh isolated marrow cells  Fig . Alternalls Fig. , there wlls Fig.  from X/Δlls Fig. . We isol+ stromal cells from control mice  and ROSA26R-Tomato (Tm/Con) mice after a 4- and a 10-day chase, and quantified Tm+ cell portions by FACS. Shown in Fig. + stromal cells from the mice chased for 10 days was significantly higher than that for 4 days . In a separate experiment, we administered tamoxifen to p13 Ctnnb1fl/+;ROSA26R-Tomato (Con), Agc1-CreERT2;Ctnnb1fl/+;ROSA26R-Tomato (iAgc/Con), and Agc1-CreERT2;Ctnnb1fl/fl;ROSA26R-Tomato (iAgc/CKO) mice, and after a 10-week chase, stromal cells were isolated and plated in culture. Flow analysis revealed a higher percentage of β-catenin– Tm+ cells from iAgc/CKO animals compared to that from iAgc/Con mice  between C-MPCs of X/CKOTomato mutant and control animals  between growth plate chondrocytesals Fig. .Tomato and control littermates, and total C-MPC RNAs were extracted from the non-trabecula-bound Tm+ stromal cells of 5-week-old X/CKOTomato and Col10a1-Cre;Ctnnb1fl/+;ROSA26R-Tomato (X/CHetTomato) control mice. The RNA-seq expression profiling of growth plate chondrocytes showed only 23 genes with equal to or more than two-fold changes in expression levels, out of over ten thousand genes detected in the experiment  and/or p53 deletions in MPCs. The qPCR confirmed that adeno-Cre (Ad-Cre) efficiently deleted Ctnnb1 and/or p53 conditional alleles  showed a 2.85-fold increase in p53 expression compared to the mock treated Ctnnb1fl/fltm MPCs . The p53 protein was slightly higher in the ß-catenin–Tm+ MPCs, in spite of incomplete ß-catenin deletion , while no signal was detected in control MPCs  assay revealed a higher number of β-gal+ cells in ß-catenin– MSPC population. This phenotype was attenuated by removal of p53  projected p53 as the top upstream regulator  mice 2. Both groups tested a variety of hypothetical causes for the declines including potential alteration in proliferation and/or apoptosis in chondrocytes and/or Chon-obs. Their findings are conflicting and are inadequate to justify the Chon-obs phenotype. In our study we did not find any evidence for the chondrocyte-derived “Osx” cells1 or for the severe defects in proliferation in both ß-catenin– chondrocytes and Chon-obs2  – C-MPCs (Tm+GFP+ cells) – Chon-obs (Tm+GFP+) shown by tamoxifen chase experiments. (3) The finding of discrete regulations by Wnt/ß-catenin substantiated by RNA-seq profiling and histological and differentiation analyses favors the stepwise idea  the C-MPCs’ progenitor cell-like properties Fig. . AlthougPCs Fig. . (2) Seqdea Fig. .– MPCs suggested that ß-catenin likely does not directly regulate Runx2 and Osx promoter activity. The partial rescue of trabecular volume in X/DKO mice solidified the key negative role of p53 in the context of Wnt/ß-catenin pro-osteogenic function.The full rescue by solely deleting p53 from ß-cateninEvaluation of whether there is any change in osteoclast differentiation compared to the X/CKO mice would prove to be an interesting follow-up.– MPCs, it is implausible that a p53-Osx physical interaction could be the reason for osteogenic inhibition28. Of great ongoing interest is further understanding of the granule-form of Osx and its transport regulation. Likewise, p53 upstream events triggered by ß-catenin signaling are equally important and yet to be elucidated.Since p53 and Osx were not concurrently localized in the same cellular compartment of ß-cateninOur study advanced understanding in two fundamental subjects: (1) identification of p53 as a key node negatively involved in Wnt/ß-catenin-mediated osteogenesis; (2) revealing of a stepwise chondrocyte to osteoblast process independently regulated by Wnt/ß-catenin signaling Fig. . Given tCol10a1-Cre29, Agc1-CreERT230, Osxfl/fl31, Ctnnb1fl/fl, Ctnnb1EX3fl/f, p53R245W25, p53fl/fl, and 2.3Col1a1-GFP mice have been described. Ctnnb1EX3fl/fl mice were provided by Dr Makoto Taketo of Kyoto University. 