Bax, Bim, and Bak) and anti-apoptotic  genes and extrinsic  pathways, DNA methyltransferase 1, 3a, and 3b, histone deacetylase inhibitors 1, 2, and 3, cell viability, and apoptosis in hepatocellular carcinoma (HCC) SK-Hep 1, colorectal cancer SW620, and pancreatic cancer PaCa-44 cell lines. The SK-Hep 1, SW620, and PaCa-44 cells were cultured and treated with TSA and zebularine. To determine cell apoptosis, cell viability, and the relative gene expression level, flow cytometry assay, MTT assay, and qRT-PCR were done respectively. The result indicated that zebularine and TSA changed the expression level of the Bax, Bak, Bim Bcl-2, Bcl-xL, Mcl-1, DR4, DR5, FAS, FAS-L, TRAIL, DNA methyltransferase 1, 3a, and 3b, histone deacetylase inhibitors 1, 2, and 3 by which induced cell apoptosis and inhibit cell growth in all three cell lines. Concluding, TSA induced its role through both extrinsic and intrinsic apoptotic pathways in three cell lines, whereas, zebularine played its role via both pathways in the SK-Hep 1cell line, it had no significant effect on Bcl-2, Bcl-xL, and Mcl-1 gene expression in SW620 and PaCa-44 cell lines. Aberrant histone modifications or promoter region hypermethylation of tumor suppressor genes (TSGs) have been recognized as the important epigenetic molecular mechanism in cancer induction. The potential anticancer activities of histone deacetylase inhibitors (HDACIs) and DNA methyltransferase inhibitors (DNMTIs) have been investigated in recent years. The current study was assigned to investigate the effect of trichostatin A (HDACI) in comparison to zebularine (DNMTI) on the intrinsic pro-apoptotic ( Aberrant histone modifications or promoter region hypermethylation of tumor suppressor genes (TSGs) have been recognized as the important epigenetic molecular mechanisms in cancer induction and progression. These mechanisms can inactivate TSGs and cancer-related genes lead to cancer induction and progression. The N-terminal domains of all core histones are subject to diverse modifications, such as methylation, acetylation, phosphorylation, and ubiquitylation at certain residues . AcetylaIn addition to histone modification, DNA methylation, primarily at CpG dinucleotides, has long been identified to play an important role in controlling gene expression. Histone modifications and DNA methylation play a significant role in modulating chromatin structure which can control gene expression. DNA methyltransferases (DNMTs), are responsible for DNA methylation, the activity of these enzymes affects chromatin structure and gene expression . AccordiBCL-2 family divided into three groups: promoters of apoptosis ; inhibitors of apoptosis ; and regulatory BH3-only proteins   and anti-apoptotic  genes and cytoplasmic/extrinsic  pathways, DNA methyltransferase 1, 3a, and 3b, histone deacetylase inhibitors 1, 2, and 3, cell viability, and apoptosis in hepatocellular carcinoma SK-Hep 1, human colorectal cancer SW620, and human pancreatic cancer PaCa-44 cell lines.In-vitro studies have demonstrated that DNMTIs (such as zebularine) and HDACIs  can induce apoptosis through two molecular mechanisms comprising extrinsic and intrinsic apoptotic pathways , 8. The nd PUMA) -11. The nd PUMA) . The curMaterialsHepatocellular carcinoma SK-Hep 1, human colorectal cancer cell lines SW620, and human pancreatic cancer PaCa-44 cell lines were purchased from the National Cell Bank of Iran-Pasteur Institute. The zebularine, TSA, and Dulbecco’s modified Eagle’s medium (DMEM) were obtained from Sigma  and dissolved in dimethyl sulfoxide (DMSO) to make a work stock solution. Further concentrations of the compounds, zebularine, and TSA were obtained by diluting the provided stock solution. Other necessary compounds comprising materials and various kits were purchased as provided for previous works , 14. TheCell culture and cell viability  The SK-Hep 1, SW620, and PaCa-44 cells were cultured in DMEM containing 10% FBS (Sigma) and 1% antibiotics which include 10,000 unit/mL penicillin G sodium(Sigma), 10,000 ug/mL streptomycin sulfate, and 25 ug/mL amphotericin B (Sigma) at 37 °C in 5% CO2 °C for 24 h and then seeded into 96-well plates (3 × 105 cells per well). After 24 h, the medium was replaced with an experimental medium containing various concentrations of zebularine , and TSA . The control groups were treated with DMSO, at a concentration of 0.05 %. After 24 h, the SK-Hep 1, SW620, and PaCa-44 cells, experimental and control, were investigated by MTT assay according to Standard protocols to determine cell viability. In this process, the MTT solution was added to each well for 4 h at 37 ℃ and then the MTT solution was changed by DMSO and shaken for 10 min to dissolve all of the crystals. Finally, the optical density was detected at a wavelength of 570 nM by a microplate reader. Each experiment was repeated three times (triplicates). IC50 was analyzed by GraphPad Prism Version 8.0. software. Non-linear regressions of log (inhibitor) vs response were selected for IC50 estimation. The bottom and top parameters were constrained to 0% and 100%.Cell apoptosis assayTo determine SK-Hep 1, SW620, and PaCa-44 cells apoptosis, the cells were cultured at a density of 3 × 105 cells/well and incubated overnight and then treated with zebularine and TSA, based on IC50 values demonstrated in Real-time Quantitative Reverse Transcription PolymeraseChain Reaction (qRT-PCR)Bax, Bak, Bim, Bcl-2, Bcl-xL, Mcl-1, DR4, DR5, FAS, FAS-L, TRAIL genes, DNA methyltransferase 1, 3a, and 3b, histone deac-etylase inhibitors 1, 2, and 3 qRT-PCR was done. The SK-Hep 1, SW620, and PaCa-44 cells were treated with zebularine and TSA, based on IC50 values demonstrated in To determine the relative expression level of the Result of cell Cell viability by the MTT assay. IC50 values are shown in The cell viability of the SK-Hep 1, SW620, and PaCa-44 cells treated with zebularine and TSA was investigated by MTT assay by which the activity of cellular enzymes reduced the tetrazolium salt MTT and produced a dark-blue formazan crystal. The crystal was dissolvable in DMSO to determine the viable cells. As shown in Result of cell apoptosis assayP < 0.001). Maximal and minimal apoptosis was seen in SK-Hep 1 cell treated with TSA and PaCa-44 cell treated with zebularine after 24 h of treatment respectively, To determine apoptosis, the SK-Hep 1, SW620, and PaCa-44 cells were treated with zebularine and TSA, based on IC50 values, for 24 h and then stained using annexin-V-(FITC) and PI to determine apoptotic cells in the early and late apoptosis stage. As indicated in Determination of genes expressionEffect of zebularine on the gene expressionBax, Bak, Bim, DR4, DR5, FAS, FAS-L, and TRAIL genes and down-regulated the expression of Bcl-2, Mcl-1, Bcl-xL, DNA methyltransferase 1, 3a, and 3b, histone deacetylase 1, 2, and 3 in SK-Hep 1cell line significantly. Additionally, zebularine up-regulated the expression of Bak, Bax, Bim, DR4, DR5, FAS, FAS-L, and TRAIL genes and down-regulated the expression of DNA methyltransferase 1, 3a, and 3b in SW620, and PaCa-44 cell lines significantly. It had no significant effect on Bcl-2, Bcl-xL, and Mcl-1 gene expression in SW620, and PaCa-44 