The PACE ‘Steps to Success’ programme is a complex educational and development intervention for staff to improve palliative care in long-term care facilities (LTCFs). In a cluster randomized controlled trial, this programme has been implemented in 37 LTCFs in 7 European countries. Alongside an effectiveness study, a process evaluation study was conducted. This paper reports on the results of this process evaluation, of which the aim was to provide a more detailed understanding of the implementation of the PACE Programme across and within countries.The process evaluation followed the Reach, Effectiveness, Adoption, Implementation, Maintenance (RE-AIM) framework and involved various measures and tools, including diaries for country trainers, evaluation questionnaires for care staff, attendance lists and interviews  with country trainers, managers, PACE coordinators and other staff members. Based on key elements of the PACE Programme, a priori criteria for a high, medium and low level of the RE-AIM components Reach, Adoption, Implementation and intention to Maintenance were defined. Qualitative data on factors affecting each RE-AIM component gathered in the online discussion groups and interviews were analysed according to the principles of thematic analysis.The performance of the PACE Programme on the RE-AIM components was highly variable within and across countries, with a high or medium score for in total 28 (out of 37) LTCFs on Reach, for 26 LTCFs on Adoption, for 35 LTCFs on Implementation and for 34 LTCFs on intention to Maintenance. The factors affecting performance on the different RE-AIM components could be classified into three major categories: (1) the PACE Programme itself and its way of delivery, (2) people working with the PACE Programme and (3) contextual factors. Several country-specific challenges in implementing the PACE Programme were identified.The implementation of the PACE Programme was feasible but leaves room for improvement. Our analysis helps to better understand the optimal levels of training and facilitation and provides recommendations to improve implementation in the LTC setting. The results of the process evaluation will be used to further adapt and improve the PACE Programme prior to its further dissemination.www.isrctn.com—ISRCTN14741671 (FP7-HEALTH-2013-INNOVATION-1 603111) July 30, 2015.The PACE study was registered at  As more and more people live out their lives in nursing and residential care homes, there are concerns about the quality of the end-of-life care given in these settings –7. To imThe PAlliative Care for older people in Europe (PACE) Steps to Success Programme is a 1-year multicomponent train-the-trainer programme for nursing homes that aims to stepwise implement a palliative care approach into the day-to-day routines in nursing homes . The corIn the first 2 months, the PACE coordinators received ‘pre-intervention training’ from the country trainers. The intervention was then rolled out to all care staff in the following 6 months, with each sequential step of the intervention being delivered in a training session by the country trainer, one step every month. Each training covered one of the six key elements of the intervention see Fig. . The proIn the cluster randomized controlled trial, the PACE Programme did not reach its intended effects . We did Process evaluations are recommended to open the ‘black box’ of interventions in trials  and are A suboptimal implementation might be one of the reasons why the PACE Programme did not reach its intended effects. Therefore, the aim of this process evaluation study was to provide a more detailed understanding of the implementation of the PACE Steps to Success Programme, across countries and within specific countries. Specifically, we sought to assess the programme’s Reach, Adoption, Implementation and the intention of staff to Maintain the PACE Steps to Success Programme in future practice, and the factors affecting these. The RE-AIM component Effectiveness will not form part of this paper, since it has already been examined separately and its results are reported elsewhere .The process evaluation study systematically monitored and evaluated the implementation of the PACE Steps to Success Programme in 37 LTCFs across seven countries , accordTable Reach, PACE coordinators used attendance lists to register how many staff members attended each training session, multidisciplinary review meeting (step 3) or reflective debriefing session (step 6) until month 18 of the intervention. In addition, to assess Adoption, PACE coordinators reported on the number of PACE documents  that were completed and archived at the end of the consolidation period (month 12). The extent to which the intervention was implemented as intended was investigated by analysing structured diaries that country trainers completed on a weekly basis during the 12 months of the intervention, in which they kept track of all the activities they performed regarding the PACE Steps to Success Programme. Additionally, we examined the quality of the training by involving the appreciation of care staff members towards the programme and trainer’s teaching competencies. Questions about their appreciation were added to an evaluation questionnaire, which was distributed to all care staff members after Step 6 of the intervention (month 8). As we could not measure Maintenance within the time scope of this research project, the evaluation questionnaire also contained questions on staff member’s intention to maintain elements and tools of the PACE Steps to Success Programme in their future daily practice and whether they would recommend the programme to other LTCFs.To measure In order to gain insight into the factors that affected the RE-AIM components, the facilitators and barriers participants encountered during the implementation period, and their recommendations for broader implementation or preferred adaptions to the programme, semi-structured group interviews using a topic-list were performed with care staff members and PACE coordinators, and individual interviews with facility managers (month 13–15). Researchers in each country were trained in conducting these qualitative interviews and were supported by the first and last authors (MOV and HRP) during monthly online meetings. They were instructed to take notice of the nursing home staff’s answers on the evaluation questionnaires, which were collected a few months earlier, and prepare some targeted questions that they could bring in if needed. The interviews were recorded and transcribed verbatim. Lastly, country trainers were invited to attend one of two online discussion groups (month 13). Organizing discussion groups online has the advantage that participants can easily join the discussion from anywhere at any convenient time . During Before the results were analysed, we established criteria for high, medium and low levels of Reach, Adoption, Implementation and intention to Maintenance during a consensus meeting with the PACE consortium, based on key elements of the PACE Programme. For example, Reach was rated ‘high’ if the mean attendance rate on all six training sessions was 70% or higher, ‘medium’ if 30-69% and ‘low’ if below 30%. Cut-off scores for Adoption were somewhat higher than for Reach, because we thought that higher rates on Adoption would be more easily achievable in practice  interviews were analysed according to the principles of thematic analysis , in a deTable Implemented and intended to be Maintained were generally higher than the levels to which the PACE Programme Reached and was Adopted by the target population. We will focus on each specific component below.Figure The mean attendance rate on all six training steps varied widely between LTCFs, from 4% in one facility in The Netherlands up to 81% in one facility in Switzerland . At these critical moments, attending the training conflicted with caring for residents. A third set of factors concerned the motivation and expectations of staff. Some staff members were very eager to learn more about palliative care, others were barely interested. Especially in times of reorganizations or when other projects were ongoing or just finished, motivation to attend training was found to be low, resulting in low attendance. This motivation sometimes increased after the first training session, because discussing wishes for future care with residents (Step 1) raised enthusiasm among staff, making them curious about the remainder of the PACE Programme (Quote 5). More often however, the attendance rate could also drop during the PACE Programme because of expectations that were not fulfilled (Quote 6), resistance to the trainer or when staff already had missed one of the former training sessions. Lastly, the qualitative information revealed that attendance could be raised by certain stimuli or incentives. For example, financial reimbursement or the prospect of receiving a certificate for attending a minimum of four training sessions contributed to staff members’ attendance (Quote 7). In some LTCFs, attending the training was compulsory. This increased the attendance rate, but often had a negative effect on staff members’ motivation and enthusiasm (Quote 8). A manager who encouraged staff to attend the training, by giving them time off their shifts, also helped in attaining a high level of reach.Qualitative information from the attendance lists, group and individual interviews and online discussion groups provided insight into the factors that both hindered and facilitated attendance. First, te Table , Quote 1The proportion of residents with a completed Looking and Thinking Ahead document (PACE step 1) archived in the residents’ care file at the end of the consolidation period ranged from 6%  to 186% (LTCF in The Netherlands)  completed and documented at the end of the consolidation period ranged from 0%  to 135%  . Second, factors related to the organization of daily care practice played a role in the level of Adoption. For example, in LTCFs where each staff member was responsible for the care of one or a few residents, the discussion regarding wishes for future care (step 1) was said to be easier, because a bond of trust was often already established between this staff member and the resident (Quote 10). This bond of trust was largely absent in LTCFs where nurses and physicians were hired from external companies, or in times of understaffing, when there is no time to sit and get to know residents. Adopting the documents was made easier in LTCFs where they were incorporated in each resident chart or made electronically available (Quote 11). Also, staff members said they were more inclined to complete the PACE documents when they were already used to doing so, i.e. when their LTCF already worked with similar documents for pain and depression, or to engage in advance care planning (ACP). However, when staff in a LCTF already worked with documents already perceived to be of good quality, the PACE documents were evaluated as duplication and causing a lot of extra work, resulting in resistance of staff members to use the documents. The large amount of paperwork, in combination with the fact that staff had little time to complete them and the documents not being electronically available, contributed to this resistance, especially in older employees who often showed little willingness to change their way of working (Quote 12). Furthermore, some staff members preferred to discuss pain or depression without an assessment scale. Anxiety that everything is verifiable played a role for a few. A more frequently mentioned reason for resistance against the documents was that care staff members did not feel skilled or competent enough to assess pain or depression in residents (Quote 13). They believed that each document should be used by a professional on that domain; i.e. a social worker for the ACP discussions (PACE Step 1), a physiotherapist for the pain assessments and a psychologist or other physician for the depression assessments (PACE Step 4). Also discussing death and dying (Step 1) was often felt to be uncomfortable, especially for care staff members in Poland and Italy, because of a cultural taboo regarding these topics (Quote 14). But also in other countries, staff members indicated that they were not prepared to discuss these issues, ‘because they were trained to act instead of talk’. Other factors influencing Adoption concerned the target group of the documents: the residents, families and their general practitioners (GPs). Barriers to adoption were residents who were not willing or able to discuss death (e.g. because of dementia), little involvement of families in residents’ care, assumptions that residents and family members expect staff members to enhance the