2.3Col1a1-GFP mice were provided by Dr David Rowe of University of Connecticut. Ctnnb1fl/fl , ROSA26R-tdTomato  (Gt (ROSA)26Sortm9(CAG-tdTomato) Hze, Stock No: 007909) and ROSA26R-YFP (B6.129 × 1-Gt (ROSA)26Sortm1(EYFP)Cos/J, Stock No: 006148) mice were purchased from the Jackson Laboratory. Tamoxifen (Sigma-Aldrich T-5648) was injected intraperitoneally at 1.5–3.0 mg/10 g body. All animals were housed in pathogen-free conditions, procedures followed the rules and regulations of AAALAC and were approved by Institutional Animal Care and Use Committee of University of Texas MD Anderson Cancer Center.32. Total bone marrow cells were cultured in alpha-MEM media containing 20% FBS under 5% O2 up to around 10 days. Attached cells were passaged for expansion.Bone marrow nonhematopoietic stromal cells were isolated as describedCell sorting experiments were performed on Arial II Cell Sorter (BD Bioscience). Flow cytometry analyses were carried out using Gallios 561 (Beckman Coulter). Services were provided by MDACC NORTH Campus Flow Cytometry and Cellular Imaging Core Facility. Data were analyzed using FlowJo or Kaluza.32. Collected cells were plated 0.5–1 × 106/well in 6-well plates and cultured in alpha-MEM media/20% FBS under 5% O2 for 10 days without changing media and were stained with crystal violet with methanol. R&D Mouse Mesenchymal Stem Cell Functional Identification Kit (R&D) was used for in vitro osteogenic, adipogenic, and chondrogenic assays.Bone marrow plugs flushed out of femurs, tibias, and humeri were treated with Collagenase I  and Dispase II  as previously describedLong bones were fixed in freshly made 4% paraformaldehyde/PBS (pH 7.2) at 4 °C overnight and changed to 14% EDTA for 2–7 days at 4 °C. Decalcified bones were immersed in 30% sucrose/PBS for 1 h before embedded in OCT compound. Then, 8–12 µm sections were prepared using CryoStar NX70 Cryostat. Hyaluronidase treatment (2 mg/ml in PBS [pH 5.0]) was used for antigen retrieval, 20’ for embryonic or 30’ for postnatal tissue at 37 °C. Primary antibodies used were anti-mouse collagen type I  and anti-Collagen X antibody . Secondary antibodies were Alexa fluor 488 goat anti-rabbit IgG and Alexa fluor 488 goat anti-mouse IgG (Molecular probes). IF sections were mounted with Prolong Gold antifade reagent with DAPI (Invitrogen P36931).Fluorescence images were captured using A1 Laser scanning confocal microscope by Nikon Instruments at Microscopy Laboratory in the Department of Genetics at MDACC.+ C-MPCs were sorted by FACS from fresh marrow of 5-week-old X/CKOTomato and X/CHetTomato mice, two of each genotype. Total RNAs were isolated using Quick-RNA Micro-prep kit (Zymo research), followed by additional DNase treatment and purification . Around 100 ng total RNAs of each sample was sent to Sequencing and Microarray Facility at MDACC for strand-specific RNA-Seq analysis. Libraries were made with Illumina’s TruSeq Stranded Total RNA Library Prep Kit and were sequenced in 76 paired-end format on Illumina Next Generation Sequencing-HiSeq4000.Tm33.X-gal staining procedure was as describedAd5-cmv-GFP and Ad5-cmv-Cre were purchased from Baylor College of Medicine’s Vector Development Lab. Primary stromal cells were transduced with Ad5-cmv-Cre or Ad5-cmv-GFP at a concentration of 5000 pv/cell (8 µg/ml polybrene). After around 24 h, fresh media was added to replace media containing adenoviruses. Lenti-ß-catenin was generously provided by Andrew Gladden of the Genetics department. Lentivirus plasmids pGIPZ2 (empty vector), pGIPZ3 (non-specific shRNA), and pGIPZ-shp53 were purchased from MDACC Functional Genomics Core.Quick-RNA Micro-prep kit (Zymo Research) was used to extract total RNAs. cDNAs were synthesized using amfiRivert cDNA Synthesis Platinum Master Mix (Gendepot). qPCR reactions were made using amfiSure qGreen Q-PCR Master Mix(2X), Low Rox (Gendepot), and QuantStudio 6 (Applied Biosystems). Primer sequences were designed using Integrated DNA Technologies’s PrimerQuest tool.t-test in GraphPad Prism 7.0. The mean values were presented. The error bars indicated SEM.Statistical analysis was calculated by two-tailed, unpaired Student’s Supplementary figure legendsSupplementary Figure S1Supplementary Figure S2Supplementary Figure S3Supplementary Figure S4Supplementary Figure S5Supplementary Figure S6Supplementary Figure S7Supplementary Figure S8Supplementary Figure S9