cell lines  and mitochondrial (intrinsic) pathways, both of which converge on the activation of the cascade of caspases . HDACIs DNMTs , HDACs  in SK-Hep 1 cell line significantly. Additionally, zebularine up-regulated the expression of the genes of the extrinsic apoptotic pathway and pro-apoptotic genes (as mentioned in the result part) and down-regulated the expression of DNMTs , HDACs  in SW620, and PaCa-44 cell lines significantly. We did not observe significant changes in the expression level of Bcl-2, Mcl-1, and Bcl-xL in SW620, and PaCa-44 cell lines treated with zebularine. Similarly, in-vitro studies have demonstrated that DNA demethylating agents zebularine and decitabine activate the mitochondrial apoptotic pathway in T cells  and down-regulated the expression of the anti-apoptotic genes,  T cells . Similar T cells . It has  T cells . In recell lines . Several HCT-116 . Experim HCT-116 .Bax, Bak, Bim, DR4, DR5, FAS, FAS-L, and TRAIL genes and down-regulated the expression of Bcl-2, Mcl-1, Bcl-xL, DNA methyltransferase 1, 3a, and 3b, histone deacetylase 1, 2, and 3 in all three cell lines significantly. Inconsistent with our result, other researchers have indicated that histone deacetylase inhibitor sodium butyrate decreases Bcl-XL protein levels by down-regulating its RNA expression in mesothelioma cell lines. Besides, depsipeptide, as an HDACI, decreases the expression of Bcl-2, Bcl-XL, and Mcl-1 in multiple myeloma cells  and pro-apoptotic (BIM)genes in pancreatic cancer cell lines CFPAC1, Miapaca2, HPAF, PSN1, Panc1 PC, Paca44, RT45P1, and T3M4  and pro-apoptotic (BIM)genes in pancreatic cancer cell lines CFPAC1, Miapaca2, HPAF, PSN1, Panc1 PC, Paca44, RT45P1, and T3M4 , Class I HDACs , and Class II HDACs  and up-regulate CIP/KIP Family  resulting in cell apoptosis in colon cancer LS 174T, and LS 180 cell lines (Meanwhile, the extrinsic and intrinsic pathways are not the only molecular mechanisms of zebularine and TSA. The results of current studies indicated that DNA methyltransferase inhibitor zebularine and histone deacetylase inhibitor TSA can induce apoptosis through both mitochondrial/intrinsic and cytoplasmic/extrinsic apoptotic pathways, in hepatocellular carcinoma SK-Hep 1, human colorectal cancer SW620, and human PaCa-44 pancreatic cancer cell lines. Our finding demonstrated that zebularine induced significant apoptosis in all three cell lines. It up-regulated the expression of  T cells . Similar T cells . It has  T cells . In recell lines . Several HCT-116 . Experim HCT-116 . As mentma cells . In huma and p53 . Resent and T3M4 . Similarand T3M4 . In pancand T3M4 . Additioand T3M4 . Meanwhill lines , 43. FinBcl-2, Mcl-1, and Bcl-xL gene expression in SW620, and PaCa-44 cell lines. Therefore, TSA can induce a stronger apoptotic effect through the activation of both pathways. Finally, both agents induced apoptosis significantly, which provided the theoretical basis for the clinical application of these compounds.In conclusion, our results indicated that histone deacetylase inhibitor TSA can induce its apoptotic effects through both mitochondrial/intrinsic and cytoplasmic/extrinsic apoptotic pathways in hepatocellular carcinoma SK-Hep 1, human colorectal cancer SW620, and human PaCa-44 pancreatic cancer cell lines. Further, zebularine can induce apoptosis by an extrinsic apoptotic pathway in three cell lines. It had no significant effect on


To model the problem of radiation resistance in prostate cancer, cell lines mimicking a clinical course of conventionally fractionated or hypofractionated radiotherapy have been generated. Proteomic analysis of radiation resistant and radiosensitive DU145 prostate cancer cells detected 4410 proteins. Over 400 proteins were differentially expressed across both radiation resistant cell lines and pathway analysis revealed enrichment in epithelial to mesenchymal transition, glycolysis and hypoxia. From the radiation resistant protein candidates, the cell surface protein CD44 was identified in the glycolysis and epithelial to mesenchymal transition pathways and may serve as a potential therapeutic target. Localis22.12 incubator at 37°C to allow colonies to form. After 10 days, colonies were stained with crystal violet  and counted. The experiments were performed three separate times.Human PCa adenocarcinoma cell lines DU145 and PC3) were purchased from the American Type Culture Collection . DU145 cells were treated with 10 Gy daily for five fractions over several weeks (DU145‐HF). Generation of DU145‐CF, cell culture, clonogenic survival, cellular proliferation, matrigel transwell invasion, soft agar, and western blot were performed following methods previously described . The gen5 and PC3Radiation therapy remains a definitive treatment modality for prostate cancer with curative intent. While it is an effective treatment for a large majority of patients, cancer relapse can occur and is challenging to salvage. Radiation resistance is seen in both conventional and hypofractionated radiation therapy treatment schedules. Potential therapeutic targets to improve radiation remain elusive. This study is the first published proteomic analysis of prostate cancer cell lines generated from clinically relevant treatment schedules. This study uses an isogenic radiation resistant cell line  to investigate the proteome and elucidate the pathways and proteins implicated in persistence of prostate cancer following radiation therapy. The cell surface protein CD44 has been implicated in radiation resistance in a range of cancer types and we suggest that it may also represent a potential target in prostate cancer.5 cells per well onto a six‐well plate overnight. After 16 h, siRNA control or CD44 siRNA  were transiently transfected into cells using SiTran . One day later, cells were harvested and seeded at 250 and 4000 cells per well onto a six‐well plate in 10% FBS DMEM in triplicates, then mock irradiated (0 Gy) or irradiated with 6 Gy dose of ionizing radiation, respectively. Cells were then placed in a humidified CO2 incubator at 37°C to allow colonies to form. After 10 days, colonies were stained with crystal violet  and counted. To confirm knockdown, the transfected cells were lysed at 48 h post‐transfection and western blotting performed using anti‐CD44 antibody . The experiments were performed three separate times.For CD44 siRNA knockdown experiments, cells  were seeded at 3 × 102.26) were washed three times with Stain Buffer . Cells were then resuspended in 100 μl volume and stained with FITC anti‐CD44  or FITC anti‐IgG1 isotype control  on ice in the dark for 30 min. Flow cytometry was performed using LSR II system  and flow cytometry analysis was done using FlowJo .DU145‐PAR, DU145‐CF, DU145‐HF, PC3‐PAR, and PC3‐CF cells , washed three times with cold phosphate buffered saline (pH 7.4) before cells were pelleted. Cell pellets were resuspended in 150μL of 50% (v/v) 2,2,2,‐Trifluoroethanol and sample preparation was as previously described  with an 2.42 iBAQ values of the total number of proteins (4410) identified across the whole cell lysates from all cell lines, in triplicate. Parameters used for consensus clustering were; max_k = 7, Euclidean P as the similarity metric, pItem = 0.8, seed = 1000, and reps = 1000. For visualisation, abundances were converted to z‐scores.Consensus clustering was performed using ConsensusClusterPlus  (v1.5.0)2.5https://www.inkscape.org). g:Profiler was run on the enriched radiation lists separately, however visualised in the same instance to enable better visualisation of protein overlaps in pathways.Proteins of interest were processed for pathway analysis using the non‐ranked method g:Profiler   . ggPlot2 (3.2.1) , Complex33.1While radiation therapy is an effective treatment modality for many PCa patients, the development of biochemical recurrence due to radiation resistance remains a clinical concern. Recent advances in the clinic have enabled the variation in radiation schedules with the two predominant ones being investigated in this paper using a cell line model; conventional fractionation (CF) radiation therapy and hypofractionated (HF) radiation therapy. To mimic the clinical scenario of resistance to both treatments, DU145 PCa cells were mock irradiated with 0 Gy (DU145‐PAR), irradiated with 2 Gy daily for 59 fractions over several weeks (DU145‐CF) as previously described  or 10 Gy3.2p = 0.0029 for 4 Gy, p = 0.0015 for 6 Gy, p = 0.0001 for 8 Gy; DU145‐HF: p = 0.0011 for 4 Gy, p = 0.0242 for 6 Gy, p = 0.0083 for 8 Gy; Figure p = 0.01 for 0 Gy and p = 0.02 for 6 Gy, Figure p = 0.0156), and proliferation increased following 6 Gy irradiation  from the 4410 proteins detected across the cell lines . These gene lists were compared to the significantly altered proteins (295 proteins for DU145‐CF cells and 194 for DU145‐HF cells) of both radiation resistant cell lines, and presented as the z‐scores of the logOxygen plays a key role in the response of irradiation‐induced reactive oxygen species, a major problem with radiation therapy is hypoxia . It has Most cancer cells display an increase in aerobic glycolysis and use this metabolic pathway for the generation of ATP as the main source of energy . This in3.52 of 0.3521269 was calculated between the fold change in DU145‐CF cells (as compared to DU145‐PAR) and the DU145‐HF cells (as compared to DU145‐PAR), indicating there is a low correlation observed in the fold changes of radiation resistant cells. There were 72 proteins  in both cell lines, as represented in the plot as circles. The significantly altered proteins have been highlighted . It was confirmed that CD44 expression was increased, and the therapeutic utility of an anti‐CD44 blocking antibody in PCa radio‐sensitisation was demonstrated. The study of radioresistant PCa has been limited by technical challenges in obtaining prostate samples following relapse (invasive procedure and very little tissue obtainable). As such, the creation of isogenic radioresistant PCa cell lines is one avenue to address this clinical problem. Although our research is primarily based on an isogenic DU145 PCa cell line model, we were able to validate increased CD44 expression in a second isogenic PC3 PCa cell line model, suggesting that our findings are not cell‐line specific. Further work investigating the therapeutic role of CD44 in an in vivo model, as well its potential as a predictive biomarker  are needThis work was partially funded through an operating grant from the National Cancer Institute Early Detection Research Network (1U01CA214194‐01) to T.K. and P.C.B. A Prostate Cancer Canada Movember Discovery Grant (D2017‐1811) to T.K. and S.K.L. and a Project Grant from the Canadian Institute of Health Research (PJT‐162384) to T.K. and S.K.L.The authors declare no conflict of interest.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.


One of the major glyconutrients in EBN is sialic acid, which has a beneficial effect on neurological and intellectual capability in mammals. The aims of this research were to study the effects of sialic acid from EBN on cell viability and to determine its effect on mitochondria membrane potential (MtMP) in Caco-2, SK-N-MC, SH-SY5Y, and PC-12 cell lines. Fourteen samples of raw EBN were collected from four different states in Malaysia. The confluency of the epithelial monolayers measurement of the tight junction for all the cell lines was determined using transepithelial electrical resistance (TEER), and the sialic acid uptake study in cell lines was determined by using ultra-high performance liquid chromatography (UHPLC). The MTT assay was conducted for cell viability study. The MtMP in cell lines was determined using the Mito Probe JC-1 Assay by flow cytometer analysis. We have recorded a statistically significant difference between the uptake of sialic acid from EBN and the standard solution. A higher amount of sialic acid was absorbed by the cells from extract of EBN compared to the standard solution. The amounts of sialic acid uptake in Caco-2, SK-N-MC, SH-SY5Y, and PC-12 cell lines were (0.019 ± 0.001), (0.034 ± 0.006), (0.021 ± 0.002), and (0.025 ± 0.000) µmol/L, respectively. The MTT results indicated that the concentration of sialic acid increased the cell viability and showed no cytotoxicity effects on cell lines when they were exposed to the sialic acid extract and sialic acid standard at all the tested concentrations. The number of active mitochondria was found to be significantly higher in SH-SY5Y cell lines with a 195% increase when treated with sialic acid from EBN. Although many researchers around the globe use SH-SY5Y and SK-N-MC for Alzheimer’s disease (AD) study, based on our finding, SH-SY5Y was found to be the most suitable cell line for AD study by  The mechanism involved here includes conversion of metabolites energy to reduced nicotinamide adenine dinucleotide (NADH) followed by electron transfer from NADH while protons are also pumped across inner membrane to intermembrane space. This process creates transmembrane potential that utilizes ATP synthase for reflow of protons across inner membrane, and finally this energy is the driving factor of adenosine diphosphate (ADP) phosphorylation to ATP . Three fvia oral administration of sialic acid during an initial postnatal period in the rodents  and it is one of the eight glyconutrients which has helped to increase cell tissue repair , promotein vitro cell line. One of the most common cell lines used is the neuroblastoma (SH-SY5Y) cells. These cells were applied as a prototype for Aβ cytotoxicity in AD  until use.Four different types of cell lines were used in this study: the colorectal adenocarcinoma (Caco-2/ATCC cat. no. HTB-37); the neuroepithelioma (SK-N-MC/ATCC cat. no. HTB-10); the neuroblastoma (SH-SY5Y/ATCC cat. no. CRL-2266); and the pheochromocytoma (PC-12/ATCC cat. no. CRL-1721). The entire cell lines were purchased from American Type Culture Collection . Each of the cell lines was seeded and maintained in 25 cml-glutamine . Only PC-12 cell was added with 15% horse serum . All the cells were maintained in the same conditions, at 37°C in an incubator with 5% carbon dioxide, 95% humidity, and air atmosphere. The medium was replaced every 2–3 days. The cells were maintained until they reached 80% confluency.The PC-12 and SH-SY5Y cells were grown in Dulbecco’s Modified Eagle’s Medium  supplemented with 20 and 15% v/v fetal bovine serum (FBS), while SK-N-MC cells were grown in Eagle’s Minimum Essential Medium  supplemented with 20% v/v fatal bovine serum. The entire medium contained 1% v/v nonessential amino acid , 1% antibiotic (penicillin–streptomycin) , and 1% v/v 2  were equilibrated and sterilized according to the manufacturer’s recommendations. Two hundred microliters of culture medium was added to the upper compartment of the cell culture system. The ohmic resistance of a blank (culture insert without cells) was measured in parallel. The blank value was subtracted from the total resistance of the sample, in order to obtain sample resistance. The final unit area resistance (Ω*cm2) was calculated by multiplying the sample resistance by the effective area of the membrane (0.33 cm2 for 24-well millicell insert plates).The cells will reach the maximal levels of differentiation after several days in incubation. To confirm this process, transepithelial resistance values (TEER) can be monitored using EVOM2™ because fully differentiated polarized cells have tight junctions with a TEER of >200 Ω*cm2 . The EVO2 into a 96-well plate. The cells were then incubated overnight in an incubator with 5% carbon dioxide (CO2), 95% air atmosphere, and 95% relative humidity at 37°C. The next day, the medium was changed to medium-free FBS following exposure to (or treatment with) sialic acid at the required concentration , and the cells were incubated for 24 h. MTT reagent at a concentration of 5 mg/ml and a volume of 10 µL was added to each well and incubated for 3–4 h until the purple precipitate was visible. Later, the medium was aspirated from the wells as completely as possible without disturbing the formazan crystals and cells on the plastic surface. Then, 100 µL of dimethyl sulfoxide (DMSO) was added to each well, followed by agitation on a plate shaker for 5 min, and finally, the optical density was read at 570 nm. The number of surviving cells is directly proportional to the level of the formazan product created -2,5-diphenyltetrazolium bromide) assay is commonly used to measure cell viability and proliferation . Adheren created .The absorbance value for the blanks should be 0.00 ± 0.1 optical density (O.D) units. The absorbance range for untreated cells should typically be between 0.75 and 1.25 O.D. units. Selection of a cell number  allows for the measurement of both stimulation and inhibition of cell proliferation. If the absorbance values of the experimental samples are higher than the control, this indicates an increase in cell proliferation. Alternatively, if the absorbance rates of the experimental samples are lower than the control, this indicates a reduction in the rate of cell proliferation or a reduction in overall cell viability.2PO4, 10 mM Mes, 0.5 mM of MgCl2, and 1.0 mM CaCl2 at pH 6.0) and incubated at 37°C for 2 min. Later, 1 ppm (∼3 μmol/L) of sialic acid extract was added to each well and incubated for 15 min at 37°C. Then, the buffer was aspirated, and cells were rinsed with cold buffer for three times before adding 1 ml of 200 mM NaOH to solubilize the cells and they were left overnight at 4°C. After an overnight incubation at 4°C, the cells were subsequently removed; the levels of sialic acid in buffer and cells were determined by using UHPLC. This procedure follows the method of Derakhshandeh and his colleagues with slight modification  was added to the tube and incubated for 5 min. After the incubation, 10 µL of JC-1 stock solution  was added to all tubes and incubated for another 30 min at 37°C. Later, all cells were washed using the warm PBS and centrifuged to obtain the cell pellet. Then, the cells were resuspended by adding 500 µL of PBS to each tube. All tubes were analyzed using a flow cytometer machine (BD FACSAria™) with 488 nm excitation using emission for Alexa Fluor® 488 dye and fluorescence microscopy. The JC-1 dye detection was used with bandpass filters centered around 529 nm (green fluorescence) and bandpass filters centered around 595 nm (orange fluorescence). The logarithmic signal amplification was used with the typical green–orange electronic signal compensation near 4% and orange–green signal compensation around 10% . The kit contains 30 µg powdered dye, DMSO, carbonyl cyanide 3-chlorophenylhhydrazone (CCCP), and 10× phosphate-buffered saline (PBS). The cell lines were fixed with 150 µL of sialic acid extract and standards for 24 h prior to staining the cells for flow cytometer measurement. In brief, all reagents were equilibrated at room temperature before beginning the experiment. Two hundred micromolar of JC-1 powder stock solution was prepared by dissolving the vial with 230 µL of the DMSO. Then, 1 × 10ound 10% . The depp < 0.05 was considered statistically significant.All data were analyzed using the SPSS software version 16 . The viability of the cell lines induced with sialic acid was analyzed using the one-way analysis of variance (ANOVA) with post hoc Tukey’s test to show significant difference between groups. 2 and 292.71 ± 80.61 Ω*cm2, respectively, while SH-SY5Y and PC-12 cell lines reached their 50% confluency at day 15 with 305.91 ± 80.61 Ω*cm2 and 280 ± 127.98 Ω*cm2, respectively. The Pearson Rank correlation analysis was performed to determine the relationship between the days of culture and TEER reading. Based on results in p < 0.01).The determination of cell cultures confluency stage for entire cell lines using EVOM2™ was shown in p < 0.05).The percentage of cell viability in epithelial and neuroblastoma cell lines was significantly higher when they were induced with sialic acid extract compared to sialic acid standard. Above the concentration of 60 μg/ml, all cell lines showed negative effect of sialic acid on cell viability.p < 0.05) and between the extraction and standard extraction (p < 0.05), respectively. However, only Caco-2 cell lines showed a significant difference between the control and standard (p < 0.05). For basal solution, the mean absolute difference (MD) of sialic acid uptake was significant between the control and the extraction in all cell lines. The MD of sialic acid uptake was also significant between the extraction and the standard. In contrast, only SH-SY5Y cell line has recorded the differences in sialic acid uptake between the control and the standard.p < 0.05). The Tukey post hoc test revealed that the number of active mitochondria in SH-SY5Y is significantly higher when induced with sialic acid compared with control (p = 0.000). However, even without treatment with sialic acid, all other cell lines have a higher number of active mitochondria at the start of the experiment.2, and as such the differentiation process is confirmed by careful monitoring of the cell’s polarization process . In addition, it should also be highlighted that translating TEER into a functional estimate of tightness is tough. This is due to the fact that composition of the tight junction complexes and the size of the compound of interest are the underlying factors that influence the endothelial monolayer tightness . In our 00 Ω*cm2 .in vivo studies, in vitro cytotoxicity and cell viability assays were shown to be more advantageous in terms of speed, lower cost, and automation potential. Due to these reasons, studies conducted with human cells are deemed more appropriate than in vivo research , and HCl. None of the sialic acid on the market is obtained from bird’s nests. Since bird’s nests naturally contain higher bioactive compounds including sialic acid, it may be one of the reasons why sialic acid obtained from bird’s nest has a slightly higher absorption rate than the commercially available sialic acid. In addition, the increase in cell viability with higher concentration might be influenced by the mitogenic properties in sialic acid extract from EBN that promoted the cell growth as manifested by previous studies which showed the enhanced cell division in rabbit corneal keratocytes using EBN . AlthougIn general, sialic acid rarely exists free in nature and is usually available as component of oligosaccharide chains of mucins, glycoproteins, and glycolipids. In terms of its position, sialic acid usually occupies terminal, nonreducing positions that are highly exposed and functionally essential. These commonly refer to nonreducing positions of oligosaccharide chains of complex carbohydrates on both outer and inner membrane surfaces, mainly to galactose, N-acetylgalactosamine, and other sialic acid moieties. The highest concentration of sialic acid is present in mammalian central nervous system, where majority of it is found in gangliosides (65%), followed by glycoproteins (32%), while the remaining exists as free sialic acid .To date, there are limited findings on digestion and mechanisms involved among sialic acid compounds. It was reported that rat intestinal walls are highly permeable to free sialic acids. In addition, it was highlighted that sialidases of bacterial origin could possibly cleave sialic acid residues from milk oligosaccharides in colon; however it was not evident if sialic acid is capable of absorption across colonic mucosa . Hence, Based on literature, it was found that the normal range of total sialic acid (TSA) level found within serum or plasma falls in the range of 1.58–2.22 mmol/L. From this, only about 0.5–3.0 μmol/L corresponds to free form of SA . Thus, wEukaryotic cells contain several types of organelles, which may include nucleus, mitochondria, chloroplasts, the endoplasmic reticulum, the Golgi apparatus, and lysosomes. Each of these organelles performs a specific function critical to the cell's survival. Since cell lines were also obtained from the eukaryotic cells, they also have the same organelles. Mitochondria study gained attention in 1970s, when research was focused on studying energy conservation mechanism as well as ATP synthesis. At the same time, chemiosmotic theory of oxidative phosphorylation was also established, which led to conferment of Nobel Prize in Chemistry awarded to Peter Mitchell in year 1978 . Any parIn this study, the JC-1 was used because it is more specific for measuring changes in mitochondria membrane potential (MtMP) . It is ain vitro, SH-SY5Y cell line is more pronounced if the study is focusing on mitochondrial dysfunction. Apart from its suitability for AD, it can also be applicable for other studies focused on mitochondrial dysfunction in other diseases.Previous study showed that the mitochondrial functionality can be restored by natural product such as herbal medicine which helps in preserving the dopaminergic neurons in Parkinson disease , and in in vitro technique. The increase observed in mitochondrial membrane potential in entire cell lines when subjected to sialic acid treatment indicated presence of healthy cells. This is vital to study on MtMP measurement for its effect on mitochondrial dysfunction. The sialic acid uptake also was noticed to occur with varying degrees in cell lines. Based on our findings among all the tested cells, SH-SY5Y was found to be the most suitable cell line especially for studies that will be focused on the expression of active mitochondria.Our finding has indicated that MtMP measurement could be used to study mitochondrial dysfunction by


H-tetrazolium bromide (MTT) microtiter assay. Euphorbia schimperiana, Acokanthera schimperi, Kniphofia foliosa, and Kalanchoe petitiana exhibited potent antiproliferative activity against A427, RT-4, MCF-7, and SiSo cell lines, with IC50 values ranging from 1.85 ± 0.44 to 17.8 ± 2.31 µg/mL. Furthermore, these four extracts also showed potent antiproliferative activities against LCLC-103H, DAN-G, A2780, KYSE-70, HL-60, and U-937 cell lines, with IC50 values ranging from 0.086 to 27.06 ± 10.8 µg/mL. Hence, further studies focusing on bio-assay-guided isolation and structural elucidation of active cytotoxic compounds from these plants are warranted.Medicinal plants have been traditionally used to treat cancer in Ethiopia. However, very few studies have reported the in vitro anticancer activities of medicinal plants that are collected from different agro-ecological zones of Ethiopia. Hence, the main aim of this study was to screen the cytotoxic activities of 80% methanol extracts of 22 plants against human peripheral blood mononuclear cells (PBMCs), as well as human breast (MCF-7), lung (A427), bladder (RT-4), and cervical (SiSo) cancer cell lines. Active extracts were further screened against human large cell lung carcinoma (LCLC-103H), pancreatic cancer (DAN-G), ovarian cancer (A2780), and squamous cell carcinoma of the esophagus (KYSE-70) by using the crystal violet cell proliferation assay, while the vitality of the acute myeloid leukemia (HL-60) and histiocytic lymphoma (U-937) cell lines was monitored in the 3--2,5-diphenyl-2 Cancer is one of the leading causes of death worldwide. Mainly due to the rapid growth in the human population and increment in the prevalence of risk factors associated with economic transition, cancer incidence and death are rising in Africa . MoreoveMedicinal plants have been traditionally used in Ethiopia for the treatment of various diseases, including cancer ,4,5. HowA. schimperi, E. schimperiana, K. foliosa, and K. petitiana—showed negative T/Ccorr. values, indicating relevant cytotoxic activity at 50 µg/mL , and two suspensions (HL-60 and U-937) cell lines. PBMCs were included to compare the results obtained by the HL-60 and U-937 to primary noncancer cells. This should allow an initial assessment of the selectivity of the extracts. Accordingly, concentration-response curves of the extracts against 10 cell lines were generated and IClculated , Table 250 ˃ 50 µg/mL).Selectivity is a desired property of active lead anticancer agents . DiffereA. schimperi: potent activity was observed when the extract of leaves of A. schimperi was tested on all ten cell lines with IC50 values ranging from 1.87 ± 0.4 to 10.31 ± 3.45 µg/mL. This plant is reported to contain acolongifloroside K as its major active principle, as well as cardiotonic glycosides ouabain and acovenoside A as minor constituents; in fact, it is used by Maasai people in east African as an arrow poison [A. schimperi have been reported for their antiviral [w poison . Young lw poison , hepatitw poison , and tonw poison  in Ethiontiviral , antibacntiviral , antiprontiviral , and in ntiviral  activitintiviral .E. schimperiana: the methanolic extract of E. schimperiana showed moderate cytotoxicity activity against the MCF-7 cell line, with an IC50 value of 25.2 µg/mL [Euphorbia (E. turcomanica) also reduced the viability of HT-29 cells, with an IC50 value of 43 µg/mL [E. umbellata latex sap showed potent cytotoxic activity on hepatocellular carcinoma tumor cells (Hepa1c1c7), at IC50 values ranging from 2-12 µg/mL [E. tirucalli, showed cytotoxic activity against a wide range of human cancer cell lines, including esophageal squamous cells (11.08 µM) and pancreatic carcinoma cells (6.84 µM) [O-β-D glucoronside, and 3-methyl-qurecetin-7-O-β-D-glucoronside), isolated from the 80% methanol extract of E. schimperiana, showed antioxidant activities [E. schimperiana showed potent cytotoxic activity against A427, SiSo, and RT-4 cell lines at concentrations ranging from 1.85 ± 0.44 to 3.28 ± 1.2 µg/mL, and moderate cytotoxic activities against A2780 (26.54 ± 18.5 µg/mL)..2 µg/mL . The met43 µg/mL . Similar12 µg/mL . Euphol,tivities . In the K. petitiana: K. petitiana, known for its traditional use as a treatment of breast and skin cancer in Ethiopia [50 values ranging from 2.09 ± 0.43 to 10.41 ± 5.59 µg/mL. Another plant from the same genus, K. crenata, has been reported to possess potent cytotoxic activity, with IC50 values of 2.33 and 28.96 µg/mL against mesothelioma (SPC212) and hepatocarcinoma (HepG2) cell lines, respectively [K. gracilis, on the other hand, showed only weak antiproliferative activity (IC50 = 136.85 ± 2.32 µg/mL) against human hepatocellular carcinoma (HepG2) [K. pinnata, was reported for its potent cytotoxic activity against the human nasopharyngeal (KB) cell line, with the ED50 value of 80 ng/mL [K. tubiflora were reported to displayed strong cytotoxic activity against A549, Cal-27 , A2058 (melanoma), and HL-60 (promyelocytic leukemia) cell lines [Ethiopia , also shectively . The chl80 ng/mL . Furtherll lines .K. foliosa: K. foliosa inhibited the proliferation of all cell lines, with IC50 values ranging from 14.54 ± 4.14 to 27.06 ± 10.8 µg/mL. Knipholone anthrone, a 4-phenylanthraquinone isolated from this plant, has been reported to possess cytotoxic activity against human acute monocytic (THP-1) and promonocytic (U-937) leukemic cell lines, with IC50 value of 0.9 ± 0.09 and 0.5 ± 0.05 µM, respectively [ectively .http://www.theplantlist.org, accessed on 15 April 2021). Each plant materials were shade dried and ground into powder.Different parts of 22 plant species  were col®®, R-200and R-210, Duisburg, Germany) with heating  at 37–40 °C, followed by freeze-drying  the aqueous concentrate. The dried powder (200 g each) was macerated in 1 L (80% methanol) and shaken for 48 h. The macerated plant material was then filtered through Whatman No.1 filter paper by using a Buchner funnel. The crude methanol extracts were concentrated with a rotary evaporator , in a humid atmosphere of 5% CO2 at 37 °C [v/v) heat-inactivated fetal bovine serum  and supplemented with 30 mg/L penicillin and 40 mg/L streptomycin. Cells were incubated in a 5% CO2 humidified incubator , at 37 °C, and passaged weekly.The cancer cell lines in this study, MCF-7, A427, RT-4, SiSo, LCLC-103H, DAN-G, KYSE-70, A2780, HL-60, and U-937 were obtained from the German Collection of Microorganisms and Cell Culture  . The A27at 37 °C . Cells w®®) with 3.2% sodium citrate as an anticoagulant. Whole blood was 1:2 diluted with phosphate-buffered saline (PBS) . Afterward, 10 mL Histopaque®®-1077  were overlaid with 10 mL of the diluted cell suspension and centrifuged without brake (400× g) for 30 min at room temperature. Subsequently, the PBMC layer was transferred to a new 50 mL centrifugal tube, washed with 10 mL PBS , and centrifuged for 10 min at 250× g. The obtained cell pellet was then washed three times with PBS (5 mL) and centrifuged at 250× g for 10 min. After isolation, the PBMCs were cultured in RPMI 1640 , supplemented with 2 mM L-glutamine  1% v/v antibiotics  and 10% v/v heat-inactivated fetal calf serum. The viability and number of cells were assessed by the trypan blue exclusion assay. Trypan blue was added in a 1:1 ratio to the cell suspension and counted with a Neubauer counting chamber. Cell viability was always >90%. Cells were adjusted to a density of 1 × 106 cell/mL for the following MTT assay. The assay was performed as described, with extract concentrations ranging from 50 to 3.13 µg/mL.Peripheral blood from healthy humans was provided by the blood bank of the University Medicine Greifswald. All donors signed informed consent forms that their blood could be used for research purposes, and all samples were randomized so as to conceal the identity of the donors. Blood samples were collected by venipuncture in tubes (BD Vacutainerv/v) was used as a solvent. The plates were incubated for an additional 72 h at 37 °C. After 96 h, the culture medium was discarded and replaced with a 1% glutaraldehyde buffer saline for 20 min and then stored under Dulbecco’s buffer solution (pH 7.4) at 4 °C. On the day of staining, the buffer solution was removed, and the cells were stained with 0.02% crystal violet in deionized water (100 µL/well) for 30 min. Excess dye was discarded by washing the plates for 15 min in fresh water. The cell-bound dye was redissolved in 70% (v/v) ethanol/water and the optical density was measured at λ = 570 nm with a Spectramax 384 Plus plate reader  or a Sunrise plate reader . The IC50 values were calculated by linear least-squares regression of the T/C corr values versus the logarithm of the added extract concentration and extrapolating to the T/C corr. values of 50% [T is the mean optical density (OD) of the treated cells, ODC the mean OD of the controls, and ODC,0 the mean OD of seeded cells at the time the drug was added.Crystal violet assay was used for both primary and secondary screening of extracts, as described previously . This asH-tetrazolium bromide (MTT) assay was used in this experiment to determine the inhibition of the viability of suspension cells by the extracts [The 3--2,5-diphenyl-2extracts . Briefly50 values were calculated with the software GraphPad Prism 7.0a by determining the inflection point of the simulated sigmoidal curves. The results are presented as means ± standard error of mean where appropriate.All tests were independently performed in triplicate. ICA. schimperi, E. schimperiana, K. petitiana and K. foliosa, showed cytotoxic activity against all ten cell lines. Moreover, these extracts possessed selective cytotoxicity toward suspension cell lines (HL-60 and U-937) when compared to their effect on PBMC, consistent with their traditional use in anticancer therapies. In addition to these four plants, C. abyssinica and G. involucrata also showed selective cytotoxic/antiproliferative activities against some of the human cancer cell lines used in this study. These encouraging results have motivated us to begin isolating and identifying the active components of these four plant extracts, which may contain novel lead compounds for the treatment of cancer.The results of this study indicate that crude extracts of 4 out of the 22 plant species have good cytotoxic activity against human cancer cell lines, as depicted in


Cell barcoding by amine-reactive fluorescent dyes enables analogous cell-based multiplex assays, but requires multiple labeling reactions and quality checks prior to every assay. Here we describe generation of stable, fluorescent protein-barcoded reporter cell lines suitable for multiplex screening of antibody to membrane proteins. The utility of this cell-based system, with the potential of a 256-plex cell panel, is demonstrated by flow cytometry deconvolution of barcoded cell panels expressing influenza A hemagglutinin trimers, or native human CCR2 or CCR5 multi-span proteins and their epitope-defining mutants. This platform will prove useful for characterizing immunity and discovering antibodies to membrane-associated proteins.Multiplex immunoassays with acellular antigens are well-established based on solid-phase platforms such as the Luminex Song et al. describe an immunoassay method, which is a multiplex, fluorescence-barcoded cell lines system that enables high-throughput screening of antibodies binding to large number of protein variants in a single-tube manner. They demonstrate the utility of this tool by assessing binding of different antibodies to a panel of influenza hemagglutinin antigens and to epitope/domain map CCR2 and CCR5 antibodies. For many multi-subunit or multi-pass transmembrane proteins, the native conformation is preserved only when the protein is properly expressed on the cell membrane2. For this reason, cell-based binding assays using protein antigens expressed on the cell surface are widely used in antibody screening and characterization3. A major limitation in these assays is that they are single analyte, or singleplex methods, and consequently time-consuming, sample-depleting, and costly when used to screen samples with high numbers (>10) of antigens. Although multiplex immunoassays based on the Luminex® technology are well-established, only acellular antigens can be immobilized on detection beads with this platform.Membrane proteins are often critical antigens in humoral responses to infection and physiologically significant in health and disease. They are, therefore, valuable targets in vaccine design and therapeutic antibody development7. Distinct cell populations are labeled with unique signatures of amine-reactive fluorescent dyes of different emission wavelengths and intensities before pooling for exposure to potentially reactive antibody followed by analysis by flow cytometry. The FCB technique improves the throughput of flow cytometry experiments and minimizes staining variability and antibody consumption. Although this FCB approach offers flexibility in barcoding cell samples from different sources, especially primary cells, barcoding, labeling, and quality assurance procedures are required prior to every experiment. In contrast, a panel of reporter cell lines stably barcoded by endogenous expression of fluorescent proteins (FPs) yet capable of expressing select membrane-associated antigens by transduction, would establish a stable and highly reproducible immunoassay platform. Once established, endogenously barcoded reporter cell lines would greatly simplify the FCB approach.Fluorescent cell barcoding (FCB) was developed as a cell-based multiplexing technique® beads. Individual reporter cell sub-lines, each bearing a unique combination of FPs, were subsequently engineered to express different cell surface proteins to permit their pooling in a cell-based multiplex immunoassay. Flow cytometry was then used to de-multiplex signals from pooled reporter cell lines by serial gating to identify FP combinations. In this proof-of-concept study, we established a 16-plex basic panel and tested the feasibility of expanding to a 256-plex panel of reporter cell lines. We successfully applied this platform to multiplex detection of antibody binding to cell panels expressing various influenza A hemagglutinin (HA) trimers or human CCR2b and CCR5, and several domain-swap or point mutants that define specific protein domains/epitopes.Here we describe a cell-based, multiplex immunoassay platform using reporter cell lines with stable barcoding by FPs. Briefly, a basal cell line was engineered to express different combinations of FPs that can be reliably detected and distinguished in different channels on flow cytometers. This resulted in a panel of FP-barcoded reporter cell lines analogous to barcoded Luminex8, simplifying the processes of genetic modification and assay processing. A drawback of K562 as a reporter cell line is its expression of the Fcγ receptor, CD32A, which binds most human IgG subclasses with sufficient affinity to raise background thresholds  but not A/Christchurch/16/2010(H1N1), an A/California/07/2009(H1N1)-like strain. Antibody HC19, specific for HA A/Hong Kong/JY2/1968(H3N2)21, bound to that HA but not to others. This assay may be an alternative to Luminex bead assays in high-throughput screening of antigen-specific and cross-reactive antibodies secreted in clonal cultures of human B cells from influenza-vaccinated subjects22.Influenza HAs are diverse trimeric membrane proteins. Recombinant HA protein preparation can be challenging as some HA ectodomains are unstable and cell surface expression can be a surer option for obtaining native structure when working with unknown or novel influenza strains. Multiplex immunoassays with collections of cell surface-expressed HA antigens may be useful in influenza vaccine design and immunological monitoring. To demonstrate, we prepared a panel of 12 reporter cell lines expressing the HA trimers of diverse influenza A subtypes and successfully validated their structural integrity with several reference antibodies  are key to many biological functions and are an important class of drug targets, constituting some 34% of US Food and Drug Administration approvals26. The combination of gain- and loss-of-binding readouts across all the ectodomains allowed a semi-quantitative determination of the contribution of each individual ectodomain  in ECL2A domain Fig.  showed tTo expand the established 16-plex reporter cell line panel, additional transductions for more FPs are necessary. Potentially, the accumulations of genetic insertions in the reporter cell genome could impact the expression and processing of preexisting FPs, other transduced proteins (antigens), and impair cell metabolism or proliferation. To address this concern, we generated a reporter expressing a fifth FP, LSSmOrange. Reporter cell lines FP0000 and FP1111, with none or four preexisting FPs, were pooled and transduced to express LSSmOrange. Similar transduction efficiencies and LSSmOrange intensities were achieved in both parental lines  remained capable of the expression, folding, and processing of analyte proteins, a complex membrane protein, human CCR5, was tested as an example analyte. Similar transduction efficiencies and surface CCR5 expression levels were detected based on the four cell lines established above .Homo sapiens sapiens) subject under Duke Institutional Review Board Committee guidelines (IRB Pro00062495). Written informed consent was obtained.Peripheral blood mononuclear cells (PBMCs) were obtained from one human (30. pLV-EF1a-IRES-Puro (Addgene plasmid # 85132) was a gift from Tobias Meyer31. pLB (Addgene plasmid # 11619) was a gift from Stephan Kissler27. pMD2.G (Addgene plasmid # 12259) and psPAX2 (Addgene plasmid # 12260) were gifts from Didier Trono. mScarlet-I-mTurquoise2 (Addgene plasmid # 98839) was a gift from Dorus Gadella32. pLenti-smURFP-T2A-mCardinal (Addgene plasmid # 80348) was a gift from Erik Rodriguez and Roger Tsien33. pMito-miRFP703 (Addgene plasmid # 80000) was a gift from Vladislav Verkhusha15. pLSSmOrange-mKate2 (Addgene plasmid # 99868) was a gift from Marc Tramier34. Plasmid mNeonGreen-C1 was provided by Allele Biotechnology and Pharmaceuticals, Inc., under a license for non-commercial use.pU6-(BbsI)_CBh-Cas9-T2A-BFP (Addgene plasmid # 64323) was a gift from Ralf Kuehn17. Coding sequences for other FPs were cloned from plasmids mentioned above, with EBFP210 from pU6-(BbsI)_CBh-Cas9-T2A-BFP, mTurquoise211 from mScarlet-I-mTurquoise2, mNeonGreen12 from mNeonGreen-C1, mCardinal13 from pLenti-smURFP-T2A-mCardinal, mKate214 and LSSmOrange16 from pLSSmOrange-mKate2, and miRFP70315 from pMito-miRFP703. Coding sequences for cytoplasmic domain truncated mouse CD4 (amino acid (aa) 1–423), CD8a (aa 1–222), and CD86 (aa 1–268) were cloned from splenocyte cDNA samples from one C57BL/6 mouse. Coding sequences for full-length human CCR2b and CCR5, and cytoplasmic domain truncated human CD4 (aa 1–424), CD8a (aa 1–209), CD86 (aa 1–276), and CD154 (aa 1–240) were cloned from PBMC cDNA samples from one healthy donor.DNA template coding FP hmKeima8.5 was synthesized (Genscript) based on published amino acid sequencesDNA templates coding influenza HA antigens A/Christchurch/16/2010(H1N1) (GISAID Accession EPI280344) and A/Texas/50/2012(H3N2) (GenBank Accession KJ942744) were synthesized  based on amino acid sequences in the databases. Plasmid containing coding sequences for influenza HA A/Solomon Islands/3/2006(H1N1)  was provided by Aaron G. Schmidt (Ragon Institute). Plasmids containing coding sequences for other influenza HA antigens used were provided by Stephen C. Harrison , including HA antigens A/Japan/305/1957(H2N2) (GenBank CY014976), A/Hong Kong/JY2/1968(H3N2) (GenBank CY147438), A/American black duck/New Brunswick/00464/2010(H4N6) (GenBank CY138045), A/Viet Nam/1203/2004(H5N1) (GenBank AY818135), A/Taiwan/2/2013(H6N1) (GISAID EPI459855), A/Taiwan/1/2017(H7N9) (GISAID EPI917065), A/northern shoveler/California/HKWF1204/2007(H8N4) (GenBank CY039588), A/Jiangxi/IPB13/2013(H10N8) (GenBank KJ406543), and A/mallard/Wisconsin/10OS3941/2010(H14N6) (GenBank CY133266).® Endo-free Plasmid DNA Mini Kit II, Omega Bio-tek) for mammalian cell transfection. Recombinant sequences in all plasmids used were verified by DNA Sanger sequencing .Standard molecular cloning procedures were followed for plasmid modification and gene cloning. Endotoxin-free plasmids were prepared  were constructed by replacing the U6-loxP-CMV-EGFP-loxP cassettes in plasmid pLB with EF1a promoter, EFS core promoter , or EF1a-IRES-Puro cassettes from plasmid pLV-EF1a-IRES-Puro, respectively. pLB-EXIP was further modified to generate bicistronic vectors pLB-EF1a-IRES-EBFP2, pLB-EF1a-IRES-mTurquoise2, pLB-EF1a-IRES-mCardinal, pLB-EF1a-IRES-mCD4, pLB-EF1a-IRES-mCD8a, and pLB-EF1a-IRES-mCD86 by replacing puromycin-resistant gene in pLB-EXIP with coding sequences for corresponding genes following IRES element. pLB-EFS-IRES-mNeonGreen was generated from pLB-EXIP by replacing EF1a promoter with EFS core protomer and puromycin-resistant gene with mNeonGreen coding sequences.Lentiviral transfer vector plasmids expressing EBFP2, mTurquoise2, mCardinal, mKate2, miRFP703, LSSmOrange, and hmKeima8.5 were generated by cloning of corresponding coding sequences into pLB-EF1a. Lentiviral transfer vector plasmid expressing mNeonGreen was generated by cloning of mNeonGreen coding sequences into pLB-EFS. Human CD4, CD8a, CD86, and CD154 coding sequences  were cloned into pLB-EXIP for puromycin-mediated selection. Human CD8a coding sequences were cloned into pLB-EF1a-IRES-mTurquoise2 and pLB-EF1a-IRES-mCardinal, and human CD86 coding sequences were cloned into pLB-EF1a-IRES-EBFP2 and pLB-EFS-IRES-mNeonGreen. These bicistronic expression vectors can co-express human CD8a/CD86 and FPs, with FPs at a lower expression level driven by IRES element. Coding sequences for influenza HA antigens, human CCR2b, CCR5, and their domain-swapped or point mutants were cloned into pLB-EXIP.HEK 293T and K562 cell lines were purchased from ATCC. HEK 293T cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10% heat-inactivated HyClone fetal bovine serum (FBS) (Cytiva), 10 mM HEPES buffer, and 55 µM 2-Mercaptoethanol . K562 and derivative cell lines were maintained in RPMI-1640 medium (Gibco) supplemented with 10% heat-inactivated HyClone FBS, 10 mM HEPES buffer, 1 mM sodium pyruvate, 1× MEM NEAA, 55 µM 2-Mercaptoethanol, 100 units/ml penicillin, and 100 µg/ml streptomycin . For all K562-derivative cell lines, monoclonal cell lines were established by single-cell sorting (see below) and used in binding assays in this study.CD32A exon 1 were designed with the online tool (http://crispr.mit.edu). sgRNAs used in this study were sgRNA-hCD32A-1 (5′-AGCAGCAGCAAAACTGTCAA-3′), sgRNA-hCD32A-2 (5′-ATGTATGTCCCAGAAACCTG-3′), and a negative control sgRNA (5′-TGTCATGCGTCACTTAGTGC-3′). Corresponding DNA oligos were synthesized and cloned into plasmid pU6-(BbsI)_CBh-Cas9-T2A-BFP. K562 cells were transfected with CRISPR-Cas9 targeting plasmids using Lipofectamine 3000 Transfection Reagent (Invitrogen). Ninety-six hours after transfection, the cells were collected for flow cytometry analysis and single-cell sorting (see below).Single-guide RNAs (sgRNAs) targeting human g for 45 min at 32 °C. For transductions with pLB-EXIP-based vectors, cells were selected with puromycin  between 3 and 7 days after transduction. Seven days after transduction, the cells were collected for flow cytometry analysis and single-cell sorting (see below).Lentiviral transfer vector plasmids were co-transfected into HEK 293T cells with packaging plasmids pMD2.G and psPAX2 using Lipofectamine 3000 Transfection Reagent (Invitrogen). Forty-eight hours after transfection, culture supernatants were collected and filtered through 0.45 µm polyvinylidene difluoride membrane filters (Millipore). K562-derivative cell lines were transduced with the filtered supernatants containing lentiviral vectors by spinoculation at 1000 × 18, S5V2-2919, CH6720, and HC1921 were prepared as recombinant human IgG1 antibodies as described35.Monoclonal antibodies used in this study included the following: FITC-conjugated anti-human CD32A , hCD32-APC , hCD16-PE , hCD32-PE , hCD64-PE , hCD4 , hCD8a , hCD86 , hCD154 , mCD8a-PerCP-eFluor710 , mCD86-PE-Vio770 , mCD4-APC-Fire750 , hCD58-PE , HA-tag , hCCR2 , hCCR2 , hCCR5 , hCCR5 , hCCR5 , hCCR5 , and hCCR5 . Isotype control antibodies included the following: Mouse IgG1 κ-isotype control (Rockland 010-001-330), Human IgG1κ , and Human IgG1 Lambda . Secondary antibodies included the following: Goat Anti-Human IgG-PE (Southern Biotech 2040-09), Goat Anti-Mouse IgG, and Human ads-PE (Southern Biotech 1030-09). