Banana is an important livelihood source for more than 12 million smallholder farmers in Uganda. Despite this contribution, its productivity continues to decline due to Banana Xanthomonas wilt (BXW). Cultural practices have been deployed to effectively control BXW but require a continuous and timely application, thus, prompting scientists to develop genetically modified (GM) bananas which display BXW resistance or tolerance. With prospects for commercialization of these GM bananas on the agenda, this paper applied a Contingent Valuation Method to assess producer acceptance of GM banana suckers among 233 banana producing households. Results show that producers were willing to pay between Ugandan shillings (UGX) 1100 to 1700 (US$0.28–0.44) per GM banana sucker Annual demand for GM banana suckers ranged from 70 to 82 million suckers. The results suggest that, in the event of not commercializing BXW-resistant GM bananas, Uganda loses an annual revenue ranging from UGX 76 to 139 billion (US$ 19.51 to 35.70 million). The Uganda’s agricultural census estimates 4 million tonnes of bananas to be grown on 807,000 ha, with approximately 68% of the crop produced in the Western Region, followed by the Central Region (23%), the Eastern Region (8%) and the Northern Region with less than 1%.4 Depending on the region, the daily per-capita consumption of bananas in Uganda is more than a 500 g cooking banana,5 conferring Uganda with the highest per-capita consumption of cooking banana in the world.6 Besides domestic consumption, the banana crop grown in Uganda contributes to farmers’ incomes through sales in fresh fruit and other value-added products such as chips, cakes, wines, juice, and flour.7 Moreover, different parts of the crop can also be used for other domestic and industrial purposes. Due to the increasing importance attached to bananas and the number of farmers benefiting from it, increasing productivity and profitability is seen as an important step toward achieving household and national food security and driving down the real price of food to accelerate economic growth, increase the sustainable management of natural resources, improve nutrition and health and reduce poverty and hunger.8In Uganda, Musa species (banana and plantain) are key crops supporting the livelihoods of millions of smallholder farmers,Xanthomonas vasicola pv. musacearum(Xvm), formely called Xanthomonas campestris pv. Musacearum9 starts with wilting of leaves or male bud and premature ripening of fruits, leading to the death of the whole plant. Where it occurs, BXW causes acute infections that can lead to the complete loss of a plantation. In the earlier years (2001 to 2004) of its onset, BXW caused about a 30–50% decrease in banana yields in Uganda.11 Recent studies, however, report yield losses resulting from BXW infections to be as high as 100% if not controlled.14 Meanwhile, economic losses of about US$2–8 billion have been reported over a decade as a result of the disease.15 As a result, average annual production declined from 10.5MT in 2002 to as low as 4.3MT in. 201616Despite its importance, years of devastating Banana Xanthomonas Wilt (BXW) infections have continued to cause massive losses in banana plantations thus reducing the food security of many households. The BXW disease, caused by 17 However, BXW control using cultural practices can be inconsistent when the value-chain actors, especially the farmers and traders, fail to comply during implementation. Among these practices, the use of clean planting materials (such as tissue culture plantlets) is the least adopted practice because most farmers rely on planting materials  from informal sources, and tissue-cultured plants are expensive. Besides, farmers do not have the technical means to verify whether the planting materials are BXW-free or not,18 yet long incubation periods of up to 24 months and latent infections have been reported.19 There is, therefore, a high risk of transmitting the disease within and across farms through infected planting materials, even when farmers apply the other control practices effectively. On the other hand, genetically modified (GM) BXW-resistant bananas have been developed through genetic engineering and field tested in Uganda.20 GM bananas aim to increase productivity and nutritional value, and could effectively contribute toward food security in the near future.21 Currently, Uganda’s experimental program with agricultural biotechnology is one of the largest in Africa,5 with reports showing some GM-lines could be released for multiplication, distribution, and commercialization in 2020.8Considerable effort has been devoted to controlling BXW since its discovery in Uganda in 2001. Currently, two major approaches exist: first, the use of cultural control practices; and second, the use of genetic engineering (modification). The use of cultural practices includes avoiding introducing the disease into new areas by using clean planting materials, removing the male buds using a forked stick immediately after the banana bunch has formed its last cluster, removing all infected stems (by cutting off at soil level), and cleaning all used tools using sodium hypochlorite (JIK) or fire flame.Evidence exists on the technical performance of the new technology against BXW as explained but nothing much has been documented as far as producer acceptance and demand for genetically modified banana planting material are concerned. Understanding producer acceptance for GM banana suckers and their perception toward the technology in this study will help inform policymakers and plant breeders on the market potential that exists, and the quantity of GM banana suckers needed to satisfy the potential market.1.2.22 suggesting the need to understand producers’ perceptions toward GM crops.Genetically Modified (GM) food products in many countries have slowly been declared as GM food. However, this has come along with several issues pertaining to health, finance, and environmental safety,23 Farmers are directly affected by the spread of GM crops and have an important responsibility for their future. Their absence from the discussions and the limited academic interest in the views of farmers regarding GM crops amount to an important missing element in the debate and policymaking in the sector.24 Silencing of this group has also allowed other interest groups to speak on their behalf for their own ends. Organizations concerned with spreading the technology, for example, tend to emphasize the benefits of GM crops for farmers, often relying on absolute figures regarding the adoption of the technology in agriculture.25 For NGOs and social movements critical of GM crops, the promised benefits preached by advocates of GMOs are illusory, particularly for producers in developing countries.26GM organisms have been at the center of a major public controversy, involving different interests and actors. While much attention has been devoted to consumer views on GM food, there have been few attempts to understand the perceptions of GM technology among farmers in developing countries. In the context of these efforts and beyond, disproportionate attention has been given to the opinions of the end consumers of GM food compared to the views of the primary consumers of this technology.27 similarly, reported that the two greatest advantages associated with GM crops are their convenience of management and increased productivity. Fernandez et al.,28 go on to reveal that farmers in the US have continued to adopt GM cotton, soybeans and maize, citing their ability to increase yields as the primary reason and further asserts that this could lower the price of food, which would boost productivity in farming and increase the supply of food for the world’s rapidly growing population. In their study,29 also reported that farmers unanimously agreed that a reduction in the cost of herbicides was the main advantage of using GM crops, where nonselective herbicides are used. Celeres  suggested that among some of the reasons for planting GM seeds are the increase in productivity, reduced pesticide use, obtaining a higher quality product and achieving better financial results, Celeres 30, meanw31 in their study revealed that widespread adoption of GM soy in the South, which requires less manpower during cultivation, led to the breakup of families and unemployment. In addition, producers seem increasingly convinced of the harmful effects of GMOs on health and the environment. They point to a series of socio-economic dangers that these plants represent for producers and consumers. They vigorously challenge the advantages stressed by advocates, arguing that the benefits claimed are illusory.26 As a result, farmers’ worries concerning GM crops go beyond financial and practical issues, despite these carrying more weight. The payment of royalties to the companies that own the technology and the ban they impose on replanting their seeds in ensuing seasons has on several occasions been mentioned as an important drawback in adopting GM varieties. Farmers also fear for the genetic crossover contamination of conventional crops, which may lead to financial penalties for the producer, and greater resistance of weeds to herbicides used in the growing process.32 Conversely, it is important to note that for some crop species, there is little or no risk of the recombined DNA leaking into the biome. Commercially grown bananas, for example, are predominantly sterile and do not produce pollen, so there is less chance of leakage. Pimbert et al.,32 also reveal that the spread of these crops will lead to a greater dependency of farmers on biotechnology. Of late, consumer resistance has also been identified as a major constraint to GM crop production by farmers.33 Clustered regularly interspaced short palindrome repeats (CRISPR) have been developed to counter such negative sentiments associated with a genetically modified organism. This is due to the fact that they are easier to design and implement, have a higher success rate, are more versatile and less expensive compared to the genetically modified organisms and other new breeding techniques(NBTs) such as zinc ﬁnger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs).34 Waltz et al.,35 also report in their study that such new breeding technologies (CRISPR), have brought about rapid positive changes in the attitude of many producers toward genetically engineered plants. However, the CRISPR technology is new and yet to be implemented in Uganda in the near future.According to declarations, the adoption of GM crops is also thought to reduce on-farm manual labor, especially with regard to the use of pesticides. However, Zambrano et al.,8 conducted a study to understand future adoption and consumption of GM banana in the Great Lakes Region (GLA). Their findings revealed that each dollar invested in the development and dissemination of a GM resistant BXW banana in Uganda generates US$ 30 per plantlet, with an average adoption rate of 54%. The authors, however, anticipated that some undesirable attributes of the GM crop could affect the overall market potential. Considering all of the above, producer behavioral trends toward GM crops have become a vital factor influencing how lucrative and attractive the future market for GM crops will be. This will impact the future course of action for the public- and private-sector investments in the development and use of GM technology. Thus, producer and consumer acceptance toward GM technology is crucial for the global market of GM products, agricultural trade, and the future development of agricultural biotechnology.In terms of benefits, Ainembabazi et al.,2.2.1.A face-to-face survey using a pre-tested questionnaire was conducted in August 2018 among banana-producing households in three administrative regions of Uganda , comprising three-distinct agro-ecological zones where cooking bananas (green bananas) are mostly grown and used as a source of staple food and income. The survey employed a simple random sampling procedure, in which each respondent had the same probability of being selected. Overall, 232 respondents were considered for this study. The first component of the survey involved collecting individual and household data. The second section contained perception and attitudinal questions regarding GM banana planting material. Lastly, the third section captured information on the respondent’s demand and willingness to pay for GM banana planting material.For the ‘Willingness to Pay’ (WTP) section, enumerators first gave a brief description of GM banana planting material to the respondents before obtaining data on their WTP. This was done to create awareness since the product was new and not yet known by all respondents within the selected sites. After full description of the product to the respondent by the enumerator, through cheap talks, six bids were assigned randomly to different respondents (UGX 2500 (US$ 0.64), UGX3000 (US$ 0.77), UGX3500 (US$ 0.90), UGX4000 (US$ 1.03), UGX 4500 (US$ 1.16) and UGX 5000 (US$ 1.28)). The respondents were then asked if they were willing to pay for GM banana sucker at the randomly assigned bid price. Those who answered “yes” were asked if they would be willing to pay at another randomly assigned higher price that had been increased by 5%, 10%, 20%, 30%, 40%, or 50%. However, each respondent was only offered a single second bid. Respondents who answered “no” to the first bid were offered another randomly assigned lower price that was discounted by either 5%, 10%, 20%, 30%, 40%, or 50%.2.2.2.2.1.36 However, for negative statements related to GM banana suckers, the scores were reversed with . Four categories of perception statements were created as indices for the study, including environment, health, food safety and risk, and government and institutional regulation. Each category constituted at least three perception statements, whose scores were later averaged to form an index . Analysis of data then involved the use of descriptive statistics including means and standard deviations to characterize producers. T-tests were also carried out to establish any significant differences in perceptions between those who were aware of the GM banana suckers and those respondents who were not aware of it at all.Producer acceptance of GM banana suckers was evaluated through their perception and attitudes. Producers’ perceptions were assessed for various GM-related parameters including its effect on the environment, health, food safety and risk and government and institutional regulation. Each perception response was measured on a five-point Likert scale with a score from  as demonstrated by those of Kimenju & Hugo.2.2.2.37 The four probabilities are then denoted as follows:A number of approaches are used in conducting the ex-ante assessment of new agricultural technologies. Some of the notable ones include Cost–Benefit Analysis (CBA), and several valuation methods, including Contingent Valuation, the Travel Cost Method, and Hedonic Pricing. For this study, double-bounded Contingent Valuation Method (CVM) was used since the GM banana planting material was still new and not yet in the markets . Farmers were presented with two bids, with the second bid being contingent upon the response to the first bid. If the individual responded yes to the first bid, the second bid was greater; if it was a no to the first bid, then the second bid was smaller than the first one. Thus, there were four possible outcomes to the questions: (a) both answers are “yes”; (b) both answers are “no”; (c) a “yes” followed by a “no”; and (d) a “no” followed by a “yes”.lnL) for the sample then takes the form:where 38where where: 2.2.3.40 Once the estimated market potential has been calculated, it would be possible to determine if the market is large enough to sustain the proposed production or sustain an additional producer in the market place.40 Market potential (demand) for GM banana suckers was estimated in terms of maximum annual total sales revenue that could be generated from GM banana suckers from the three regions under study. The following formula derived by40 was used to estimate the market potential.The estimation of market potential for a product is critical in evaluating its viability as it provides an estimate of the maximum total sales potential for a given market.P = Mean Willingness to pay or average selling price and A = Average annual consumption.Where: MP = is the market potential, N = Number of possible buyers at price P, 3.3.1.Individual and household characteristics of respondents are shown in Self-sufficient producers were also characterized by large acreages of land, equivalent to an average area threefold that of self-insufficient producers allocated to banana production. Self-sufficient producers have more years of banana farming experience than the self-insufficient producers. Overall, producers had an average age of 47 years, with an average household size of approximately 5 people. No significant differences were observed based on the gender and age of the producer.3.2.41 who found out that the awareness level of consumers toward GM food concept was still low. Similarly, Chen and Chern,41 also showed in their study that the majority of consumers are still not very well-informed about the GM foods.Banana producers’ perceptions were considered very important to the process of making GM banana planting material acceptance decisions. Perceptions were generally compared across two producer groups: (1) those who were aware of GM technology, that is, those who have received any kind of information or have already heard about anything related to GM technologies, comprising of 27%, and (2) those who were not aware of GM technologies, making up 73% of the sample. The low awareness of GM technology among producers is in line with Tanius & Seng,43, with those aware of the technology being even more negative compared to those who are unaware. This finding could be attributed to negative information being disseminated in public by anti-GM advocates. This implies that there is a great need for stakeholders involved in GM technology deployment and promotion to provide, sensitize, and disseminate adequate and relevant science-based information concerning GM to the general public.Generally, the findings still reflect negative producers’ perception of the GM technology in relation to the purported threat of adverse influence on health, environment, and food safety, similar to Kikulwe et al. (2011)3.3.A bivariate probit model was estimated to assess the mean willingness to pay value for GM banana suckers. The results showed that the mean willingness to pay per GM banana sucker ranged from Ugandan shillings (UGX) 1092 to UGX1702 (US$ 0.28 to 0.44) per sucker. The Western region had the highest percentage (80%) of respondents willing to pay for suckers at the mean price of UGX 1092 (US$ 0.28) per GM banana planting material, while the Eastern region had the least (44%) willing to pay for the GM banana planting material. At the upper mean willingness to pay price (UGX 1702- US$ 0.44), the percentage of respondents willing to pay for GM banana sucker dropped in all the three regions .10.1080/3.4.Data for the banana producers were estimated in a bivariate probit model to obtain the factors that influenced producer willingness to pay for GM banana planting materials. The results of the modeling efforts are as shown in Bid prices as usual had a negative and statistically significant influence on producers’ willingness to pay for GM banana suckers. This implies that an increase in the amount of the bid price offered by UGX 1 reduced the likelihood of a producers’ willingness to pay for GM banana suckers at less than 10%. This finding perfectly mimics the theory of demand, which shows an inverse relationship between price and quantity demanded of a commodity.44 who reported that farmers considered their local crop varieties to be more adaptable to drought and pests compared to improved and modified varieties.The soil fertility and pest management technology perception index stood out to be the most important factor influencing producers’ willingness to pay decisions for GM suckers. An increase in soil fertility and pest management technology perception index by one unit decreased the likelihood of a producer’s willingness to pay for GM suckers by 0.1878. This could because farmers perceive their indigenous varieties to be more tolerant to soil deficiency and pest and disease infections than improved or new varieties. This finding is in line with Kansiime and Mastenbroek,36 who assert that income influences willingness to pay positively. A unit increase in the area under banana production as well increased the probability of the producer willingness to pay for GM banana suckers by 0.0245. This is consistent with the findings of Schnurr & Addison,5 that show that the larger the farm, the more likely respondents are to hold positive attitudes toward GM crops.On the other hand, annual expenditure on bananas and the total area of land allocated to banana production had a positive and significant influence on producers’ willingness to pay for GM banana suckers. An increase on annual expenditure on bananas by UGX 1 increased the likelihood of the producers’ willingness to pay for GM banana suckers by 0.1493. It was noted that a positive correlation exists between annual expenditure on bananas and annual income. Individuals with high annual income earnings were likely to be willing to purchase GM banana suckers. This finding resonates with those of Kimenju & De–Groote,3.5.4 The number of households engaged in banana farming was estimated at 459,555 in central, 696,102 in western and 209,283 in eastern Uganda, respectively. Out of these households, on average, 56% of the sample was willing to pay the price of UGX 1092 (US$ 0.28) and above per GM banana sucker. At the regional level, 45% of the households in the Central, 80% in the Western and 44% in the Eastern were willing to pay the mean price of UGX 1092 (US$ 0.28) and above per GM banana sucker. This results into a potential buying population of 209,098, 556,882 and 91,561 for the Central, Western, and Eastern regions, respectively. On the other hand, at the mean willingness to pay the value of UGX 1702 (US$ 0.44) and above per GM banana sucker, 52% of the household on average were willing to pay. Specifically, 39% of the households in Central, 78% in the Western and 39% in the Eastern regions were willing to pay that mean price and above. This resulted in an equivalent buying population of 176,929, 539,479 and 81,094 for the Central, Western, and Eastern regions, respectively. The average proportions of 56% and 52% households willing to pay for GM banana sucker at the mean price of 1092 (US$ 0.28) and 1702 (US$ 0.44) and above, respectively, is in line with the mean adoption rate of 55% reported by Ainembabazi et al.8Demand and market potential for GM banana suckers were estimated based on information extracted from the Uganda census of Agriculture report that was documented by Uganda Bureau of Statistics.Using the generated statistics, the overall potential annual demand for each region was then derived by multiplying the average potential number of GM banana suckers demanded annually per household for each region by the number of households willing to pay at mean prices and above. The market potentials were computed by multiplying the overall potential annual quantities of GM banana suckers demanded for each region by the mean willingness to pay prices.From the computations, the overall potential annual quantity demanded of GM banana suckers stood at around 70 and 82 million suckers at the mean price of UGX 1092 and 1702, respectively. The Western region had the highest potential annual demand for GM banana planting material being a major production hub in the country, while the Eastern region had the least overall potential annual demand for GM banana suckers.The total market potential for GM banana suckers was UGX 76 billion (US$ 19.51 million) at the mean price of UGX 1092 (US$ 0.28) and UGX 139 billion (US$ 35.70 million) at a mean price of UGX 1702 (US$ 0.44) see . This im4.This study assessed acceptance of GM planting material among banana-farming households in Uganda. The findings show that more than 62% of banana producers are self-insufficient with about one acre of land allocated to banana production. The producers genuinely agree that if the majority of the population is in support of GM, it should be legalized. Apparently, a huge market potential, valued at a tune of UGX 76 billion (US$ 19.51 million) to 139 billion (US$ 35.70 million) for GM banana suckers have been estimated. However, some negative perceptions that may affect the overall market potential and future acceptance of GM products were also elicited from the producers. Such concerns were mainly related to possible threats that GM products may pose to health, environment, food safety and risk and costs linked to government and institutional regulations.35 have revealed a rapid positive change in attitude toward genetically engineered plants using CRISPR-editing. Thus, a study looking at how farmers in Uganda perceive CRISPR and NBTs and how this affects their attitudes and perceptions toward genetic engineering technology needs to be undertaken in the future. Finally, to effectively predict the market, there is also a need to carefully project the market potential for GM banana planting materials beyond 1 year. However, since the crop is vegetatively propagated, on average each planting material procured in year one will produce at least six extra suckers for use in the subsequent year. This implies that the projected first-year market has a possibility of either declining or expanding in subsequent years.The existence of these negative perceptions, therefore, points to the need to conduct sensitization campaigns for banana producers and masses at large on GM technology as most of the information that ignites the negative perceptions are mainly false and not based on scientifically proven evidence. Similarly, new breeding technologies (NBTs) such as CRISPR have shown positive results, and recent studies, for example, Waltz,


To evaluate the expression of tripartite motif-containing 33 (TRIM33) in ccRCC tissues and explore the biological effect of TRIM33 on the progress of ccRCC.  The Cancer Genome Atlas (TCGA) database was used to examine the mRNA expression levels of TRIM33 in ccRCC tissues and its clinical relevance. Immunohistochemistry (IHC) was performed to evaluate its expression in ccRCC tissues obtained from our hospital. The correlation between TRIM33 expression and clinicopathological features of the patients was also investigated. The effects of TRIM33 on the proliferation of ccRCC cells were examined using the CCK-8 and colony formation assays. The effects of TRIM33 on the migration and invasion of ccRCC cells were explored through wound healing and transwell assays, along with the use of Wnt signaling pathway agonists in rescue experiments. Western blotting was used to explore the potential mechanism of TRIM33 in renal cancer cells. A xenograft model was used to explore the effect of TRIM33 on tumor growth. β-catenin, cyclin D1, and c-myc, and inhibited tumor growth in ccRCC cells in vivo.  Bioinformatics analysis showed that TRIM33 mRNA expression in ccRCC tissues was downregulated, and low TRIM33 expression was related to poor prognosis in ccRCC patients. In agreement with this, low TRIM33 expression was detected in human ccRCC tissues. TRIM33 expression levels were correlated with clinical characteristics, including tumor size and Furman's grade. Furthermore, TRIM33 overexpression inhibited proliferation, migration, and invasion of 786-O and ACHN cell lines. The rescue experiment showed that the originally inhibited migration and invasion capabilities were restored. TRIM33 overexpression reduced the expression levels of  TRIM33 exhibits an abnormally low expression in human ccRCC tissues. TRIM33 may serve as a potential therapeutic target and prognostic marker for ccRCC. According to 2018 global cancer statistics, renal cell carcinoma (RCC) is one of the most common cancers in the world, with more than 400,000 patients newly diagnosed, and more than 170,000 deaths due to RCC each year , 4. Therβ/Smad pathway. TRIM33 has been identified as an effective regulator of TGF-β, which plays a positive or negative regulatory role in this pathway [β/Smad pathway [β-catenin pathway [The TRIM family is a class of proteins with E3 ubiquitin ligase activity. Its members participate in many important biological processes, including autophagy, carcinogenesis, intracellular signal transduction, protein ubiquitination and degradation, and innate immunity. Recent studies have found that dysregulation of TRIM protein expression can cause a variety of diseases, such as tumors, neuropsychiatric diseases, immune diseases, chromosomal abnormalities, and developmental diseases . The TRI pathway . In rena pathway . However pathway .β-catenin, cyclin D1, and c-myc molecules in the Wnt signaling pathway. In other words, TRIM33 can inhibit the progress of ccRCC by inhibiting the activity of the Wnt/β-catenin pathway. This is very important for elucidating the function of TRIM33 in ccRCC. TRIM33 plays a role as a tumor suppressor gene in ccRCC and is expected to become a new ccRCC therapeutic target and prognostic marker.Unlike previous studies of renal clear cell carcinoma, for the first time, it was found that upregulation of TRIM33 inhibited the expression of With the patients' informed written consent, we collected 80 samples of human ccRCC tissues and normal kidney tissues from the Shandong Provincial Hospital during the period between 2013 and 2018. The pathological samples were first fixed with the corresponding fixative, then embedded in paraffin, and sectioned for immunohistochemical detection. The study was approved by the ethics committee of the Shandong Provincial Hospital. The principles of the Declaration of Helsinki and that of the World Medical Association were followed to conduct the study.β-actin , anti-E-cadherin , anti-N-cadherin , anti-vimentin , anti-β-catenin , anti-cyclin D1 , and anti-c-myc  antibodies. Wnt agonist 1 was purchased from Selleck Chemicals  and was used at a final concentration of 10 μM at 37°C for 24 h.The cell line used in this study was purchased from the Chinese Academy of Sciences cell bank. The antibodies being used included rabbit anti-TRIM33 , anti-UALCAN, Human Protein Atlas, and Gene Expression Profiling Interactive Analysis (GEPIA) are three online analysis websites based on the TCGA database –16. We eH score.First, the paraffin-embedded tissue specimens were sectioned and then subjected to routine deparaffinization and gradient alcohol processing. Then, we used citrate buffer (pH 6.0) for antigen retrieval. Afterward, the specimen on the section was incubated with anti-TRIM33 antibody at 4°C overnight. On the second day, the specimen on the section was incubated with the secondary antibody for 30 minutes in a 37°C incubator, and the DAB kit (Abcam) was used to detect the expression of the target protein and hematoxylin (Abcam) was used to stain the nucleus. Finally, two independent pathologists evaluated and calculated the corresponding μg RNA using the Quantscript RT Kit (Takara). The reaction conditions for qRT-PCR were predenaturation at 94°C for 5 min, 94°C for 45 s, and 60°C for 60 s, for a total of 40 cycles. Three replicate wells were set for each sample. The primer sequences used for real-time PCR were as follows: TRIM33 forward, 5′-TGGACCAAAGGAAATGTGAACG-3′ and TRIM33 reverse, 5′-TGTGTGTCTGCATAAACTTGAACA-3′.Total RNA was extracted with the TRIzol Reagent (Takara). Then, cDNA was synthesized from 2 First, we extracted total protein from the cell line, followed by protein denaturation, electrophoresis, membrane transfer, and incubation with primary antibody overnight in a shaker at 4°C. The next day, we incubated the Polyvinylidene Fluoride (PVDF) membrane with the secondary antibody for 1 hour at room temperature, and finally, we developed it according to the instructions of an ultrasensitive chemiluminescence kit (Millipore).μg) was added to the medium/ml. After 12 hours, the old medium was replaced with freshly completed medium, followed by incubation with 2 μg/ml puromycin (Sigma-Aldrich) to select kidney cancer cell lines stably expressing TRIM33. After 72 hours, real-time PCR and western blotting were used to evaluate the transfection efficiency, thereby establishing two kidney cancer cell lines with stable TRIM33 overexpression.To overexpress TRIM33 in kidney cancer cell lines, we commissioned OBiO Technology  to design a TRIM33 lentiviral vector for the expression of TRIM33 and an eGFP control lentiviral vector. We inoculated human kidney cancer cells into a 24-well plate. When the cells reached 40-50% confluency, a certain amount of lentivirus with polybrene (5 μl of Cell Counting Kit-8 (Dojindo) solution was added to each well. After incubating at 37°C for 1 h, absorbance was measured at 450 nm using a microplate reader.The cells in both the control group and the experimental group were inoculated into 96-well plates at a density of 5,000 cells/well, and each group was provided with 3 double wells. After incubating the cells for a certain period of time, 10 Kidney cancer cells were seeded in 6-well plates at a density of 500 cells/well after incubating for 14 days at 37°C, fixing with 4% paraformaldehyde for 30 minutes and staining with 0.1% crystal violet for 10 minutes at room temperature. Image-Pro Plus software was used to count the colonies.μl sterile pipette tip. Then, the cell layer was washed thrice with PBS, and 2 ml of serum-free culture medium was added. Pictures were taken at certain times. The wound healing rate was evaluated using Image-Pro Plus.Kidney cancer cells were seeded in a 6-well plate, and when confluency reached about 95%, the cell layer was scraped using a 200 We mixed the melted Matrigel gel (Corning) with serum-free medium according to a certain ratio (1 : 6), spread it evenly on the bottom of the Transwell chamber (upper chamber surface), and placed it in a 37°C incubator for coagulation for about 2 to 4 hours to make it appear gel-like. Subsequently, the cells that grew to the logarithmic phase were digested, counted, and the serum-free medium was diluted to a certain ratio and evenly inoculated to the bottom of the Transwell chamber, and then the Transwell chamber was cultured in a well plate with 10% FBS medium. After culturing the cells for 24 hours, we took out the Transwell chamber, wiped the cells inside the chamber and the remaining Matrigel glue with a cotton swab, washed it 3 times with PBS, and fixed the cells passing through the bottom and back of the chamber with paraformaldehyde, stained them with crystal violet, counted and analyzed them, and then drawn the corresponding histogram. The only difference between the cell migration experiment and the invasion experiment is that Matrigel is not used, and the rest of the steps are exactly the same.7/ml. Subsequently, 100 μl of cell suspension was injected into the left armpit of nude mice in the form of a subcutaneous injection. Then, we payed close attention and measured the changes in tumor volume every three days. Finally, at 35 days of rearing, the mice were sacrificed, and the tumors were excised for corresponding evaluation.First, we use Matrigel to make ACHN cells in logarithmic growth into a cell suspension with a concentration of 5 × 10χ2 test. Statistical significance of differences between groups was assessed using a two-tailed t-test and one-way ANOVA, respectively. A value of P < 0.05 was considered to indicate statistical significance.The data are expressed as the mean ± S.E.M. Statistical analysis was performed using Prism software (GraphPad 8.0). The relationship between immunohistochemical staining and pathological parameters was determined by the Pearson P = 0.0084) (P < 0.0001) (P = 0.042) (P = 0.0036) . Finally, the overall survival (P = 0.0034) curve and disease-free survival curve (P = 0.035) of TRIM33 in KIRC, which was drawn through the use of GEPIA website, also showed statistical significance  . Further 0.0001) . Similar= 0.042) . Then, t Figures . We also Figures . It was  Figures .H score of these three typical samples (H score of these 80 samples (P < 0.0001) (P = 0.0233) (P = 0.031) and Furman's grade (P = 0.036)   were dra 0.0233) . In addi= 0.036) .In order to select the renal cancer cell lines suitable for this study, we used qRT-PCR and western blotting to determine the expression of TRIM33 in HK-2 and the three renal cancer cell lines. It was observed that the expression of TRIM33 in the three kidney cancer cell lines was significantly lower than that in normal renal tubular epithelial cells. Since the expression of TRIM33 in 786-O and ACHN cell lines was lower than that in the A498 cell line, we chose 786-O and ACHN cell lines for further experiments Figures . In ordeTo investigate whether TRIM33 overexpression affects the proliferation of renal cancer cell lines, we used the CCK-8 assay Figures  and perfβ-catenin, cyclin D1, and c-myc protein  is one of the most common malignant tumors of the urogenital system worldwide. Although a variety of treatments are now available to treat ccRCC, the global mortality rate of ccRCC has remained high in the past few decades. A large number of existing studies have confirmed that the occurrence and swelling of ccRCC is the result of the accumulation of cellular and molecular aberrations, including abnormalities in epigenetics, transcriptomics, miRNA, proteomics, and metabolomics –19. All analysis . At presβ signaling pathway by binding to phosphorylated SMAD2/3 or monoubiquitinated SMAD4 in hepatocellular carcinoma, human chronic myelomonocytic leukemia, and pancreatic cancer [β signaling pathway may not be the main pathway through which TRIM33 inhibits tumorigenesis. Previous studies have mainly focused on the TGF-β signaling pathway, but now more attention is being paid to the Wnt signaling pathway [β-catenin pathway, a process not related to its E3 ligase activity of its RING domain. In human glioblastoma, it has been shown that TRIM33-mediated β-catenin destabilization is mediated through a dual effect. On the one hand, TRIM33 can inhibit the activity of Wnt3a, and on the other hand, it can inhibit EGF-induced transactivation of β-catenin [β-catenin pathway, thereby promoting tumor cell proliferation and tumorigenesis. These results support the key role of TRIM33 in nuclear degradation of β-catenin in human glioblastoma. In addition, there are two review articles showing that TRIM33 plays a key role in the Wnt signaling pathway by degrading β-catenin [β-catenin pathway in ccRCC is still unknown.Tripartite motif-containing 33 (TRIM33) is a protein with multiple biological functions. It has a RING domain at the N-terminus, two B-boxes, and a coiled-coil domain . TRIM33 c cancer , 10, 26.c cancer . This in pathway , 32. Stu-catenin . It shou-catenin . Howeverβ-catenin, cyclin D1, and c-myc in the Wnt signaling pathway. At the same time, incubation with a Wnt signaling pathway agonist restored migration and invasion as well as the levels of β-catenin, cyclin D1, and c-myc. Finally, in the mouse xenograft model, ACHN renal cancer cells overexpressing TRIM33 produced significantly smaller tumors in mice. In summary, these findings indicate that TRIM33 overexpression can exhibit antitumor activity by inhibiting the Wnt/β-catenin pathway in renal clear cell carcinoma.To explore the potential role of TRIM33 in clear cell carcinoma, we used the TCGA database to reveal the mRNA expression levels of TRIM33 in human ccRCC tissues and its clinical relevance. TRIM33 showed significantly lower expression in kidney cancer tissues than in normal tissues, and its expression levels were significantly associated with multiple clinical features. The use of bioinformatics to identify targets that may play a role in tumor progression is becoming more and more popular . Subsequβ-catenin pathway plays an important role in regulating cell proliferation, differentiation, self-renewal, adhesion, and migration in tumors [β-catenin pathway can affect tumor growth and metastasis by regulating the expression of downstream genes such as cyclin D1 and c-myc [β-catenin pathway has been observed in many types of human tumors, and it is thought to promote tumor development. Inhibiting the Wnt/β-catenin pathway has become an effective way for a variety of tumor-targeted therapies. For example, in esophageal squamous cell carcinoma, silencing SALL4 has been shown to inhibit EMT by inhibiting the activation of the Wnt/β-catenin pathway, thereby inhibiting the survival, migration, invasion, and drug resistance of cancer cells in vitro, as well as the tumorigenic ability in vivo [β-catenin pathway to promote cancer cell proliferation, cell cycle progression, and tumorigenicity in vivo [β-catenin pathway [β-catenin pathway activation [β-catenin pathway was downregulated and proliferation, migration, and invasion were inhibited [β-catenin pathway. This is important for understanding the role of the Wnt/β-catenin pathway in cancer.As we all know, the Wnt/n tumors –40. The nd c-myc . Abnorma in vivo . In hepa in vivo . In brea pathway . In cervtivation . In renanhibited . Similarβ-catenin pathway by TRIM33. The limitation of this study is that we did not perform an in-depth analysis of the regulatory mechanism of TRIM33 on the Wnt/β-catenin pathway. Our study indicates that TRIM33 plays a role as a tumor suppressor gene in ccRCC and may become a potential target and prognostic marker for kidney cancer treatment in the future.In this study, we found that TRIM33 expression is reduced in ccRCC tissues and cells and is related to the poor prognosis of ccRCC. In vitro, TRIM33 overexpression inhibited the proliferation, colony formation, migration, invasion, and EMT ability of renal cancer cells. In vivo, the growth rate of TRIM33-overexpressing renal cancer cells was significantly inhibited. This may be due to the inhibition of the activity of the Wnt/


The remainder were modified with activated carbon (C). Each modifying agent, or filler, was added at a rate of 1 g, 2 g, or 3 g per 100 g of epoxy resin. The effect of the hygrothermal exposure (82 °C temperature and 95% RH humidity) was examined. The effects of thermal shocks, achieved by cycling between 82 °C and −40 °C, on selected mechanical properties of the filler-modified epoxy compounds were investigated. Strength tests were carried out on the cured epoxy compound specimens to determine the shear strength, compression modulus, and compressive strain. The analysis of the results led to the conclusion that the type of tested epoxy compounds and the quantity and type of filler determine the effects of climate chamber aging and thermal shock chamber processing on the compressive strength for the tested epoxy compounds. The different filler quantities, 1–3 g of calcium carbonate (CaCO3) or activated carbon (C), determined the strength parameters, with results varying from the reference compounds and the compounds exposure in the climate chamber and thermal shock chamber. The epoxy compounds which contained unmodified epoxy resin achieved a higher strength performance than the epoxy compounds made with modified epoxy resin. In most instances, the epoxy compounds modified with CaCO3 had a higher compressive strength than the epoxy compounds modified with C (activated carbon).The objective of this paper is to analyze the effects of hygrothermal exposure on the mechanical properties of epoxy compounds modified with calcium carbonate or carbon fillers. In addition, comparative tests were carried out with the same parameters as hygrothermal exposure, but the epoxy compounds were additionally exposed to thermal shocks. The analysis used cylindrical specimens produced from two different epoxy compounds. The specimens were fabricated from compounds of epoxy resins, based on Bisphenol A  and a polyamide curing agent. Some of the epoxy compounds were modified with calcium carbonate (CaCO Epoxy materials  have found widespread use in many industries ,2,3,4,5.It is difficult to choose the best of the many commercially available fillers. There is no universal filler that can optimize all performance properties of plastic materials. The selection of a suitable filler should be conditioned by the requirements for the cured resin and its processing. For any specific filler, it should be considered that some performance properties of the plastic material will decrease as other performance properties increase. The addition of a filler to a resin usually improves its mechanical properties. An epoxy resin has a higher tensile strength with the addition of a filler than without it. The bending strength is negatively affected by granular fillers. The compressive strength depends on the filler type ,11.3) filler [One of the popular fillers of epoxy-based polymers is the calcium carbonate (CaCO) filler ,23,24,25) filler ,12,23,26) filler ,25 use c) filler ; e.g., p) filler ; e.g., 4) filler . He and ) filler ,28,29 an) filler ,31, amon) filler  reported3 to the epoxy resin improves the mechanical properties and the thermal resistance. Yang et al. [3 is the amount of filler in the epoxy matrix [3 (rod-like and cube-like) with 