condition of a resident rather than talk about death, and GPs who are not in favour of measuring pain in a structured manner. In contrast, residents who promoted ACP discussions among other residents, family who were keen on such discussions and grateful afterwards, and the experience that a pain scale facilitated communication with GPs helped in adopting the tools (Quote 15). Lastly, the qualitative data revealed that Adoption could be raised by certain stimuli from others. For example, in a few LTCFs, completing the documents was mandatory. In others, staff members were actively reminded to use the tools or the manager even announced that an audit would be performed (Quote 16). An external stimulus in Belgium came from the Flemish government, who reports the number of documented ACP discussions as an indicator of the quality of care in a LTCF.The qualitative data shed light on the factors affecting the level of Adoption. First, the The rating for Implementation consisted of two elements; fidelity (the extent to which the six steps were delivered as intended) and the care staffs’ appreciation of the trainer’s teaching competencies and the overall programme. First, fidelity scores ranged from 5 to 8 (out of 8) and were generally high in all countries. The intervention was fully implemented as intended in 28 out of 37 LCTFs in terms of number, order and timing of training sessions; all six PACE Steps were taught, in the right order and within 8 months. In seven other LTCFs , the six PACE Steps were taught, but not in the right order and/or not within 8 months. Only in two LTCFs (in Belgium and England) were not all six PACE steps taught, but still training was completed on five steps. Second, the combined score for satisfaction with the trainer’s teaching competencies and with the overall PACE Programme ranged, on a scale from 0-8, from 3.2 (LTCF in Finland) to 7.8 (LTCF in Poland) . In addition, scheduled meetings were sometimes cancelled due to illness among staff or due to the LTCF being placed in quarantine because of a viral outbreak in the LTCF. Regarding the characteristics of the PACE trainer, we found that satisfaction among staff members was higher when the trainer had experience in palliative care, so that he/she could incorporate examples from his/her own practice and understood what was relevant in daily practice. Experience in teaching was also considered crucial (Quote 18 and 19), since the teaching style of some trainers was perceived as too simplistic and did not engage staff, thus decreasing motivation to attend training sessions. Also, characteristics of the training programme influenced the satisfaction of staff members. Some felt the time between training sessions was too long, for instance because they had forgotten what was discussed with the previous step, while others experienced it as too short with more time being needed to digest all the information before continuing with another subject in another step (Quote 20). Remarks were also made on the number and length of each training session.The qualitative data showed that The rating for Maintenance is a combination of both the manager’s and the care staffs’ intention to continue working with PACE steps and tools. Managers of 26 LTCFs stated in the interviews that they were willing to continue with PACE in their own LTCF. The other managers had no intention to continue with PACE (managers of five LCTFs) or declined to be interviewed (managers of six LTCFs) which we also interpreted as ‘having no intention to work with PACE anymore’.The score for staff member’s intention to work with PACE steps/documents in the future, together with their recommendation regarding PACE to other LTCFs ranged, on a scale from 0 to 8, from 2.5 (LTCF in Poland) to 7.9 (LTCF in England)  (Quote 21), whereas reasons to quit were the high workload, the large number of documents, the preference for other or no documents at all or the PACE documents being viewed as not clear and not simple enough for severely demented residents (Quote 22). Secondly, the future availability of PACE coordinators played a role in the intention of managers and staff to continue with PACE (Quote 23 and 24). Appointing staff who would be responsible for the continuation of PACE was regarded as important. However, this was difficult in some LTCFs, as PACE coordinators were not always committed to consolidate PACE, no longer working in the facility or blocked by unsupportive fellow staff members in performing their tasks. In some other LTCFs, the enthusiasm for PACE faded after the trainer had left the LTCF, leaving no-one to organize any follow-up. Lastly, aspects around the organization and policy of the LTCF influenced the intention to continue with PACE. Changes within management or minimal involvement of the management in PACE was found to hinder its maintenance. In another instance, the size of the LTCF was found too large to facilitate the continuation of PACE. In contrast, having a budget available to further roll out PACE or having worked already on the electronic availability of the PACE tools within the residents’ care plans, helped to maintain PACE (Quote 25). Specifically in England, being able to use the PACE Programme in care home inspection registration and to help nurses revalidate their registration were mentioned as factors positively influencing the maintenance of PACE (Quote 26).The qualitative data revealed why managers and care staff did or did not intend to work with PACE in the future. A first set of reasons were related to the Some country-specific challenges in implementing the PACE Programme were identified. For example, in Poland, LTCFs are characterized by a strong sense of hierarchy between the different professions and a clear determination of fields of expertise and division of tasks . Being ‘task oriented’ made it more difficult to embrace the steps that required a holistic approach to residents. In addition, in Poland as well as in Italy, prevailing cultural taboos on discussing death impacted on staffs’ ability to implement the steps. Limited knowledge on palliative care was especially seen in Italy, where palliative care was often equated with euthanasia or seen as an approach only belonging to cancer patients and hospice care; some doctors therefore refused to take notice of the PACE Programme. In Finland and Switzerland, the PACE Programme was—more often than in the other countries—perceived as to not fit the level of knowledge and needs of the facility and the nurses. Staff indicated that a good level of palliative care was already established in their facility and that they did not learn that much from the PACE Programme. Instead, they had hoped for a more technical training. In England in particular, the attendance rates on the training sessions were low. The long shifts that some staff members worked here (up to 12 h) created difficulties for them to attend training sessions or made staff members openly admit that they would not come to training on their day off. Some LTCFs therefore had requested the country trainer to shorten the training sessions to 45 min, so that staff could rotate to access training sessions in normal working hours, or to give one-on-one training sessions. In the Netherlands and Belgium, reorganizations and transformations of residential care homes into nursing homes were every day’s business, causing anxiety and instability within teams. On top of that, in a few Belgian LTCFs, there was a tense atmosphere between PACE coordinators (often head nurses) and palliative care specialist nurses, who were perceived to be jealous because they believed palliative care was only their specialty. Palliative care specialist nurses did sometimes not attend training and were in return not invited by PACE coordinators to multidisciplinary review meetings. Lastly, a specific challenge encountered in the Netherlands was working together with a large number of GPs. The fact that the Dutch country trainers were GPs themselves helped somewhat to convince GPs to attend the multidisciplinary review meetings in the LTCF.The aim of this process evaluation study was to provide a more detailed understanding of the implementation of the PACE Steps to Success Programme, across countries and within specific countries. By examining its Reach, Adoption, Implementation and intention to Maintenance, we have shown that the performance of the PACE Programme on the different components was highly variable within and across countries, with generally a better performance on the components Implementation and Maintenance than on the components Reach and Adoption. Our study sheds light on areas where suboptimal implementation of the PACE Programme may have led to limited effects found in the PACE trial, as well as helps to better understand the optimal levels of training and facilitation and provides recommendations to improve implementation in the complex LTC setting.Whereas the interplay of several factors within all RE-AIM components should be taken into account when explaining why the PACE Programme did not reach its intended effects within the trial , the proAll factors that we found to affect the different RE-AIM components could be classified into three major categories, namely (1) the PACE Programme itself and its way of delivery, (2) people working with the PACE Programme, and (3) contextual factors. The first category entails factors like the high amount of paperwork, the time between training sessions, the vocabulary used in the PACE documents and the practical experience of the trainer and his/her way of teaching. According to our process evaluation, key recommendations for future implementation are to reduce the amount of paperwork , to allow a flexible length of time between training sessions, to provide clear materials and to ensure that trainers are well qualified in palliative care as well as teaching (see Table The three categories described above largely correspond with the domains in the Consolidated Framework For Implementation Research (CFIR) , with thThese are not isolated categories, but interrelate with each other in a way that corresponds to the findings from a realist process evaluation within the Facilitating Implementation of Research Evidence (FIRE) study performed in care homes . This stAlthough the barriers and facilitators we identified may not be all novel , and mosOur implementation activities were shaped and to some extent constrained because the PACE Steps to Success intervention was tested in a cluster randomized controlled trial, in which there was limited space for flexibility to adapt the PACE Programme to the specific context . HoweverVery few published examples of process evaluation studies examining the implementation of palliative care programmes in nursing homes exist, and none at the scale of this study set in multiple country contexts. Major strengths of our process evaluation study were that it was set up using a rigorous study design, embedded within the PACE cluster randomized controlled trial and performed in a similar manner in seven countries. Whereas the implementation of most interventions, including the implementation of the precursor of the PACE Programme—the Six Steps to Success Programme—are evaluated in a less robust way , 39, we The large amount of information we collected posed challenges on the key informants of the PACE trial, who indicated they were overburdened by all PACE related activities (the intervention and the research), which might have decreased their enthusiasm for the PACE Programme. To reduce the workload of PACE coordinators, we chose to provide attendance lists that only asked for the number of care staff members attending each session and the total number of care staff members working in the LTCF at that moment. However, this compromised our ability to describe whether the participants were representative for the total mix of care staff members working in the LTCF and our ability to describe whether the same individuals attended several sessions or not. To be able to reflect on whether over-representation of certain groups and under-representation of others, as well as degree of consistency of attendance over time may have affected the implementation process and intervention success, we would recommend a more extensive examination of Reach in future studies.The implementation of the PACE Steps to Success Programme was feasible, but also highly variable within and across countries. The results of the process evaluation will be used to further adapt and improve the PACE Steps the Success Programme before its further dissemination. We recommend that future implementation of the PACE Programme is guided by close and ongoing monitoring of the fit between the programme and the context.


Epoxy bonded steel plates  for the strengthening of concrete structures were introduced to the construction industry in the late 1960s, and the use of fibre reinforced polymers (FRPs) was introduced in the 1990s, which means that these techniques have already been used in construction for 50 and 25 years, respectively. In the first part of the paper, a historical survey of the development and introduction of these strengthening techniques into the construction industry are presented. The monitoring of such applications in construction is very important and gives more confidence to this strengthening technique. Therefore, in the second part of the paper, two long-term monitoring campaigns over an extraordinarily long duration will be presented. Firstly, a 47-year monitoring campaign on a concrete beam with an epoxy bonded steel plate and, secondly, a 20-year monitoring campaign on a road bridge with epoxy bonded CFRP (carbon fibre reinforced polymers) strips are described. The paper is an expanded version of the paper presented at the SMAR2017 Conference. The first ideas on strengthening of concrete with epoxy bonded steel plates were presented in the 1960s by L’Hermite et al. . A figurIn the 1970s and 1980s, besides the investigations mentioned above, research on the performance and on a better understanding on the epoxy bonded steel plates was also performed at Empa in City, Switzerland. An overview of these investigations at Empa is given in . Bond teCarbon fibre reinforced polymers (CFRP) are used for load carrying components in the air and space industry since the 1970s. Empa researchers developed the idea of using CFRPs instead of steel in the 1980s. An analysis showed that the employment of advanced composites  reduced the costs for the scaffolding in the subsequent strengthening of bridges. Since reinforcement plates made of advanced composites were extremely light, scaffolding was not necessary . FurtherThe Kattenbusch Bridge in Germany was the world’s first bridge where the EBR strengthening technique Glass Fibre Reinforced Polymers (GFRP) were applied . It is aAs a world premiere, the Ibachbridge near Lucerne in Switzerland was successfully strengthened with CFRP strips in 1991 ,18. See The idea of prestressing the CFRP laminates was firstly studied in a PhD thesis work by Deuring . The prefib model code 2010 [For the transfer of new products and techniques to the industry, it is essential that guidelines exist. The first code in Switzerland on externally bonded reinforcement was published in 2004, , and alsode 2010 ,26 and wThe FRP strengthening technique for reinforced concrete structures is nowadays state-of-the-art and is introduced into the industry. Several companies sell the FRP strips or fabrics and the suitable epoxy adhesive. The second author predicted in 1997 the worldwide demand of CFRP for EBR as given in As described, this material is used already for 25 years in construction. However, the application of this material requires not only knowledge of the ultimate and serviceability limit states, but also information on the durability of the CFRP over the remaining lifetime of the structures. Many investigations on the durability and long-term behaviour of unstressed and prestressed externally bonded reinforcements are available in the literature. For example, Correia et al.  investigMoreover, in a study by Kim et al. , carbon In addition, Foraboschi  presenteIn the following, two different long-term monitoring campaigns over an extraordinary long time will be presented. Monitoring of CFRP strips provides indications about their long-term behaviour, and, consequently, confidence in the use of this material for strengthening civil structures.Firstly, in a long-term laboratory test that has been ongoing at Empa since 1970, a concrete beam has been strengthened in flexure using a steel plate. A two-component epoxy adhesive was used. The beam was and is still loaded to 87% of the mean ultimate load and is still in a good state. Secondly, a monitoring campaign on a road bridge at the boarder of Switzerland with epoxy bonded CFRP strips has been going on since 1996. Displacements are measured manually by using a mechanical strain gauge.Prefabricated reinforced concrete (RC) elements of the roof of an industrial building  had to bAs discussed before, in 1970, the knowledge on the strengthening technique of bonding steel plates with epoxy adhesive to concrete was at the beginning. Therefore, in this year, a test program was performed at Empa to investigate this technique. Several prefabricated RC elements strengthened by epoxy bonded steel plates were tested statically in a four-point bending test, one element was tested under fatigue and one was used for a long-term sustained loading test. This test is still running today and will be presented in this paper. See also ,33.c,cube,28 ≈ 28 MPa; in 2002, the concrete strength was fc,cube ≈ 58 MPa, the steel plate is likely to be an ordinary construction steel S235 with a yielding strength of fy ≈ 236 MPa, and the adhesive is a two-component epoxy adhesive probably from the company Ciba-Geigy  [The RC elements for the experiments were produced from a Swiss prefabrication company  and were delivered to Empa. Unfortunately, only limited documents from the investigation today exist. However, the material properties were reconstructed in the year 2002: the concrete compressive strength after 28 days was fzerland) .The beam is loaded as a four-point bending test  and consThe mid-span displacement of the long-term test is measured with a dial gauge  and the After 47 years, the beam is still in a good condition. The steel plate has some corrosion at the surface, which is considered as not relevant . The str2, Existing maximum flexural moment from loading: 30 × 0.945 = 28.4 kNm ,Flexural resisting flexural moment: 159 × 0.95 × 0.19 = 28.7 kNm.The tensile stress in the steel plate can be estimated from the measured steel strain of 0.9% by using an elastic modulus of 210 GPa as 189 MPa. The corresponding tensile force is therefore 159 kN  with a cross-section of 80 × 1.2 mm and a length of 4.2 m were applied in order to strengthen the positive bending moment in the bridge cross-direction . In totaDisplacement measurements with a mechanical strain gauge  on two oT = 10−5, a displacement variation of ΔT × αT × L ≈ 0.05 mm has to be expected. Therefore, the measurements in Some measurements of the variations of the displacements are given in No water wetting,FRPs should be used only for Exposition Classes X0, XC1 (dry) and XC3,For other exposition classes: special measures such as coatings are necessary,Moderate sustained loading ,Tg)Service temperatures ≤ 50 °C . The foIn the first part of the paper, the impressive history of the development and introduction of the EBR strengthening technique to the construction industry is described. In the 1960s and 1970s, bonded steel plates were used to strengthen reinforced concrete. Then, in the 1980s, the FRPs were introduced and the first site applications were in the 1990s. although very highly loaded, the strengthening of an RC beam with externally bonded steel plates is, after 47 years of sustained loading in the laboratory, still in good condition, and no failure was observed,the increase of the displacements happens mainly due to creep of concrete,the epoxy adhesive for the bonding of the steel plate to the concrete is still in good condition.In the second part of the paper, two extremely long-term monitoring campaigns are presented. The following conclusion from these investigations can be made. The 47-year-old test on a beam with an epoxy bonded steel plate and a high sustained load showed that:the CFRP strips are still in good condition,the measurements show only seasonal temperature deformations.The 20-year-old CFRP strips glued on the bottom side of the Rhine Bridge in Oberriet showed that:Lastly, recommendations on how FRPs are correctly applied are given, with the aim to get durable strengthening solutions.


PP2A-B56 is a serine/threonine phosphatase complex that regulates several major mitotic processes, including sister chromatid cohesion, kinetochore-microtubule attachment and the spindle assembly checkpoint. We show here that these key functions are divided between different B56 isoforms that localise to either the centromere or kinetochore. The centromeric isoforms rely on a specific interaction with Sgo2, whereas the kinetochore isoforms bind preferentially to BubR1 and other proteins containing an LxxIxE motif. In addition to these selective binding partners, Sgo1 helps to anchor PP2A-B56 at both locations: it collaborates with BubR1 to maintain B56 at the kinetochore and it helps to preserve the Sgo2/B56 complex at the centromere. A series of chimaeras were generated to map the critical region in B56 down to a small C-terminal loop that regulates the key interactions and defines B56 localisation. Together, this study describes how different PP2A-B56 complexes utilise isoform-specific interactions to control distinct processes during mitosis. The cells in our body are a hive of activity, but that activity must be kept under control. This is never more critical than when a cell divides, because unchecked cell division can lead to cancer. Fortunately, enzymes called kinases and phosphatases exist to control the countless proteins in a cell; these enzymes help ensure that each step of cell division is complete before moving on to the next.Kinases control other proteins by adding bulky phosphate groups to them, while phosphatases remove those groups. For a long time, phosphatases were assumed to be less specific than their kinase counterparts. Yet it has now become clear that phosphatases achieve specificity by interacting with a range of regulatory subunits.A phosphatase called PP2A oversees a number of key steps in cell division by working together with its regulatory B56 subunit. In human cells, there are five separate genes that encode B56 subunits, and all of these B56 ‘isoforms’ were thought to exert the same influence on the PP2A phosphatase. The fact, however, that different isoforms are found at different locations within the cell suggested otherwise.To investigate this, Vallardi et al. focused on a particular stage of cell division when the activity of the PP2A-B56 complex is essential. Before a cell divides it duplicates its genetic material and the two copies of each chromosome are held together until the cell is ready to pull them apart. The experiments compared two representative B56 isoforms: one that concentrates at the centromere, the region where the copied chromosomes are held together; and another found at the kinetochore, a nearby structure that is involved in pulling the two chromosomes apart. By eliminating all but one isoform and measuring the ensuing activity of the PP2A-B56 complex, Vallardi et al. could differentiate between the main regulatory roles of each isoform. These experiments showed that B56 isoforms control separate processes during cell division, which mirrors their different locations within the cell.Next, Vallardi et al. looked at the receptor proteins that recruit each isoform to its position. Removing or relocating different receptors showed how they anchor select B56 isoforms in different positions while the associated PP2A enzymes get to work on different processes. Further experiments using ‘hybrid’ subunits made from parts of two different B56 isoforms then helped to reveal the site on the B56 subunits that determines which receptors they bind to. Together these findings show that slight differences between each B56 isoform ultimately dictate where they localise and what processes they control when cells divide. Protein Phosphatase 2A (PP2A) is a major class of serine/threonine phosphatase that is composed of a catalytic (C), scaffold (A) and regulatory (B) subunit. Substrate specificity is mediated by the regulatory B subunits, which can be subdivided into four structurally distinct families: B (B55), B’ (B56), B’ (PR72) and B’’ (Striatin) .In humans, the B subunits are encoded by a total of 15 separate genes which give rise to at least 26 different transcripts and splice variants; therefore, each of the four B subfamilies are composed of multiple different isoforms . AlthougWe addressed this problem by focussing on prometaphase, a stage in mitosis when PP2A activity is essential to regulate sister chromatid cohesion , kinetocThe kinetochore is a multiprotein complex that assembles on centromeres to allow their physical attachment to microtubules. This attachment process is stochastic and error-prone, and therefore it is safeguarded by two key regulatory processes: the spindle assembly checkpoint (SAC) and kinetochore-microtubule error-correction. The SAC preserves the mitotic state until all kinetochores have been correctly attached to microtubules, whereas the error-correction machinery removes any faulty microtubule attachments that may form. The kinase Aurora B is critical for both processes because it phosphorylates the kinetochore-microtubule interface to destabilise incorrectly attached microtubules and it reinforces the SAC, in part by antagonising Knl1-PP1, a kinetochore phosphatase complex needed for SAC silencing . These tAs well as localising to the outer kinetochore, PP2A-B56 also localises to the centromere by binding to shugoshin 1 and 2 (Sgo1/Sgo2) . The cryIn human cells, B56 isoforms are encoded by five separate genes . The interaction interfaces involved in BubR1 and Sgo1 binding are extremely well conserved between all of these B56 isoforms . This exHowever, one crucial observation throws doubt over this issue of redundancy: individual B56 isoforms localise differentially to either the kinetochore or centromere in human cells . It is tPP2A-B56 isoform localisation to the centromere and kinetochore was visualised in nocodazole-arrested HeLa Flp-in cells expressing YFP-tagged B56 subunits. This revealed that while some B56 isoforms localise predominantly to the sister kinetochore pairs marked by Cenp-C (B56γ and δ), others localise mainly to the centromere defined by Sgo2 (B56α and ε), and one isoform displayed a mixed localisation pattern (B56β) . B56 isoCentromeric PP2A-B56 is important for maintaining sister chromatid cohesion during mitosis in human cells . In agreTo examine which B56 isoforms can support kinetochore functions, we first focussed on SAC signalling. The SAC is activated at kinetochores by the phosphorylation of ‘MELT’ repeats on Knl1 by the kinase Mps1 . These pAs expected, simultaneous depletion of all B56 isoforms enhanced basal Knl1-MELT phosphorylation in nocodazole, delayed MELT dephosphorylation upon Mps1 inhibition with AZ-3146 , and preOverexpression of GFP-B56α has previously been shown to rescue kinetochore-microtubule attachment defects following the depletion of all PP2A-B56 isoforms in human cells . To undeWe next sought to determine the molecular explanation for differential B56 isoform localisation. This was difficult to reconcile with existing structural data mapping the interaction between PP2A-B56 and the reported kinetochore and centromere receptors - BubR1 and Sgo1 – since these demonstrate that the key interacting residues are well conserved between all B56 isoforms  Wang et. FurtherAt the centromere, Sgo1 and Sgo2 can both bind to PP2A-B56 , but SgoDepletion of Sgo2, but not Sgo1, caused a significant reduction in B56α levels at the centromere . It is iWe next examined whether specific binding to Sgo1 and/or Sgo2 could explain differential B56 isoform localisation. To address this, we artificially relocalized Sgo1 or Sgo2 to the inner kinetochore, by fusing it to the kinetochore-targeting domain of CENP-B (CB). This location was chosen, even though it partially overlaps with the endogenous centromeric B56 pool, because it is still accessible to Aurora B. This may be important because phosphorylation of Sgo2 by Aurora B has been proposed to be needed for B56 interaction . WhereasH187A) reduced but did not completely remove B56γ from kinetochores/centromere , which we confirmed was defective in binding CB-Sgo1 in vivo  was sufficient to relocalise B56α from centromeres to kinetochores . We cannot, however, exclude the possibility that the corresponding TKHG sequence in B56γ positively regulates LxxIxE interaction and kinetochore localisation. Considering that this region also controls Sgo2 and centromere binding, a simple explanation could be that Sgo2 interaction obscures the LxxIxE binding pocket. However, this appears unlikely for four reasons: 1) Sgo2 depletion does not relocalise B56α to kinetochores  Sgo2tochores , 3) centtochores , and 4) tochores . AlthougAn important additional finding of this work is that Sgo1 contributes to the B56γ signal observed at the kinetochore . This liIn summary, the work presented here explains how different members of the PP2A-B56 family function during the same stage of mitosis to control different biological processes. This is the first time that such sub-functionalisation has been demonstrated between isoforms of the same B family. It is currently unclear why such specialisation is necessary or at least preferable to a situation whereby all B56 isoforms operate redundantly, as initially suggested . One posHeLa Flp-in cells , stably WT (ΔSgo1) was created by site directed mutagenesis to create three mutations: Y391F, L394S and M398Q. The dCas9-DARPIN-flag was created by digesting pHAGE-TO-dCas9-3xmCherry (Addgene #64108) with BamHI and XhoI to remove 3xmCherry and replace with a synthesised DARPIN-flag that binds to GFP with high affinity  and subcloned into pCDNA5-LAP-BubR1WT  through WT  was usedaffinity . The gRNaffinity ) into thCells were transfected with 20 nM siRNA using Lipofectamine RNAiMAX Transfection Reagent (Life Technologies) according to the manufacturer’s instructions. For simultaneous knockdown of all B56 isoforms (B56pool) the single B56 isoform siRNA were mixed at equimolar ratio of 20 nM each. The siRNA sequences used in this study are as follows: B56α (PPP2R5A), 5’-UGAAUGAACUGGUUGAGUA-3’; B56β (PPP2R5B), 5’-GAACAAUGAGUAUAUCCUA-3’; B56γ (PPP2R5C), 5’-GGAAGAUGAACCAACGUUA-3’; B56δ (PPP2R5D), 5’-UGACUGAGCCGGUAAUUGU-3’; B56ε (PPP2R5E), 5’-GCACAGCUGGCAUAUUGUA-3’; Sgo1, 5’-GAUGACAGCUCCAGAAAUU-3’; Sgo2, 5’-GCACUACCACUUUGAAUAA-3’; BubR1, 5’-AGAUCCUGGCUAACUGUUC-3’; Knl1, 5’-GCAUGUAUCUCUUAAGGAA-3’; Bub1 5’-GAAUGUAAGCGUUCACGAA-3’; Control (GAPDH), 5’-GUCAACGGAUUUGGUCGUA-3’;. All siRNA oligos were custom made and purchased from Sigma, except for Sgo1, which was ordered from Dharmacon (J-015475–12).For reconstitution of B56 isoforms or mutants, HeLa Flp-in cells were transfected with 100 nM B56pool or mock siRNA and, in some experiments, 20 nM additional control, Sgo1, Sgo2, BubR1, Bub1 or Knl1 siRNA. Cells were transfected with the appropriate siRNA for 16 hr, after which they were arrested in S phase for 24 hr by addition of thymidine. Subsequently, cells were released from thymidine for 8–10 hr and arrested in prometaphase by the addition of nocodazole. YFP-B56 expression was induced by the addition of doxycycline during and following the thymidine block. For BubR1 knockdowns and for all chromosome alignment assays, cells were released from thymidine for 6.5 hr and arrested at the G2/M boundary with RO3306 for 2 hr. Cells were then released into nocodazole (BubR1 experiments) or normal growth media  for 15 mins before MG132 was then added for 30 mins to prevent mitotic exit. For alignment assays, this is critical to analyse the synchronous alignment of mitotic cells over a 45 min period.2, 20 mM KCl, 2 mM CaCl2) for 10 min at room temperature before being spun onto slides using a Cellspin cytocentrifuge (Tharmac). Slides were airdried for 1 min and then fixed in 4% formaldehyde in PBS for 10 min at room temperature. Blocking and immunofluorescence staining  was carried out as described below. The percentage of cells with at least one split sister kinetochore pair was quantified.Hela-FRT cells were transfected with B56pool, B56βγδε, B56αγδε or control siRNA for 16 hr, treated with thymidine for 24 hr and released into normal growth media for 6.5 hr. Cells were then arrested at the G2/M boundary with RO3306 for 2 hr before release into nocodazole for 1 hr. Mitotic cells were isolated and incubated with hypotonic buffer (20 mM Hepes (pH7.0), 1 mM MgClCells were transfected with dCas9-DARPIN-flag and a guide RNA that targets a repetitive region on chromosome 7 (at 1:3 ratio of dCas9:gRNA). Doxycycline was added to induce YFP-B56 isoform expression and 48 hr later cells arrested in mitosis with nocodazole were fixed, stained and imaged for co-localisation of YFP-B56 isoforms and Sgo2. Only cells containing defined Flag-dCas9 spots that also co-recruited YFP-B56 were imaged. The majority of these spots recruited YFP-B56, but the dCas9 spots themselves were only readily detectable in mitotic cells. For the CB-Sgo1/2 expression experiments, the endogenous Sgo1/2 was still present during these assays.http://crispr.mit.edu/) so that their complementary sequences are interrupted following successful homologous recombination. Flp-in HeLa Cas9 cells were generated and transfected with the YFP-homology arm vector and guide RNAs (B56α: gatgtcgtcgtcgtcgccgccgg B56γ: gtcaacatctagacttcagcggg) in a 1:1 ratio. Cas9 expression was then induced by addition of doxycycline and FACS was performed 2 weeks later to sort cells and enrich for the YFP-expressing population.800 base pair homology arms that span left and right of the start codon of B56α and B56γ were custom synthetized by Biomatik. A NaeI (B56γ)/SwaI (B56α) restriction site was place between the homology arms and used to insert a YFP tag by Gibson assembly. Guides were designed to span the start codon (using https://www.micro-manager.org/). Images were acquired with a Hamamatsu ORCA-ER camera every 4 min using 2 × 2 binning. For immunofluorescence, cells were plated on High Precision 1.5H 12 mm coverslips (Marienfeld). Following the appropriate treatment, cells were pre-extracted with 0.1% Triton X-100 in PEM  for 1 min followed by addition of 4% PFA/PBS for 2 min; cells were subsequently fixed with 4% paraformaldehyde in PBS for 10 min. Coverslips were washed with PBS and blocked with 3% BSA in PBS + 0.5% Triton X-100 for 30 min, incubated with primary antibodies for 16 hr at 4°C, washed three times with PBS and incubated with secondary antibodies plus DAPI for an additional 2–4 hr at room temperature in the dark. Washed coverslips were then mounted on a glass slide using ProLong antifade reagent (Molecular Probes). All images were acquired on a DeltaVision Core or Elite system equipped with a heated 37°C chamber, with a 100x/1.40 NA U Plan S Apochromat objective using softWoRx software (Applied precision). Images were acquired at 1 × 1 binning using a CoolSNAP HQ2 camera (Photometrics) and processed using softWorx software and ImageJ . All images displayed are maximum intensity projections of deconvolved stacks. All displayed immunofluorescence images were chosen to most closely represent the mean quantified data.For time-lapse analysis, cells were plated in 24-well plates, transfected and imaged in a heated chamber (37°C and 5% CO2) using a 10x/0.5 NA on a Zeiss Axiovert 200M Imaging system, controlled by Micro-manager software . Kinetochore quantification was performed as previously . For qua3VO4, 5 mM ß-glycerophosphate, 25 mM NaF, 10 nM Calyculin A and complete protease inhibitor containing EDTA (Roche)) on ice. The lysate was incubated with GFP-Trap magnetic beads (from ChromoTek) for 2 hr at 4°C on a rotating wheel in wash buffer  at a 3:2 ratio of wash buffer:lysate. The beads were washed 3x with wash buffer and the sample was eluted according to the protocol from ChromoTek. Samples were them processed for SDS-Page and immunoblotting using standard protocols.Flp-in HeLa cells were treated with thymidine and doxycycline for 24 hr and subsequently released into fresh media supplemented with doxycycline and nocodazole for 16 hr. Mitotic cells were isolated by mitotic shake off and lysed in lysis buffer . A rectangle of the same size was drawn around each band and the intensity within the band (minus the background) was calculated. The immunoprecipitated protein was used as a control, and each band was normalized to it.All antibodies were diluted in 3% BSA in PBS. The following primary antibodies were used for immunofluorescence imaging : mouse α-GFP , chicken α-GFP , mouse α- Sgo1 , rabbit α-Sgo2 , mouse α-BubR1 , mouse α-VSV , rabbit α-Knl1 , rabbit α-Bub1 , mouse α-FLAG  guinea pig α-Cenp-C  and rabbit α-pMELT-Knl1 directed against T943 and T1155 of human Knl1 , 1:1000)The following antibodies were used for western blotting : rabbit α-GFP , mouse α-B56γ , mouse α-B56α , mouse α-B56δ , rabbit α-B56ε , mouse α-PPP2CA  and rabbit α-PPP2R1A , rabbit α-BubR1 , rabbit α-Axin , rabbit α-GEF-H1 , rabbit α-Kif4a , rabbit α-RepoMan  and rabbit α-Actin  and mouse α-alpha-Tubulin . Secondary antibodies used were goat α-mouse IgG HRP conjugate  and goat α-rabbit IgG HRP conjugate .Mann-Whitney U test was performed to compare experimental groups in all kinetochore/centromere quantification graphs, whereas two-tailed, unpaired t-test with Welch’s correction was performed to compare experimental groups in all other graphs (using Prism seven software). The n numbers for kinetochore/centromere quantification statistics were derived from the individual cells , which were always from at least three separate experiments  with similar results. The n numbers for the statistics in all other graphs were defined by the number of experimental repeats. The SD bars displayed in each graph shows the variation between the means of the experimental repeats. The statistical comparisons most pertinent for the conclusions are shown in the figures and legends. The original data for all experiments displayed in graphs can be found in the raw data source file, which also contains the actual statistical values. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by Andrea Musacchio as the Senior Editor and Reviewing Editor, and three reviewers. The reviewers have opted to remain anonymous.Thank you for submitting your article "Division of labour between PP2A-B56 isoforms at the centromere and kinetochore" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:PP2A occurs mostly as a trimeric enzyme, consisting of a catalytic C-subunit, a scaffolding A-subunit and a regulatory B-subunit. The literature describes four types of structurally unrelated B subunits and their diversity is further augmented by the expression of isoforms. Five different genes encode for the B' (B56) isoform of PP2A. They are highly similar in amino acid sequence suggesting that they share redundant functions. Yet, recent studies in human cells revealed that individual B56 isoforms display distinct localization patterns, i.e. they localize to either the kinetochore or the centromere. In the submitted manuscript, Vallardi et al., investigate the molecular mechanisms underlying this differential localisation and function. They identify a domain in B56 that is responsible for this and show that it modulates the interaction with proteins containing a short linear motif (SLiM). This is probably the most novel observation reported here. Other observations, for instance the role of Sgo2 as the main receptor for centromeric PP2A-B56, are known from previous work , but the present study is systematic and provides a more comprehensive picture. Collectively, the work was praised for being systematic and because it sheds light on the molecular basis of a poorly understood phenomenon. While the analysis is generally strong, a major concern and several other points were discussed.Essential revisions:eLife revision.A point emerged strongly in the post-review discussion, namely that quantitative evidence corroborating claims on the role of the newly identified sequence motifs would not simply be nice to have but would strongly reinforce the paper. An analysis in vitro with purified B56a and B56g and peptides containing the relevant sequence motifs should aim to assess whether Sgo2 reduces the binding of B56a to BubR1, whether Sgo1 binds directly to Bubr1-B56, and whether these interactions are affected by mutation of the EPVA or TKHG motifs. Based on a significant body of previous work, we believe that these experiments are feasible and can be carried out in a time compatible with an Other important points:For some intensity plots, the authors indicate if the observed differences are significant or not  but for others they do not . Please add this information to all the plots.In some of the presented images, a clear-cut centromeric or kinetochore localization of B5a and B56g, respectively, as compared to the marker proteins Sgo2 and CENP-C, was not immediately recognisable . This is particularly true for endogenously tagged B56g, which is almost equally abundant at kinetochores and centromeres . At the very least, the partially overlapping localisation should be acknowledged.Kinetochore associated B56g, but not centromeric B56a, was sufficient to maintain the kinetochore functions of PP2A-B56 after the knockdown of all other B56 isoforms. The authors should explicitly state here that this cannot be explained by a lower expression level of B56a in these cells, as is only shown later in Figure 4A (but never commented upon). The reverse experiment, addressing whether B56a alone, but not B56g, can maintain the centromeric functions of PP2A-B56 is lacking and the authors could elect to carry it out.The relative kinetochore intensity  apparently refers to the combined kinetochore + centromere targeting. The normalised kinetochore intensity then shows the distribution between kinetochores and centromeres . The y-axes should be re-defined to make this difference clear.Figure 1—figure supplement 2. Please include the WB for the other B56 isoforms.In Figure 2 and Figure 2—figure supplement 1Figure, the authors aim to demonstrate that specific binding of B56a to Sgo2 contributes to its centromeric localization. With these figures, the following problems were identified:Figure 2C: Upon Sgo1 depletion, B56a seems to change its localization from a primarily centromeric localization to a primarily kt localization. Yet, this is not reflected in the quantifications shown in Figure 2D and 2E. Furthermore, Figure 2D gives the impression that Sgo1 is very efficiently depleted in Sgo1RNAi cells. The IF image shown in C gives a different impression. Could the authors please comment on this?Subsection “Sgo2 provides specificity for centromeric B56 recruitment”: "…, CB-Sgo2 was only able to recruit B56a ." This statement is puzzling. Figure 2J shows hardly any YFP-B56g localization. Why is the localization so different compared to Figure 1 and how was the quantification based on such IF images performed? Importantly, it should be clarified whether this figure and the related Figure 2—figure supplement 1 are carried out under conditions of depletion of Sgo1 and/or Sgo2, as this does not seem to be stated anywhere.Subsection “Sgo2 provides specificity for centromeric B56 recruitment”: "…CB-Sgo1 was able to localize both B56a and B56g to the inner kinetochore ." This statement is not supported by the data. Under wt conditions, B56g already colocalizes with CENP-C as shown in Figure 1A and 1B. Why are the quantifications  so different for control conditions?And given that B56g colocalizes with CENP-C under wt conditions, how do the authors come to the conclusion that CB-Sgo1 recruits B56g to inner kts?5) Along the same line, B56a shows a stronger co-localization with CENP-C under control conditions than B56g .The figure legend of Figure 2—figure supplement 1 (CB-Sgo1 recruits both B56a and B56g to centromeres) may be wrong. Apparently, what is meant is.….to inner Kts.Figure 3—figure supplement 2: There is hardly any signal for wt B56g  that colocalizes with CENP-C. Could the authors please comment on this?One of the reviewers raised a concern that the data are presented in a very compact manner that may not be readily accessible to a broad readership. While no specific suggestion was provided, the authors should consider this point and consider changes that will improve readability and raise the paper's general interest. A point emerged strongly in the post-review discussion, namely that quantitative evidence corroborating claims on the role of the newly identified sequence motifs would not simply be nice to have but would strongly reinforce the paper. An analysis in vitro with purified B56a and B56g and peptides containing the relevant sequence motifs should aim to assess whether Sgo2 reduces the binding of B56a to BubR1, whether Sgo1 binds directly to Bubr1-B56, and whether these interactions are affected by mutation of the EPVA or TKHG motifs. Based on a significant body of previous work, we believe that these experiments are feasible and can be carried out in a time compatible with an eLife revision.eLife review. This decision was reached for the following reasons:We thank the reviewers and editors for their very careful review and valuable comments/suggestions. In general, we agreed that all of the proposed changes would add value to the manuscript. However, as discussed in a subsequent email exchange with the editor, we also believe that the suggestion to use purified components could not be achieved in a timeframe that is compatible with an The proposed work would require the purification of 7 different components  followed by the phosphorylation of one of these components (BubR1) with two kinases (Plk1/Cdk1) in an attempt to generate two stable complexes: (1) Sgo1:pBubR1:PP2A-B56γ, and (2) Sgo2:PP2A-B56α. This second complex could then be tested for its ability to bind to phospho-BubR1. These in vitro assemblies would also then need to be carried out in the presence of peptides against our identified motif to see if complex assembly was prevented. In addition to the obvious time constraints, we were concerned that assembling phospho-dependent interactions in the presence of a purified phosphatase may be difficult, and we would also have no indication as to whether the peptides could adopt a conformation that binds to Sgo1 or Sgo2 with sufficient strength to prevent the key interactions.Nevertheless, although these in vitro experiments did not seem feasible to us, we did still clearly see the value of trying to address the main questions set out in the review. Therefore, as discussed in our previous email exchange, we have attempted to address these key questions (paraphrased below for clarity) by alternative means:1) Does Sgo2 inhibit the ability of B56α to bind to BubR1?2) Does Sgo1 bind directly to the BubR1-B56 complex?3) Are any effects identified in 1 or 2 dependent on the EPVA/TKHG motif?4) Can centromeric cohesion be maintained by only B56α, and not B56γ, as our localisation data would predict.Below is a summary of the new data that is now included in the manuscript to address these key questions:1) Does Sgo2 inhibit the ability of B56a to bind to BubR1?We have now examined the effect of Sgo2 depletion on the ability of B56α to bind to BubR1 and other LxxIxE-containing proteins. This new data  shows that efficient Sgo2 depletion does not enhance the ability of B56α to bind to either BubR1 or other proteins with an LxxIxE motif. Furthermore, we are unable to detect clear Sgo2 signal in any B56 IPs, even though LxxIxE interactions are clearly different between B56α and B56γ. We now have four independent pieces of data to suggest that Sgo2 does not inhibit B56α binding to BubR1. We copy a paragraph from the new Discussion section that expands on this point:“…a simple explanation could be that Sgo2 interaction obscures the LxxIxE binding pocket. However, this appears unlikely for four reasons: (1) Sgo2 depletion does not relocalise B56α to kinetochores , (2) Sgo2 depletion does not enhance the ability of B56α to bind to BubR1 or other LxxIxE motifs during mitosis , (3) centromere and kinetochore binding can occur together in certain B56α-γ chimaeras , and (4) the regions that define each of these localisations do not fully overlap . Although we believe these results suggest that Sgo2 is unlikely to block LxxIxE interaction, in vitro experiments with purified components would ultimately be needed to formally rule this out.”2) Does Sgo1 bind directly to the BubR1-B56 complex?Current structural data suggests that Sgo1 and B56 could interact simultaneously with BubR1, and our data indicates that a BubR1-B56-Sgo1 complex could potentially exist at kinetochores. Considering that Sgo1 binds directly to the PP2A-B56 complex, we reasoned that we could address this question by immunoprecipitating BubR1-wt or -dKARD (which cannot bind B56). If Sgo1 is detected in BubR1-wt, but not BubR1-dKARD IPs, then this would suggest that BubR1 requires B56 for Sgo1 interaction; thus, implying the existence of a BubR1-PP2A-B56:Sgo1 complex.We therefore immunoprecipitated BubR1-wt or dKARD from nocodazole-arrested cells and probed for Sgo1 and B56. The figure below shows that we could detect B56γ, but not B56α, in immunoprecipates from only BubR1-wt cells, as predicted. Unfortunately, however, we were unable to detect Sgo1 in any of these IPs, even after scaling up the input and performing rapid purifications (with a GFP-nanobody) to try to detect transient interactions. Therefore, we cannot currently state whether Sgo1 does not form a complex with BubR1-B56 in cells, or whether this complex is simply not stable enough to be detected following immunoprecipitation. We suggest to keep this as “results not shown” and have added the following new paragraph to the discussion:“We were unable to detect Sgo1 in YFP-BubR1 immunoprecipitates (results not shown), however, this could simply reflect an interaction that is either transient or unstable away from kinetochores. It will be important in future to clarify exactly how Sgo1 collaborates with BubR1 to control B56 localisation and, in particular, to determine whether Sgo1 can interact with BubR1:PP2A-B56 complexes directly.”We would like to stress that we do not see this question as critical to our study at all. It follows on from a novel observation that we made regarding kinetochore B56 recruitment, but this does not impact on isoform-specific localisation at all . That is why we believe this question is suitable to be left as a future direction.3) Are any effects identified in 1 or 2 dependent on the EPVA/TKHG motif?As outlined in point 1 above, Sgo2 is very unlikely to affect LxxIxE/BubR1 binding. Therefore, although the EPVA motif is definitely required for Sgo2 binding , this is unlikely to impact on LxxIxE binding. In addition, we believe that the EPVA/TKHG motif cannot strongly influence Sgo1 binding because Sgo1 can bind to both B56α and B56g .4) Can centromeric cohesion be maintained by only B56α, and not B56γ, as our localisation data predicts.We were actually very surprised that this was not considered an essential revision, given than it is not possible to formally demonstrate a true division of labour between B56 isoforms – as our title states – without this information. Furthermore, in our opinion, this was perhaps the most novel aspect of our study given that there are no previous demonstration that B56 isoforms can control separate cellular processes. This particular advance was not commented on in the summary by the reviewers, which we suspect is the main reason that this point was not seen as essential.Nevertheless, we now include this important new data in Figure 1c, which demonstrates that, as predicted previously, depletion of all B56 isoforms leads to premature sister chromatid splitting, and this can be fully rescued if B56α is retained, but not if only B56γ remains.Other important points:For some intensity plots, the authors indicate if the observed differences are significant or not  but for others they do not . Please add this information to all the plots.Thank you for pointing out this omission. The correct stats have now been added to all the Figures.In some of the presented images, a clear-cut centromeric or kinetochore localization of B5a and B56g, respectively, as compared to the marker proteins Sgo2 and CENP-C, was not immediately recognisable . This is particularly true for endogenously tagged B56g, which is almost equally abundant at kinetochores and centromeres . At the very least, the partially overlapping localisation should be acknowledged.We agree that it is not possible to stringently categorise localisation based on the results presented. The text has now been modified to make careful statements such as predominantly or mainly centromeric/kinetochore.Kinetochore associated B56g, but not centromeric B56a, was sufficient to maintain the kinetochore functions of PP2A-B56 after the knockdown of all other B56 isoforms. The authors should explicitly state here that this cannot be explained by a lower expression level of B56a in these cells, as is only shown later in Figure 4A (but never commented upon).A statement has been added to the text to explain that B56α and B56γ are both consistently expressed in HeLa cells . Furthermore, the new data indicates that preserving B56α can only retain centromeric functions, whereas retaining B56γ only preserves kinetochore functions. This removes any concerns that the observed effects might be due to low level expression of one particular isoform.The reverse experiment, addressing whether B56a alone, but not B56g, can maintain the centromeric functions of PP2A-B56 is lacking and the authors could elect to carry it out.Indeed, we agree that this was a very important omission given that the title of the manuscript refers to shared labours between B56 isoforms at the kinetochore and centromere. New data has now been included to demonstrate that B56γ cannot support centromeric cohesion, whereas B56a can .The relative kinetochore intensity  apparently refers to the combined kinetochore + centromere targeting. The normalised kinetochore intensity then shows the distribution between kinetochores and centromeres . The y-axes should be re-defined to make this difference clear.The axes and graph titles have now been changed to clarify this point.Figure 1—figure supplement 2. Please include the WB for the other B56 isoforms.A new western has now been inserted to include all B56 isoforms, except for B56β which was undetectable.In Figure 2 and Figure 2—figure supplement 1, the authors aim to demonstrate that specific binding of B56a to Sgo2 contributes to its centromeric localization. With these figures, the following problems were identified:Figure 2C: Upon Sgo1 depletion, B56a seems to change its localization from a primarily centromeric localization to a primarily kt localization. Yet, this is not reflected in the quantifications shown in 2D and 2E.The graph in Figure 2D shows relative intensity of centromere and kinetochore staining combined, which is why this does not change following Sgo1 depletion (B56a simply redistributes from centromere to kinetochore under this condition). The previous axis indicated “kinetochore intensities”, which was incorrect since the centromere/kinetochore cannot be distinguished with this type of analysis. All of these axes have therefore now been changed to read “Cen/Kt intensity” to avoid confusion.The lineplots in Figure 2E allows the Cen/Kt can be visualised separately, and this demonstrates that B56a redistributes towards the kinetochore under this condition: the new Figure 2E now contains additional repeats to reduce variability and this does indeed demonstrate redistribution towards the kinetochore.Furthermore, Figure 2D gives the impression that Sgo1 is very efficiently depleted in Sgo1RNAi cells. The IF image shown in C gives a different impression. Could the authors please comment on this?We try to always choose images that most closely represent the mean data (a statement is included in the methods to indicates this). In the previous Figure 2D the punctate staining was actually background staining that do not consistently align with kinetochores and can also be seen in the siCon image. However, the punctate pattern visible in the zoom was indeed confusing since it did partially overlap with kinetochore. Therefore, a different region has now been selected to avoid confusion and to better represent the minimal kinetochore staining seen under this condition.Subsection “Sgo2 provides specificity for centromeric B56 recruitment”: "…, CB-Sgo2 was only able to recruit B56a ." This statement is puzzling. Figure 2J shows hardly any YFP-B56g localization. Why is the localization so different compared to Figure 1 and how was the quantification based on such IF images performed?Thank you for pointing out this error. This image was not representative of the data because it had high background cytosolic levels and poor kinetochore staining. A new example image has now been included that better represents the mean data.Importantly, it should be clarified whether this figure and the related Figure 2—figure supplement 1 are carried out under conditions of depletion of Sgo1 and/or Sgo2, as this does not seem to be stated anywhere.The CB-Sgo1/2 experiments were all performed in the presence of endogenous Sgo1/2. This has now been clarified in the Materials and methods section.Subsection “Sgo2 provides specificity for centromeric B56 recruitment”: "…CB-Sgo1 was able to localize both B56a and B56g to the inner kinetochore ." This statement is not supported by the data. Under wt conditions, B56g already colocalizes with CENP-C as shown in Figure 1A and 1B. Why are the quantifications  so different for control conditions?And given that B56g colocalizes with CENP-C under wt conditions, how do the authors come to the conclusion that CB-Sgo1 recruits B56g to inner kts?Regarding points 3 and 4:Under wt conditions, B56a and B56g already colocalise with the centromere or kinetochore, respectively . The later figures with CB-Sgo1/2  still contain this baseline Cen/Kt localisation, but we then quantify the additional localisation induced by CB-Sgo1/2 expression. The same strategy was previously used by Meppelink et al., 2015 to show recruitment of B56d to CB-Sgo1. Our data indicate that CB-Sgo2 increases B56a , but not B56g , whereas CB-Sgo1 causes a large increase in both B56a and B56g at the Cen/Kt . Therefore, although the background Cen/Kt does complicate the overall interpretation (this is now clarified in the text), we still believe that our previous statements were supported by the data; given that we observe statistically significant increases only in certain conditions . Please note, that we chose to use the inner kinetochore for these experiments in spite of these difficulties, because it may be important given that Aurora B activity was previously reported to be required for Sgo2:B56 interaction .Along the same line, B56a shows a stronger co-localization with CENP-C under control conditions than B56g .Thank you for pointing this out. The image in S6D did not demonstrate the clear kt localisation in B56g controls. This image has now been changed for a more representative alternative .The figure legend of Figure 2—figure supplement 1 (CB-Sgo1 recruits both B56a and B56g to centromeres) may be wrong. Apparently, what is meant is.….to inner Kts.As stated above, we have now correctly labelled all these axes to indicate “Cen/KT intensities” since we cannot distinguish between these two localisations in this type of analysis.Figure 3—figure supplement 2: There is hardly any signal for wt B56g  that colocalizes with CENP-C. Could the authors please comment on this?The cells imaged in Figure 3—figure supplement 2A are in interphase. Although B56γ is present in the nucleus in interphase, its main kinetochore receptor, BubR1, is not.: BUBR1 is only recruited to the kinetochore following nuclear envelope breakdown . As such, in the wt situation prior to NEB, B56γ does not colocalize with CENP-C at kinetochores.One of the reviewers raised a concern that the data are presented in a very compact manner that may not be readily accessible to a broad readership. While no specific suggestion was provided, the authors should consider this point and consider changes that will improve readability and raise the paper's general interest.We thank the reviewer for this comment. We have now expanded the text slightly in key areas to explain things better to readers who do not have experience in kinetochore signalling. Hopefully this will now help to raise interest from a general audience.


We previously reported pro‐survival effects of Wnt3a and Wnt5a proteins in vascular smooth muscle cells (VSMCs). Wnt5a achieved this through induction of Wnt1‐inducible signalling pathway protein‐1 (WISP‐1) consequent to β‐catenin/CREB‐dependent, TCF‐independent, signalling. However, we found that as atherosclerosis advances, although Wnt5a protein was increased, WISP‐1 was reduced. We hypothesized this disconnect could be due to aging. In this study, we elucidate the mechanism underlying Wnt3a pro‐survival signalling and demonstrate the differential effect of age on Wnt3a‐ and Wnt5a‐mediated survival. We show Wnt3a protein was expressed in human atherosclerotic coronary arteries and co‐located with macrophages and VSMCs. Meanwhile, Wnt3a stimulation of primary mouse VSMCs increased β‐catenin nuclear translocation and TCF, but not CREB, activation. Wnt3a increased mRNA expression of the pro‐survival factor WISP‐2 in a TCF‐dependent manner. Functionally, β‐catenin/TCF inhibition or WISP‐2 neutralization significantly impaired Wnt3a‐mediated VSMC survival. WISP‐2 was upregulated in human atherosclerosis and partly co‐localized with Wnt3a. The pro‐survival action of Wnt3a was effective in VSMCs from young (2 month) and old (18–20 month) mice, whereas Wnt5a‐mediated rescue was impaired with age. Further investigation revealed that although Wnt5a induced β‐catenin nuclear translocation in VSMCs from both ages, CREB phosphorylation and WISP‐1 upregulation did not occur in old VSMCs. Unlike Wnt5a, pro‐survival Wnt3a signalling involves β‐catenin/TCF and WISP‐2. While Wnt3a‐mediated survival was unchanged with age, Wnt5a‐mediated survival was lost due to impaired CREB activation and WISP‐1 regulation. Greater understanding of the effect of age on Wnt signalling may identify targets to promote VSMC survival in elderly patients with atherosclerosis. This causes inhibition of the β‐catenin destruction complex, which contains casein kinase‐1α, glycogen synthase kinase‐3β, AXIN and adenomatous polyposis coli. This permits β‐catenin accumulation, nuclear translocation and binding of other transcription factors such as members of the T‐cell factor (TCF)/lymphoid enhancer‐binding factor family to modify gene transcription  signalling by Wnt proteins is implicated in the regulation of VSMC survival . Adjacent to the plaque core, Wnt3a protein was observed in areas containing α‐smooth muscle actin‐positive VSMCs and CD68‐positive macrophages .To test whether β‐catenin/TCF signalling was necessary for Wnt3a‐mediated survival, VSMCs were treated with β‐catenin/TCF inhibitors: CCT031374 hydrobromide ; however, IGF‐1 and WISP‐2 mRNAs were upregulated by Wnt3a alone and with H2O2 , establishing a direct connection between TCF and WISP‐2 following Wnt3a treatment.To investigate the role of IGF‐1 and WISP‐2 in Wnt3a‐mediated survival, neutralizing antibodies that significantly retarded the suppression of apoptosis by recombinant IGF‐1 and WISP‐2 proteins   Figure .2.5et al. found that only two Fzds, Fzd1 and Fzd6, were consistently expressed at detectable levels in murine aortic VSMCs  mice; however, only Wnt3a rescued VSMCs from old (18–20 month) mice . Wnt5a‐mediated CREB activation was also assessed by quantification of the CREB‐responsive gene HAS1. Wnt5a significantly induced HAS1 expression in VSMCs from young, but not old, mice  was only observed in VSMCs from young, not old, mice . For experiments relating to the Wnt3a pro‐survival pathway, primary VSMCs were isolated from explants of aortic tissue from TOPGAL mice, as previously described . We confirmed that VSMCs from 18–20 month mice had significantly lower levels of klotho mRNA compared to cells from 2 month mice confirming the age of the VSMCs in vitro . VSMCs between passages 2 and 10 were seeded according to the densities in Supporting Information Table 2. VSMCs were then quiesced in serum‐free DMEM (SFM) for 24 hr and stimulated with 400 ng/ml recombinant human Wnt3a protein  or Wnt5a protein  in the absence or presence of 100 μM H2O2  or with 100 ng/ml recombinant mouse IGF‐1 protein  or 500 ng/ml recombinant human WISP‐2 protein . To investigate the role of TCF, two β‐catenin/TCF inhibitors were employed; 1 µM CCT031374 hydrobromide  or 25 µM iCRT‐14 . For experiments using CCT031374 hydrobromide, the inhibitor was added in combination with Wnt3a ± H2O2; however, for experiments using iCRT‐14, an additional one hour pre‐incubation with iCRT‐14 was employed. In both cases, DMSO was used as a vehicle control, 0.002% DMSO was used for CCT031374 hydrobromide or 0.05% DMSO for iCRT‐14. For neutralization experiments, 10 µg/ml of antibodies raised against IGF‐1  or WISP‐2  was also added.Housing, care and all procedures involving mice were performed in accordance with the guidelines and regulations of the University of Bristol and the United Kingdom Home Office (PPL30_3064). The investigation conforms to the 4.2To quantify β‐catenin/TCF signalling, VSMCs were transfected with either TOPFlash plasmids  or negative control mutated FOPFlash plasmids, in combination with renilla luciferase plasmids to normalize for transfection efficacy . Full protocol is given in the 4.35 cells (WISP‐1) or 6 x 105 cells (Fzd1/6) were included in each nucleofection, then VSMCs were seeded at 8 x 104 or 6 x 104 cells/well in 24‐well plates and incubated overnight (WISP‐1) or for 6 hr (Fzd1/6) at 37°C, 5% CO2 to adhere. The knockdown efficiency of these protocols has been previously reported  for WISP‐1 (S100212702 and S102673370), Fzd1 (SI02674252 and SI00218771) or Fzd6  was introduced into VSMCs using the method described previously  in 1% BSA/PBS or 2.5 µg/ml β‐catenin antibody  in PBS was added overnight at 4°C. Bound antibodies were detected with biotinylated goat anti‐rabbit IgG  or biotinylated goat anti‐mouse IgG  and then DyLight‐488 Streptavidin  diluted 1:200 in PBS. Coverslips were mounted in ProLong Gold and DAPI .Immunocytochemistry for CC3 and β‐catenin was performed to analyse cell death after 24‐hr stimulation with Wnt ± H4.5n = 4), and donors for atherosclerotic vessels were aged 34–63 years (n = 11).Human coronary arteries were isolated from cadaveric hearts donated to the Bristol Coronary Artery Biobank under National Research Ethics Service approval (08/H0107/48). Informed consent was given by relatives of the deceased. The investigation conformed to the principles outlined in the Declaration of Helsinki. Donors for clean vessels were aged 11–29 years , thus supporting the findings of the first observer.Immunohistochemistry was performed for Wnt3a  and WISP‐2  plus CD68 to identify macrophages  and α‐smooth muscle actin to identify VSMCs  . Western blots were performed as previously described  were normalized to the corresponding β‐actin  or stain‐free band .To investigate changes in protein expression, VSMCs were incubated with Wnt ± H4.8SEM. Where N was sufficient, normal distribution was tested by the Kolmogorov and Smirnov test for normality. The following tests were then used; a one‐sample t test for data presented as a fold change compared to 1, a Student t test for comparing the means of two groups and an ANOVA with a Student–Newman–Keuls multiple comparisons post hoc test for comparing means of more than two groups. Paired or unpaired analysis was used as appropriate. An output of p < 0.05 was accepted as significantly different in all tests.Results are expressed as mean ± BAB was responsible for the acquisition, analysis and interpretation of most of the data presented. GMC optimized Wnt3a immunohistochemistry and helped analysis and interpretation of staining. CEJM acquired, analysed and interpreted data showing Wnt5a‐mediated survival with age and was responsible for optimizing many protocols. HW helped with data acquisition and interpretation by sacrificing mice, helping to isolate VSMCs, optimizing protocols and providing advice. GDA helped with steering project direction. JLJ helped with design of the work, data interpretation and steering project direction. SJG was responsible for the conception and design of the work, data interpretation and steering project direction. These authors have been involved in drafting or revising this paper and have given approval for publication and accountability for this work. Click here for additional data file.


APC tumor suppressor gene is considered an initiating event and the key oncogenic driver in most CRCs; disruption of APC protein function drives activation of the Wnt/β-catenin signaling pathway. CRCs also harbor a high incidence of other mutations that often arise as a secondary event after Wnt/β-catenin dysregulation (multistep somatic evolution model) such as in KRAS, BRAF, TP53, P13K and/or SMAD4 that cooperate with APC loss to further drive tumor progression [KRAS/BRAF mutations and yield a relatively low response rate. MEK inhibitors are also being explored as a treatment for patients with KRAS/BRAF mutant tumors who are not candidates for EGFR-directed therapies, however, these compounds have yielded limited efficacy to date. A triple-combination regimen of BRAF, MEK, and EGFR inhibitors showed improved progression free survival (PFS) compared to the individual agents, but even this potent cocktail yielded a PFS of only 4.2 months in refractory CRC [Colorectal cancer (CRC) is the third most commonly diagnosed cancer among both men and women in the United States and remains an area of significant clinical need. Inactivation of the KRAS/BRAF mutant CRCs [APC . Tankyrase (TNKS) inhibitors, which are believed to act by countering the effects of APC loss on β-catenin stability, may be more broadly applicable but affect a number of unrelated cellular processes. The broad biodistribution of TKNS and similar inhibitors also increases the potential for mechanism-based toxicities associated with Wnt/β-catenin inhibition in normal tissues including intestine and bone marrow [Given the status of Wnt/β-catenin signaling as the primary driver of most CRCs and its role as a resistance mechanism in ant CRCs , adding APC and other Wnt/β-catenin pathway mutations. DCR-BCAT in combination with MEK inhibition (MEKi) also displayed synergistic efficacy and overcame MEKi-mediated drug resistance [Nucleic acid-based therapeutics such as antisense and RNA interference (RNAi) have been studied in oncology trials for almost 30 and 15 years, respectively, with limited success. However, recent regulatory approvals in diverse non-oncology indications may rekindle interest in these approaches in oncology research. RNAi, which bypasses the challenge of drugging the β-catenin protein by targeting β-catenin mRNA, has demonstrated favorable results in multiple CRC preclinical models. Furthermore, RNAi drug products generally lack broad intracellular delivery to non-hepatic tissues, potentially limiting mechanism-based off-target effects. While several early clinical trials using first-generation RNAi drug products for oncology applications have faltered, recent regulatory approvals and striking advances in both delivery technology and oligonucleotide chemistry have increased the potential for future success. In one example of this approach, our laboratory has developed DCR-BCAT, a chemically-optimized Dicer-substrate siRNA (DsiRNA) formulated in a tumor-selective lipid nanoparticle (LNP) [Finally, it is worth noting that recent years have uncovered an important role of β-catenin in tumor immunology. Wnt/β-catenin signaling functions to exclude cytotoxic T-cells from accumulating in tumors, therefore causing resistance to immune checkpoint inhibitors . Wnt sig


Hepatocellular carcinoma (HCC) is among the deadliest cancers due to its heterogeneity, contributing to chemoresistance and recurrence. Cancer stem-like cells (CSCs) are suggested to play an important role in HCC tumorigenesis. This study investigates the role of Wnt/β-catenin pathway in CSC enrichment and the capabilities of these CSCs in tumor initiation in orthotopic immunocompetent mouse model.6 Hepa1–6 CSC spheroids or control cells in upper left liver lobe.HCC-CSCs were enriched using established serum-free culture method. Wnt/β-catenin pathway activation and its components were analyzed by western blot and qRT-PCR. The role of β-catenin in enrichment of CSC spheroids was confirmed using siRNA interference. Tumorigenic capabilities were confirmed using orthotopic immunocompetent mouse model by injecting 2 × 10The serum-free cultured Hepa1–6 cells demonstrated self-renewal, spheroid formation, higher EpCAM expression, increased Hoechst-33342 efflux, and upregulated Wnt/β-catenin signaling. Wnt/β-catenin pathway upregulation was implicated with the downstream targets, i.e., c-MYC, Cyclin-D1, and LEF1. Also, we found that GSK-3β serine-9 phosphorylation increased in Hepa1–6 spheroids. Silencing β-catenin by siRNA reversed spheroid formation phenotype. Mice injected with Hepa1–6 CSC spheroids showed aggressive tumor initiation and growth compared with mice injected with control cells.Successfully induced Hepa1–6 spheroids were identified with CSC-like properties. Aberrant β-catenin upregulation mediated by GSK-3β was observed in the Hepa1–6 spheroids. The β-catenin mediated CSC enrichment in the induced spheroids possesses the capability of tumor initiation in immunocompetent mice. Our study suggests plausible cell dedifferentiation mediated by β-catenin contributes to CSC-initiated HCC tumor growth in vivo.The online version of this article (10.1186/s12885-018-4683-0) contains supplementary material, which is available to authorized users. Hepatocellular carcinoma (HCC) is the fifth most common cancer in men and the seventh in women worldwide, and is the third major cause of cancer-related deaths , 2. HCC The CSC model proposes a hierarchical population in the tumor microenvironment, where apex CSCs are the least-differentiated subpopulation retaining self-renewal capability with asymmetric division and having the highest tumorigenic potential. Subsequently differentiated cancer cells in the hierarchy lose tumorigenic potential in decremental order, ending with terminal cancer cells with little to no tumorigenic potential , 7. The The canonical Wnt/β-catenin signaling is considered as fundamental pathway in stem-cell biology which regulates several cellular events including cell proliferation . In the In this study, we investigated the role of Wnt/β-catenin in HCC-CSCs spheroids generation and maintenance. Our study for the first time successfully induced spheroids in regular tissue-culture plates with an adherence environment, providing a spontaneous spheroid formation which is closely related to the in vivo condition. The function of HCC-CSCs spheroids in tumor formation was further studied in an immunocompetent orthotopic HCC mouse model, for the very first time.Routine methods, antibody catalog numbers , Hep3B (HB-8064) and HepG2 (HB-8065), were obtained from American type culture collection . All cell lines are validated every 6 months or obtained a new ATCC stock every 6 months. All experiments were performed between 5th and 20th passage. Hepa1–6 cells were grown in DMEM with 4.5% Glucose, supplemented with 10% FBS and 1× antibiotic-antimycotic. HepG2 and Hep3B cells were grown in MEM, supplemented with 10% FBS, 1× non-essential amino acids, 1× sodium pyruvate, and 1× antibiotic-antimycotic.For in vitro enrichment of CSCs, cells were grown in a serum-free condition (SF) in DMEM/F12 , supplemented with 2 mM L-Glutamine, 20 ng/mL EGF , and 10 ng/mL bFGF , 1× antibiotic-antimycotic and 1% B-27 supplement .Hepa1–6 cells were seeded in SF media at a density of 20,000 cells/well in a 6-well plate, and incubated for 4 days to allow the development of spheroids. After confirming formation of spheroids under the microscope, these spheroid were collected, treated with trypsin-EDTA for 4 min, and prepared in single-cell suspension with 500 cells/mL density with SF media. Seed single-cell suspension was placed at 2 μL/well in 96-well plate. The wells were marked with one or two cells. Additional 150 μL SF media was added in each marked well and monitor for 20 days. Images were taken daily to track and record progress.6 cells/mL was prepared for each sample, centrifuged at 200 RCF for 5 min, washed 2 times and resuspended in 2% FBS/PBS. Hoechst-33342 was added to 5 μg/mL final concentration in 2% FBS/PBS and cells were incubated for 90 min at 37 °C, followed by 10 min on ice, followed by a single wash of ice cold 1X PBS. These cells were immediately mounted on glass slides and analyzed within 1 h on a fluorescence microscope with UV excitation settings. Cells with dye exclusion property were identified by overlapping UV filter images with bright field images.After 7 days in culture, Hepa1–6 control or spheroid cells were harvested and 10n = 3 times independently, and transfection was carried out using combination of two siRNAs targeting different exons  at the University of Louisville (UofL). All mice were housed in the UofL Research Resources Center (RRC) at 22 °C with 12-h light/dark cycles with free access to food and water. In vivo experiments using the Hepa1–6 cell line were performed in 12-week-old male C57L/J mice . Each experimental group had n ≥ 3 per group). Comparisons were performed using Microsoft Excel-2013 suite by two-tail student’s t-test with equal variance . Results with p ≤ 0.05 were considered statistically significant.Data are presented as mean ± S.D. , an important property of self-renewal. We have confirmed and quantitated the self-renewal capacity of Hepa1–6 spheroids by minimal dilution assay using a 96-well plate and found that 24% of Hepa1–6 cells acquired self-renewal capability and form spheroids, while the rest were either in quiescence (51%) or non-spheroid differentiated stage (25%)  by flow cytometry  and CD90  were upregulated in the Hepa1–6 spheroids compared with non-sphere Hepa1–6 cell control  than non-sphere Hepa1–6 cell control  and nucleus  compared to control Hepa1–6 cells by western blot anlysis , Hepa1–6 cells lost the spheroid phenotype and shifted to the differentiated phenotype, similar to their parent cells growing in the medium with serum  using siRNA to further study implication of loss of function of β-catenin in spontaneous spheroid formation. After 48–72 h of Ctnnb1 knockdown  and monitored for 2 weeks. Mice injected with Hepa1–6 spheroids developed more aggressive tumors with overall higher tumor volume (6593 ± 2615 mm3) compared with mice injected with control cells   , which is likely due to aggressive growth and higher tumor mass in the spheroid injected group. This justifies the increased overall body weight by about 3 g in the spheroid group (25.68 ± 3.2 g) compared to control   Fig. . Also, H 6) Fig. . The agg 6) Fig. . These f 6) Fig. . Increas 6) Fig. .Fig. 5CSIn this study, for the first time we showed that HCC CSC spheroids were induced using only serum-free media and adherent tissue-culture treated plates. These HCC spheroids showed significant increased expression of CSC surface markers and functional markers, indicating that CSCs were enriched in the HCC spheroids. Increased Wnt/β-catenin components were found in the HCC spheroids compared with control cells grown in the presence of serum. We also showed that β-catenin upregulation, at least partly, was GSK-3β dependent, caused transcription activities of the downstream targets including Cyc-D1, c-MYC, and LEF-1. Upon silencing β-catenin by siRNA, CSCs lost spheroid phenotype and shifted to differentiated phenotype even in serum-free media. The HCC orthotopic mouse model confirmed the higher in vivo tumorigenic potential of Hepa1–6 CSCs in immunocompetent liver microenvironment, for the first time.Serum-free conditions plus growth factors allow cells to grow in an anchorage-independent manner, and is a well-documented standard method for maintaining undifferentiated cells , 35–37. The existence CSCs is no longer debatable area since definitive evidence recently identified CSCs by three independent lineage tracking studies –41. HoweIn conclusion, successfully induced Hepa1–6 spheroids were identified with CSC-like properties. Aberrant β-catenin upregulation mediated by GSK-3β was observed in the Hepa1–6 spheroids. The β-catenin mediated CSC enrichment in the induced spheroids possesses the capability of tumor initiation in immunocompetent mice. Without genetic manipulation, our study suggests plausible cell dedifferentiation mediated by β-catenin contributes to CSC-initiated tumor growth in vivo in HCC.Additional file 1:Figure S1. Spheroid formation capability of HCC cell lines. Figure S2. β-catenin expression in HepG2 cell line. Figure S3. Knockdown efficiency of siRNA transfection. Routine Methods: Immunocytochemistry (ICC) Staining, Flow-Cytometry Analysis, Protein Extraction and Western Blot. Table S1. Primary antibodies . Table S2. HRP conjugated secondary antibodies . Table S3. Important reagents . Table S4. Sequence details for RNA interference experiments. (DOCX 573 kb)Supplementary experiments and routine methods.