2% difference in their investigations. Zotti et al. [3 fillers on the tensile strength of epoxy resin in the range up to 3%.Calcium carbonate also improves thermal properties, especially thermal stability ,27,33. Ag et al. ,25,33), g et al. . One of y matrix ,22. Sciey matrix . The fory matrix  showed ty matrix  presentei et al.  demonstri et al.  studied Another filler of epoxy-based polymers is the carbon (C) filler, especially as carbon fiber reinforced resin (CFRP) ,37,38,39Based on many studies conducted, it is noticed that different results are obtained depending on the amount of the filler, its form, and operating conditions. As epoxy materials are used in various forms  and are subjected to various factors, it seems that research on modified materials and their properties when exposed to various environmental factors is still needed. Many researchers ,44,45,463). The other group was modified with activated carbon (C). Each modifying agent, or filler, was added at a rate of 1 g, 2 g, or 3 g per 100 g of epoxy resin. The selection of the filling quantity was made on the basis of the work presented by He [Two epoxy compounds were analyzed, based on Bisphenol A  with a polyamide curing agent. One group of the epoxy compound was modified with calcium carbonate (CaCOed by He  and Yanged by He . The eff3) , added in various quantities, resulting in three versions for each of the two filler types used. The typical concentration of CaCO3 is 98.23% and its molecular weight is 100.09 g/mol. Activated carbon was used in the form of dust, with a molecular weight of 12.01 g/mol. A total of 12 modified epoxy compounds were tested (2 base epoxy compounds × 2 filler types × 3 filler quantities); see For these tests, 12 modified epoxy compounds were selected to create different versions. The versions varied by epoxy resin type, filler type, and filler quantity. The epoxy compounds compounded with Bisphenol A were as follows: unmodified epoxy resin, epoxy number between 0.49 and 0.52 mol/100 g , and modified epoxy resin (a mixture of Epidian 5 and a modified polyester resin), epoxy number 0.40 mol/100 g , with the unmodified and modified epoxy resins cured with a polyamide curing agent with an amine number between 290 and 360 mg KOH/g . The modified epoxy compounds were fabricated with either of two filler types: activated carbon (C)  or calcium carbonate , followed by addition of the these materials in the order listed, and at three different weight ratios: 1% filler content per 100 g of the epoxy resin (version 1); 2% filler content per 100 g of the epoxy resin (version 2); 3% filler content per 100 g of the epoxy resin (version 3). The components were hand-mixed with the fillers. The mixing stage continued for approximately 30 s to blend the epoxy resin thoroughly with the respective filler. The second preprocessing stage consisted of adding 80 g of the curing agent per 100 g of each epoxy resin. The resulting compound was mixed for 90 s with a disk agitator at 128 m/min to obtain a homogeneous material. The epoxy compound components were batched with an electronic scale  in a polymer–plastic container. The epoxy compound preprocessing was completed at 21 °C ± 2 °C and 21% ± 3% RH.d = 15 ± 0.5 mm and L = 60 ± 0.5 mm. The inner surfaces of the polypropylene molds required suitable pretreatment before casting by application of an adhesion-reducing agent, POLSIFORM . The adhesion-reducing agent was sprayed on the inner surfaces from a distance of approximately 30 cm. Three minutes after applying the adhesion-reducing agent, the liquid epoxy compounds (the epoxy compounds) were cast into the molds. The casting was performed at 21 ± 2 °C and 21% to 23% RH. Following the curing process (see d = 15 ± 0.5 mm and length L = 40 ± 1 mm (The epoxy compound preprocessing was followed (with due consideration of the pot life) by casting them into polyethylene molds, each of which was cylindrically hollow, with the internal dimensions cess see , the as-0 ± 1 mm a. FigureThe cured specimens were machined to an equal height on an FNB26 milling machine. The machining process used a six-blade milling cutter (Ø35 mm) made from high speed steel.The tested specimens were cured in a single run at 21 °C ± 2 °C and 21% ± 3% RH. The overall curing time was 7 days for all specimens. Three batches of the epoxy specimens were fabricated in total and for the variants listed in The scanning electron microscope (SEM) images of examples of modified epoxy compounds samples (reference samples) after the curing process were presented in The microscopic images allowed visualization of the obtained structure of the modified epoxy composition after the curing process . The noticeable heterogeneous distribution of the filler is probably the result of the mixing method used, as well as the lack of preparation of the filler surface and the type of filler. In the case of the addition of carbon as filler, smaller clusters (agglomerates) were observed in the epoxy matrix (resin and curing agent) than in the case of calcium carbonate. Further analysis of the structure of the epoxy composite will be discussed.The other two specimen batches were aged. The second batch of epoxy specimens was placed in a climate chamber, Espec SH-661  for 10 weeks. The third batch of epoxy specimens was placed in a thermal shock chamber, Espec TSE-11 , for 10 weeks. The test conditions in the thermal shock chamber presented in Strength tests were performed following the curing of the reference specimens and both the high-temperature aging and thermal-shock aging of the remaining specimens (second and third lots). All fabricated specimens were tested on a Zwick/Roell Z150 tester . The compressive strength was determined with the test method specified in EN ISO 604 standard. The test parameters were: preloading = 100 N, test load = 10 mm/min, maximum deformation 15%. The tests were focused on the analysis and comparison of compression modulus, compression strength, and compressive strain. The specimens were strength-tested in a special fixture with a sleeve shape, in which cylindrical samples of cured epoxy compounds samples were placed, which, at the same time, ensured the perpendicularity of the sample face to the cylindrical surfaces of the holder.The results for the versions of the reference specimens made from E5/PAC/100:80 and E57/PAC/100:80 and modified with calcium carbonate are shown in 3/3%).The compression modulus of the E5/PAC/100:80 epoxy compound was the highest for the specimens with 3 g of calcium carbonate (806 MPa), with the highest result of the two epoxy compounds . It was 3/1%), with a 6% difference between the results. The lowest compression modulus value (142 MPa) was found for the E57/PAC/100:80/CaCO3/3% epoxy version, which was 75% lower than for the Epidian 57 epoxy resin compound.The highest compression modulus for the E57/PAC/100:80 epoxy compound was achieved for the 2% calcium carbonate version (554 MPa), although the compression modulus achieved for the 1% filler content was only slightly lower (521 MPa for E57/PAC/100:80/CaCO3/3% and the highest for E5/PAC/100:80/CaCO3/3%. It is probably due to the nature of the interaction (properties) of the matrix and the filler. In the first case, the epoxy resin (Epidian 57) had a higher viscosity than the Episian 5 epoxy resin. This probably contributed to better dispersion and fewer calcium carbonate agglomerates in the lower viscosity resin. Therefore, it can be assumed that the type (properties) of the epoxy resin, which is the basis of the matrix , plays an important role in the creation of epoxy composites. Moreover, Li et al. [The results presented in i et al.  underlin3/3%. Of all the specimens made from E5/PAC/100:80, the lowest compressive strength (37 MPa) was found for the E5/PAC/100:80/CaCO3/1% specimens. The lowest compressive strength (14 MPa) was found for E57/PAC/100:80/CaCO3/3% specimens, and the epoxy compound with the 2% filler content had the highest compressive strength.The results in 3/3%. E5/PAC/100:80/CaCO3/1% achieved a slightly lower compressive strain (8.8%). Note that the remaining results for E5/PAC/100:80 and E57/PAC/100:80 were similar, between 4.8% and 5.1%. The obtained strength results are also confirmed by the results of the stress values presented in An analysis of the results in The results for the versions of the reference specimens made from E5/PAC/100:80 and E57/PAC/100:80 and modified with activated carbon (C) are shown in CE (697 MPa) was developed for E5/PAC/100:80/C/1%; the lowest was developed for the version with the 2% filler content (435 MPa), or 62% of the highest tested value. The compression modulus for the 3% filler version was 608 MPa, at 13% lower from the highest tested value. The widest spread of compression modulus occurred with the E5/PAC/100:80/C/3% version. For E57/PAC/100:80, the lowest compression modulus (229 MPa) occurred for E57/PAC/100:80/C/2% (2% activated carbon-filler version). E57/PAC/100:80/C with 1% and 3% of the same filler achieved similar results . The difference between the minimum and the maximum values for the epoxy compound (E57/PAC/100:80) was 30%.The results in An analysis of the results  shows thThe compressive strain test results presented in 3 and E57/PAC/100:80/CaCO3) after aging in the climate chamber.CE (339 MPa) was found for E5/PAC/100:80/CaCO3/3%, at the highest filler content. The lowest value of the same parameter (267 MPa) was developed for E5/PAC/100:80/CaCO3/1%, at the lowest filler content. The lowest compression modulus was found for E57/PAC/100:80/CaCO3/3% (175 MPa), and the highest value of the same parameter was found for E57/PAC/100:80/CaCO3/1%, at the lowest filler content. For the Epidian 5-based epoxy compounds, the compression modulus values increased with increasing filler content; the trend was inversed for the Epidian 57-based epoxy compound.The compression modulus values  do not s3 and E57/PAC/100:80/CaCO3 after aging in the climate chamber. The compressive strength test results . The compression strengths for E57/PAC/100:80/CaCO3 was less than half that for E5/PAC/100:80/CaCO3, between 25 MPa and 33 MPa. results  for E5/P3 and E57/PAC/100:80/CaCO3 after aging in the climate chamber.3/3% specimens. The remaining values in 3/1% and E57/PAC/100:80/CaCO3/2%. They had the lowest values of this parameter and reached 6%. The highest standard deviation of compressive strain was found for the specimen lots made from E5/PAC/100:80/CaCO3/3%, at a value of about 31%. After aging in a climate chamber (3 epoxy compounds containing modified epoxy resin (Epidian 57) than in compounds in which Epidian 5 epoxy resin was used as a matrix. These differences were observed in compared to reference samples  showed wide differences of up to 50%. The E57/PAC/100:80/C specimens achieved poorer results for compression strength. These were between 20 MPa and 33 MPa. As a matter of comparison, the difference between the highest and the lowest compression strengths in The results  show a t3 filler, aged in the thermal shock chamber.3/2% (420 MPa) and E5/PAC/100:80/CaCO3/1% (400 MPa), respectively. The difference was 5%. The results for the E57/PAC/100:80/CaCO3 specimens were lower than for E5/PAC/100:80/CaCO3. The lowest compression modulus value found was 229 MPa (E57/PAC/100:80/CaCO3/2%), or 60% of the maximum for the parameter. The standard deviation of compression modulus for E5/PAC/100:80/CaCO3/2% was relatively high (33%) in comparison to the other results.The results presented in 3 and E57/PAC/100:80/CaCO3.3 and E57/PAC/100:80/CaCO3 shows large differences . The lowest result for the same epoxy compound (49 MPa) was found for E5/PAC/100:80/CaCO3/3%, and the difference between the maximum and the minimum value was 38%. The lowest spread (8%) of the compressive strength test results was found for E57/PAC/100:80/CaCO3/3%.A comparison of the compression strengths of the epoxy specimens made from E5/PAC/100:80/CaCOferences . The com3 and E57/PAC/100:80/CaCO3.3/1% and E57/PAC/100:80/CaCO3/1%). E57/PAC/100:80/CaCO3/2% achieved the highest compressive strain (8.5%). The lowest result (5.6%) was found for E5/PAC/100:80/CaCO3/2%. The standard deviation of compressive strain was the highest (30%) for E57/PAC/100:80/CaCO3/1%. It can be seen  content on the tested epoxy compounds. The results for compressive strength shown in 3/1%, the exposure to both high temperature and thermal shocks (cycling) reduced the compressive strength in comparison to the reference specimens. The compressive strength was reduced by 27% post-high temperature and 35% post-thermal shocks.For E57/PAC/100:80/CaCOThe high temperature did not reduce the compressive strength for E57/PAC/100:80/C/1%, which was 33% higher than for the reference specimens. There was also no decrease in the compressive strength value of this modified epoxy compound subjected to the thermal shocks.It is believed that this specific epoxy version benefited in terms of resistance to the tested operating exposures from the modification with activated carbon.3 modified epoxy compound had a higher compressive strength after high temperature exposure than the reference specimens (+37%); the post-thermal shock of the same specimen compound had an even higher compressive strength (+54% vs. the reference specimens). Here, CaCO3 was beneficial to the compressive strength of the epoxy compounds exposed to high temperatures and thermal shocks. An inverse relationship was found for the same E5/PAC/100:80 epoxy compound when modified by activated carbon. In this case, the heat temperature and thermal shocks reduced the compression strength.For E5/PAC/100:80 (based on unmodified Epidian 5), the type of filler (at 1%) produced completely different relations. The CaCOThe modified E5/PAC/100:80 epoxy compounds had a higher compressive strength in comparison to E57/PAC/100:80 for every compared version of the filler type and quantity and the effects of the operating exposure.3 and C) are shown in The list of the compressive strength test results for the E5/PAC/100:80 and E57/PAC/100:80 epoxy specimens modified with the 2 g filler content  (with the exception of one epoxy version).The epoxy compounds modified with CaCO3 demonstrated a negative effect of the filler after aging. Compared to the reference specimens, the high-temperature aging reduced the compressive strength by 30%; the thermal shocks reduced the compressive strength by 52%. The modification with activated carbon did not negatively affect the compression strength, but slightly improved it. However, for most Epidian 57-based epoxy resin compounds with activated carbon as the filler, the compressive strength was lower than for the compounds modified by calcium carbonate.The compressive strength for E57/PAC/100:80 with 2% of CaCOA comparison of the E5/PAC/100:80 specimens aged in the climate chamber (high-temperature aging) to the reference samples showed a reduction in compressive strength for both modifiers.For the same type of E5/PAC/100:80, the thermal shocks did not reduce the compressive strength in comparison to the reference specimens, irrespective of the modifier. The compressive strength of the same epoxy compound exposed to thermal shocks was higher than for the epoxy compounds exposed to high-temperature aging.An analysis of the results listed in (1)(a)The compressive strength of the unmodified E5/PAC/100:80 specimens was much higher than for the E57/PAC/100:80 specimens.(b)3/3% .The highest compressive strength (77 MPa) was found for E5/PAC/100:80/CaCO(c)3.The lowest compressive strength (14 MPa) was found for E57/PAC/100:80/CaCO(d)The difference between the highest and the lowest compression strengths was 82% in the reference specimens.For the reference samples:(2)(a)3/3%. A slightly lower value (53 MPa) was found for the E5/PAC/100:80/C/3% specimens.The highest compressive strength (61 MPa) was found (yet again) for E5/PAC/100:80/CaCO(b)The E57/PAC/100:80 specimens also achieved poorer results. The lowest value (20 MPa) was found for the E57/PAC/100:80/C/3% (modified with activated carbon).(c)The plotted difference between the highest and the lowest compressive strength was 67%.For the high-temperature (climate chamber) aged specimens:(3)(a)The highest compressive strength (59 MPa) was found for E5/PAC/100:80/C/3% (modified with activated carbon).(b)3/3%, modified with calcium carbonate).A lower value (49 MPa) was found for the same epoxy compound (E5/PAC/100:80/CaCO(c)3/3%) achieved a compressive strength of 21 MPa, while the specimens modified with activated carbon (E5/PAC/100:80/C/3%) achieved 14 MPa.Low compressive strengths were identified for the specimens based on E57/PAC/100:80. The epoxy specimen lot modified with calcium carbonate (E57/PAC/100:80/CaCO(d)The maximum to minimum difference was 76%.Comparison of the epoxy specimens aged in the thermal shock chamber:An analysis of the results listed in ▪The epoxy compounds modified with calcium carbonate had a higher compressive strength in most instances (10 out of 12 epoxy versions), irrespective of the aging method.▪The epoxy compounds with the basic unmodified Epidian 5 resin had a compressive strength over two times higher than any of the compared specimen versions, irrespective of the modifier type or aging method.▪3 versions (−20%) and the C versions (approximately −16%).For E5/PAC/100:80, the high-temperature aging reduced the compressive strength for the 3% CaCO▪3 reduced the compressive strength by 6% and 34%, respectively, in comparison to the reference compounds.The aged E5/PAC/100:80 modified with activated carbon and CaCO▪3, the high-temperature and thermal shock aging methods did not affect the compression strength; the high-temperature aging increased the parameter value by a factor of two.For E57/PAC/100:80/CaCO▪The epoxy compounds based on Epidian 57 and modified with activated carbon suffered from thermal shock aging, with an approximately 30% reduction in compressive strength compared to the reference specimens.To conclude on the compressive strength test results for the 3% filler epoxy compounds after aging under different conditions:The visual inspection of the behavior of the specimens during the strength tests demonstrated that unmodified epoxy resin (Epidian 5) contained polyamide curing agent fabricated an epoxy compounds which frequently cracked during leveling of the edges and in the strength tests. The epoxy specimens made from E57/PAC/100:80 were much more elastic. It was necessary to fabricate several lots of the specimens, as their edges suffered from strong deformations when leveled. The aging of the specimens improved their hardness. In general, it can be concluded that in the case of the modified epoxy compounds, the observed failure of the samples, in most cases, showed the brittle failure characteristics. These results were also noticed in other works by the author .Summarizing the results presented on compression stress–strain curves, it can be noted that for some samples, these curves exhibit a large discrepancy. Probably, one of the reasons for such a discrepancy could be some fluctuations in the climatic conditions during the tests or the different curing process  of epoxy compositions containing a different type of epoxy resin (unmodified epoxy resin Epidian 5 and modified epoxy resin Epidian 57). These results require further analysis.3 toughened epoxy resin composite increases with an increasing nano-calcium carbonate content in the range of less than 6 wt. %, while impact of strength of the composite declines when filler content is more than 6 wt. %. He et al. [3/epoxy composites were enhanced by increasing the amount of calcium carbonate added, but they decreased when the filler content reached 2%. Jin and Park [Okba et al.  stated te et al.  concludee et al.  indicateand Park  revealedAlthough the study did not analyze the method of surface preparation of fillers, many scientists emphasize this aspect of modification of polymer composites (including epoxy) as important due to the obtained mechanical properties. He at al.  demonstrStrength testing was performed on specimens made from four different epoxy compounds. The specimens were made from unmodified epoxy resin and modified epoxy resin cured with polyamide curing agent. The resulting epoxy compounds (E5/PAC/100:80 and E57/PAC/100:80) were modified with calcium carbonate and activated carbon as the filler. An important exposure factor in the tests was the seasoning of some of the epoxy specimens in a climate chamber and the remainder in a thermal shock chamber for approximately 10 weeks in both cases.3 had a higher compressive strength than the epoxy compounds modified with C (activated carbon). The different quantities of the fillers, 1–3 g of calcium carbonate (CaCO3) and activated carbon (C), largely determined the strength parameters, with results differing from the reference specimens and the epoxy specimens aged in the climate chamber and the thermal shock chamber. The influence of aging in a climatic chamber and thermal shocks chamber on the change of the mechanical properties of modified epoxy compounds containing unmodified resin was noticed. After aging in the chambers, these epoxy compounds showed features of a material with reduced stiffness (elastic with forced flexibility), and before aging, they showed features of a more plastic material.An analysis of the results led to the conclusion that the type of tested epoxy compound and the quantity and type of filler determine the effect of the climate chamber aging and the thermal shock chamber processing on the compressive strength for the tested epoxy compounds. It was difficult to explicitly determine the effect of high-temperature aging (in the climate chamber) and thermal-shock aging  on the strength parameters of the specimens. In some of the examined cases, the effects of both high temperature and thermal shocks negatively affected the compressive strength of the epoxy compounds, while it was increased for some of the modified epoxy compounds. The observations discussed here apply to other mechanical parameters such as compression modulus and compressive strain. The epoxy compounds which contained unmodified epoxy resin (Epidian 5) achieved a higher strength performance than the epoxy compounds made with modified epoxy resin (Epidian 57). It can be assumed that this is related to the properties of the epoxy resin. The lower viscosity resin (Epidian 5) produced better filler dispersibility than the higher viscosity resin (Epidian 57). In most instances, the epoxy compounds modified with CaCOIn summary, proper compounding of an epoxy adhesive compound significantly determines its strength. Aging can also affect the mechanical properties of epoxy compounds.


ST6Gal-I, an enzyme upregulated in numerous malignancies, adds α2-6-linked sialic acids to select membrane receptors, thereby modulating receptor signaling and cell phenotype. In this study, we investigated ST6Gal-I’s role in epithelial to mesenchymal transition (EMT) using the Suit2 pancreatic cancer cell line, which has low endogenous ST6Gal-I and limited metastatic potential, along with two metastatic Suit2-derived subclones, S2-013 and S2-LM7AA, which have upregulated ST6Gal-I. RNA-Seq results suggested that the metastatic subclones had greater activation of EMT-related gene networks than parental Suit2 cells, and forced overexpression of ST6Gal-I in the Suit2 line was sufficient to activate EMT pathways. Accordingly, we evaluated expression of EMT markers and cell invasiveness (a key phenotypic feature of EMT) in Suit2 cells with or without ST6Gal-I overexpression, as well as S2-013 and S2-LM7AA cells with or without ST6Gal-I knockdown. Cells with high ST6Gal-I expression displayed enrichment in mesenchymal markers  and cell invasiveness, relative to ST6Gal-I-low cells. Contrarily, epithelial markers  were suppressed in ST6Gal-I-high cells. To gain mechanistic insight into ST6Gal-I’s role in EMT, we examined the activity of epidermal growth factor receptor (EGFR), a known EMT driver. ST6Gal-I-high cells had greater α2-6 sialylation and activation of EGFR than ST6Gal-I-low cells. The EGFR inhibitor, erlotinib, neutralized ST6Gal-I-dependent differences in EGFR activation, mesenchymal marker expression, and invasiveness in Suit2 and S2-LM7AA, but not S2-013, lines. Collectively, these results advance our understanding of ST6Gal-I’s tumor-promoting function by highlighting a role for ST6Gal-I in EMT, which may be mediated, at least in part, by α2-6-sialylated EGFR. Most patients are diagnosed after metastatic lesions have formed, making treatment difficult and often unsuccessful. It is thought that PDAC metastasizes early during the carcinogenic process owing to the emergence of stem-like cancer cells with high metastatic potential, termed cancer stem cells (CSCs)  remains one of the most lethal malignancies, with a dismal 5-year survival rate of less than 9% (s (CSCs)  and actss (CSCs) , 5, 6, rs (CSCs) , serum ds (CSCs) , and hypnd colon , 12, 13,nd colon , 12, 13.nd colon . Activatnd colon , and thend colon . LikewisN-glycosylated proteins, understanding its function in cancer is critical. ST6Gal-I imparts a malignant cell phenotype by regulating, via sialylation, key receptors that control tumor-associated signaling networks. For example, ST6Gal-I-mediated sialylation of the β1 integrin promotes cell migration and invasion  conferred a phenotype similar to that of the metastatic subclones, evidenced by enrichment in EMT, stem cell, and cell motility networks. To further establish a role for ST6Gal-I in EMT, ST6Gal-I expression was knocked down in the metastatic lines, complementing the overexpression of ST6Gal-I in the parental line. In all three of the cell models, high expression of ST6Gal-I led to increased α2-6 sialylation and activation of EGFR, as well as upregulated expression of mesenchymal markers and cell invasiveness. Moreover, the EGFR inhibitor, erlotinib, neutralized ST6Gal-I-dependent differences in EGFR sialylation and activation, EMT marker expression, and invasiveness in Suit2 and S2-LM7AA cells. In the aggregate, these results highlight the importance of ST6Gal-I activity in driving EMT, a critical process that promotes metastatic disease.A). Suit2 cells have relatively low metastatic potential in animal models, and in order to study the metastatic process, other investigators have developed metastatic subclones of the Suit2 line. For example, the metastatic S2-LM7AA line was generated through iterative in vivo selection, yielding a population that reliably metastasizes to the liver following injection into the pancreas (B).Immunoblotting for ST6Gal-I in multiple human PDAC cell lines revealed that, although most lines had considerable ST6Gal-I expression, the Suit2 line had negligible levels of ST6Gal-I A. Suit2 pancreas . A seconpancreas , 38. Sigpancreas B.Figure C). We first compared the RNA-Seq data generated from the two metastatic subclones. D and E, a strong correspondence was noted between the two subclones, illustrated by the fact that 17 of 20 of the Biological Functions were shared (denoted by red bars). These included cancer, gastrointestinal disease, developmental pathways , and cancer-associated cell functions . Gene Set Enrichment Analysis (GSEA) of the normalized gene expression showed that, compared with Suit2 EV cells, S2-LM7AA and S2-013 cells were enriched in stemness-associated networks , EMT, and processes related to EMT such as hypoxia (F and G). We also utilized the IPA Upstream Regulator module to determine that the two metastatic subclones exhibited activation of regulators central to stem cell networks, EMT, cell growth and proliferation, and cell migration  or cells alternatively transduced with an empty vector (EV) construct C. We fir hypoxia , F and Gigration .A) revealed that forced expression of ST6Gal-I in Suit2 cells altered 14 of 17 of the pathways that were modulated in both of the metastatic subclones (red bars). Another two of the Functions were common to one of the two metastatic lines (green bars). Functions shared with both of the metastatic subclones included cancer, gastrointestinal disease, developmental pathways , and tumorigenic cell functions . GSEA indicated that ST6Gal-I overexpression activated Wnt, Hedgehog, EMT, and hypoxia networks . In addition, the IPA Upstream Regulators activated in Suit2 OE cells were very similar to those that activated in the metastatic subclones, specifically, regulators involved in EMT, stemness, hypoxia, and cell motility , ST6Gal-I was knocked down (KD) in the metastatic S2-LM7AA and S2-013 lines . A nontargeting shRNA sequence was used as the control (shC). The expression of EMT markers was examined in cells with modulated ST6Gal-I expression. A switch between E-cadherin, an epithelial cadherin, and N-cadherin, a mesenchymal cadherin, is a hallmark of EMT. In addition, cells that have undergone EMT exhibit an upregulation in the mesenchymal transcription factors, slug and snail. As compared with Suit2 EV cells, Suit2 OE cells had increased expression of the mesenchymal markers, snail and N-cadherin, although slug expression was unchanged (A). Contrarily, the epithelial marker, E-cadherin, was reduced in OE cells. Consistent with these results, knockdown of ST6Gal-I in the metastatic S2-LM7AA and S2-013 lines strongly suppressed the expression of snail, slug, and N-cadherin, whereas E-cadherin expression was enhanced in KD cells (B and C).Based on the RNA-Seq results, we further investigated the role of ST6Gal-I in EMT. To supplement the Suit2 OE cell model A, ST6Galnchanged A. ContraKD cells , B and CD). Staining of the two metastatic subclones did not yield any overt differences in E-cadherin localization between shC and KD cells; however, there was little junctional staining in any of these lines (data not shown). We hypothesize that, since shC cells are already highly metastatic, knockdown of ST6Gal-I alone was insufficient to restore strong cell–cell junctions .An important event in EMT is the breakdown of E-cadherin-containing cell–cell junctions, an event that promotes a more mesenchymal phenotype and facilitates cell motility. E-cadherin localization was examined by immunocytochemistry in Suit2 EV and OE cells. Suit2 EV cells displayed strong E-cadherin localization at the cell–cell junctions, whereas the staining in OE cells was intracellular and not distinctively membrane bound D. StainiE), whereas ST6Gal-I KD in the S2-LM7AA and S2-013 lines caused an increase in occludin expression (E). An opposite expression pattern was noted for fibronectin, where fibronectin mRNA levels were augmented in Suit2 cells with forced OE but reduced by ST6Gal-I KD in the two metastatic lines (F).We next assessed downstream transcriptional targets of slug and snail using qRT-PCR. Slug and snail regulate the transcription of numerous genes involved in EMT including occludin, an epithelial marker suppressed during EMT, and fibronectin, a mesenchymal marker upregulated during EMT. In comparison with Suit2 EV cells, OE cells had decreased levels of occludin mRNA E, whereapression E. An oppic lines F.A–C, quantification in Fig. 4D). Upon forced ST6Gal-I OE, Suit2 cells displayed a significant increase in invasion (A and D). In contrast, KD of ST6Gal-I suppressed the invasive capabilities of the metastatic S2-013 and S2-LM7AA lines (B–D). Interestingly, the overall degree of cell invasiveness was correlated with the levels of ST6Gal-I mRNA  and protein . Cells with the highest amount of ST6Gal-I  were the most invasive.As cell invasiveness is a seminal phenotypic feature of EMT, invasion assays were conducted using Matrigel-coated transwell chambers. Cells were allowed to invade for 24 h, and cells migrating to the underside of the matrigel-coated filters were stained with crystal violet. Stained cells were solubilized in 10% acetic acid and invasion quantified by absorbance spectroscopy. S2-013 and S2-LM7AA shC cells were inherently more invasive than Suit2 EV cells, consistent with the metastatic phenotype of these two subclones (representative images in A). The total amount of EGFR was comparable in Suit2 EV and OE cells, indicating that manipulation of ST6Gal-I expression did not alter overall levels of EGFR expression. Importantly, the overexpression of ST6Gal-I caused an increase in the basal activation of EGFR, evidenced by immunoblotting for phosphorylated EGFR . SNA precipitation assays were next conducted with the S2-LM7AA and S2-013 lines. As shown in B and C, EGFR was sialylated in S2-LM7AA and S2-013 shC cells; however, levels of α2-6 sialylated EGFR were greatly reduced in the ST6Gal-I KD lines. Knockdown of ST6Gal-I also resulted in a decrease in basal EGFR activation, whereas total levels of EGFR were equivalent in EV and KD cells. These results suggest that α2-6 sialylation of EGFR by ST6Gal-I is sufficient to induce EGFR activation, independent of ligand binding.EGFR is an important driver of EMT, and our prior studies demonstrated that ST6Gal-I-mediated sialylation of EGFR promotes both basal and ligand-dependent EGFR activation in pancreatic and ovarian cancer cells . To evalFig. 5A, erlotinib treatment of Suit2 EV and OE cells not only diminished the overall activation of EGFR but also eliminated the ST6Gal-I-dependent differences in EGFR activation . Similarly, ST6Gal-I-mediated differences in EGFR activation were ablated by erlotinib in the S2-LM7AA line . On the other hand, erlotinib treatment did not eliminate ST6Gal-I-dependent differences in S2-013 cells; shC cells maintained substantially higher levels of activated EGFR than KD cells in the presence of erlotinib, although overall levels of EGFR activation were reduced by the drug in both lines .In view of the enhanced EGFR activation in cells with high ST6Gal-I expression, we evaluated the role of sialylated EGFR in directing EMT. Cells were treated with the EGFR tyrosine kinase inhibitor, erlotinib, and probed for EGFR activation by immunoblotting. As shown in Fig. 6D, quantification in Fig. 6E). Likewise, the enhanced invasiveness of S2-LM7AA shC cells, as compared with KD cells, was eliminated by erlotinib treatment (D and E). However, erlotinib seemed to have little effect on the invasiveness of S2-013 cells (D and E). Because S2-013 invasiveness was not inhibited by erlotinib, the role of EGFR in EMT was not examined further in this line.To determine whether inhibiting EGFR suppressed the invasive capabilities imparted by ST6Gal-I, invasion assays were conducted in the presence or absence of erlotinib. As observed previously, untreated Suit2 OE cells were more invasive than untreated EV cells; however, this difference was abolished by erlotinib treatment . Consistent with data in Fig. 7A, long exposure). In the S2-LM7AA line, untreated shC cells exhibited higher expression of both slug and snail in comparison with ST6Gal-I KD cells. Treatment with erlotinib abrogated ST6Gal-I-dependent differences in expression of these proteins .The expression of EMT markers was next assessed in Suit2 and S2-LM7AA cells treated with or without erlotinib , A and BC). Interestingly, erlotinib treatment increased the expression of occludin in all of the cell lines, consistent with the concept that blocking EGFR activity promotes a more epithelial phenotype. Furthermore, the differential effects of ST6Gal-I activity on occludin expression were abolished by erlotinib treatment in the Suit2 and S2-LM7AA lines. As with occludin, differences in fibronectin expression imparted by ST6Gal-I were eliminated by erlotinib treatment in the Suit2 line (D). In S2-LM7AA cells, ST6Gal-I-dependent differences in fibronectin expression were diminished, but not eliminated, by erlotinib. The combined results in We also evaluated the expression of occludin and fibronectin in erlotinib-treated cells. As shown previously, untreated cells with high ST6Gal-I expression (Suit2 OE and S2-LM7AA shC) had diminished occludin levels in comparison with cells with low ST6Gal-I expression (Suit2 EV and S2-LM7AA KD) C. Intereit2 line D. In S2-St6gal1 gene in the murine PyMT breast cancer model caused tumor cells to acquire a more differentiated phenotype  while repressing epithelial markers . ST6Gal-I overexpression also promoted cell invasion. Conversely, knockdown of ST6Gal-I in the metastatic S2-LM7AA and S2-013 lines suppressed expression of mesenchymal markers and cell invasiveness while upregulating epithelial markers. These data suggest a causal role for ST6Gal-I in driving EMT.in vitro invasion assays. Although further studies will be needed to understand the effects of EGFR inhibitors on the S2-013 line, it is noteworthy that in all three cell models  ST6Gal-I activity consistently promoted EGFR activation, expression of mesenchymal markers, and cell invasion, confirming the importance of receptor sialylation in EMT.Although ST6Gal-I has been reported to promote TGFβ-dependent EMT , the conIn summary, results from this investigation add to an accumulating body of literature highlighting a role for ST6Gal-I in endowing tumor cells with more stem-like, mesenchymal characteristics. The transition from an epithelial to mesenchymal phenotype is central to the acquisition of tumor metastatic potential, and thus, elucidating mechanisms underlying EMT is key to the development of more effective cancer treatments. Furthermore, in addition to carcinogenesis, EMT is an essential process in normal human development, as well as wound healing. Although intensive research efforts have focused on EMT, the functional involvement of cell surface sialylation  in EMT remains underinvestigated. Our results showing that ST6Gal-I promotes EMT, combined with prior studies by others, underscore the need for further research aimed at delineating sialylation-dependent mechanisms that regulate the phenotype of both normal and malignant cells.RNA was extracted from S2-013, S2-LM7AA, Suit2 EV, and Suit2 OE cells using the Qiagen RNeasy Mini Kit according to the vendor protocol .RNA concentration was quantified and submitted to the Hugh Kaul Genomics Core at UAB for bulk RNA sequencing. Three biological replicates were generated for each of the four cell lines. mRNA-sequencing was performed on the Illumina NextSeq500 as described by the manufacturer . Briefly, RNA quality was assessed using the Agilent 2100 Bioanalyzer. RNA with an RNA Integrity Number of ≥7.0 was used for sequencing library preparation. RNA passing quality control was converted to a sequencing ready library using the NEBNext Ultra II Directional RNA library kit as per the manufacturer’s instructions . The cDNA libraries were quantitated using qPCR in a Roche LightCycler 480 with the Kapa Biosystems kit for Illumina library quantitation  prior to cluster generation. Cluster generation was performed according to the manufacturer’s recommendations for onboard clustering . We generated between 30 and 35 million paired end 75-bp sequencing reads per sample for gene level abundance.http://www.bioinformatics.babraham.ac.uk/projects/fastqc, accessed September 1, 2020). STAR (version 2.7.3a) was used to align the raw RNA-seq fastq reads to the human reference genome (GRCh38 p13 Release 32) from Gencode using parameters --outReadsUnmapped Fastx; --outSAMtype BAM SortedByCoordinate; --outSAMattributes All  . Each identifier was mapped to its corresponding object in Ingenuity’s Knowledge Base. A fold change cutoff of ±2 and p-value < 0.05 was set to identify molecules whose expression was significantly differentially regulated. These molecules, called Network Eligible molecules, were overlaid onto a global molecular network developed from information contained in Ingenuity’s Knowledge Base. Networks of Network Eligible Molecules were then algorithmically generated based on their connectivity. The Functional Analysis identified the biological functions and/or diseases that were most significant to the entire data set. Molecules from the dataset that met the fold change cutoff of ±2 and p-value < 0.05 and were associated with biological functions and/or diseases in Ingenuity’s Knowledge Base were considered for the analysis. Right-tailed Fisher’s exact test was used to calculate a p-value determining the probability that each biological function and/or disease assigned to that data set is due to chance alone. For the IPA analyses shown in Z scores >1.5 were considered, and at least one of the three cell lines had a Z score of ≥2.0.IPA Biological Functions and Canonical Pathways were used, along with the Upstream Regulator module . For genGSEA was performed on the full normalized gene expression data from RNA-Seq experiments, using the GSEA software (version 4.1.0) and the Molecular Signature Database (MSigDB) (version 7.1) , 53. TheSuit2 parental and S2-013 cells were donated by Dr Michael A. Hollingsworth at the University of Nebraska . The S2-LM7AA subclone was developed by Drs Lacey McNally and Donald Buchsbaum at the University of Alabama at Birmingham . Suit2 parental and metastatic subclones were maintained in RPMI medium containing 10% fetal bovine serum (FBS) and 1% antibiotic/antimycotic supplements (Gibco). Suit2 parental cells were transduced with either ST6Gal-I overexpression vector  or empty vector (Sigma) using lentivirus. Metastatic cell lines were transduced with lentivirus encoding shRNA targeting ST6Gal-I , whereas control lines were developed using nontargeting lentivirus bearing shRNA against GFP . Following transduction, stable polyclonal populations were generated by selection with puromycin. Successful overexpression or knockdown was confirmed by immunoblotting using anti-ST6Gal-I goat polyclonal antibody .Cells were lysed using radioimmune precipitation assay buffer supplemented with protease and phosphatase inhibitors (Sigma). Total protein concentration was measured using BCA (Pierce). Samples were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Following transfer, membranes were blocked with 5% nonfat dry milk in tris-buffered saline containing 0.1% Tween20. Immunoblots were probed with antibodies to ST6Gal-I , pEGFR , total EGFR , slug , snail , E-cadherin , and N-cadherin . Protein loading was verified using anti-β-actin  or anti-β-tubulin . Membranes were incubated with horseradish peroxidase-coupled secondary antibodies , and protein was detected by enhanced chemiluminescence (Pierce).Cell lysate, 250 μg, was incubated with 150 μg of SNA-agarose . Samples were incubated overnight at 4 °C on a rotator. α2-6 Sialylated proteins were precipitated with centrifugation and washed with ice-cold PBS three times. Precipitates were resolved by SDS-PAGE and immunoblotted for EGFR as described above.p < 0.05 using a Student’s t test from at least three independent experiments, with each independent experiment performed in triplicate.RNA was extracted using the Ambion RNA Extraction Kit (Life Technologies) in accordance with the manufacturer’s protocol. Total RNA concentration was measured, and cDNA was synthesized using M-MLV reverse transcriptase (Promega). qRT-PCR samples were generated using TaqMan Fast Advanced Master Mix (Thermo). Primers for occludin (Hs05465837_g1) fibronectin (Hs01549976_m1), and ST6Gal-I (Hs00949382_m1) were acquired from Applied Biosystems. Data were normalized to GAPDH , and significance was determined as Cells were plated in 24-well plates on sterile glass coverslips with 50,000 cells per well and allowed to attach overnight. Cells were washed with PBS, fixed using 4% paraformaldehyde, and then incubated with E-cadherin antibody  at a 1:100 dilution at 4 °C overnight. Following incubation with primary antibody, slides were incubated with an anti-rabbit secondary antibody conjugated to Alexa Fluor 488  at a 1:400 dilution for 1 h at room temperature. Cells were then counterstained with Hoescht nuclear stain at a 1:10,000 dilution for 10 s. The coverslips were mounted on slides using Prolong Gold mounting medium . Cells were viewed using NIS elements software at 20× magnification.In vitro cell invasion was evaluated using growth factor reduced invasion chambers . Prior to use, invasion chambers were brought to room temperature and matrigel was rehydrated for 2 h at 37 °C using serum-free medium in a humidified tissue culture incubator. Cells were serum deprived (1% FBS-containing medium) for 24 h and then seeded into the upper well of the invasion chamber at a density of 100,000 cells in 500 μl of 1% FBS-containing media. A 5% FBS-containing medium was added to the bottom chamber and used as a chemoattractant for Suit2 and S2-LM7AA cells; a 2.5% FBS-containing medium was used as the chemoattractant for S2-013 cells. Cells were allowed to invade through matrigel for 24 h at 37 °C. After incubation, the medium containing noninvaded cells, as well as the matrigel layer, were carefully removed from the upper portion of the chamber. The underside of the transwell membrane, containing the adherent cells that had successfully invaded the matrigel, was fixed with 4% paraformaldehyde and stained with 0.5% (w/v) crystal violet. Crystal violet was dried overnight and then solubilized using a 10% acetic acid solution. Absorbance of the solution was quantified at 590 nm on a Biotek plate reader. Absorbance was normalized to that of controls (cells plated in chambers with no chemoattractant). All invasion assays were conducted at least three independent times, and each independent experiment was performed in triplicate. Significance was determined as p < 0.05 using a Student’s t test.For immunoblotting and qRT-PCR experiments using erlotinib, cells were serum deprived for 2 h using a 1% FBS-containing medium before treatment with 20 μM erlotinib for 24 h. Lysates were then collected for protein or RNA quantification as described above. Invasion assays using erlotinib-treated cells were performed as described above. Prior to plating in invasion chambers, detached cells were incubated with 20 μM of erlotinib in suspension on a rotator at 37 °C for 2 h. Cells were then plated, with inhibitor, in invasion chambers, and the invasion assay was completed as described previously.All of the data described in this report are contained within the manuscript or supplementary figures, with the exception of the RNA-Seq results. The RNA-Seq data are available under NCBI GEO Accession# GSE158527.The authors declare that they have no conflicts of interest with the contents of this article.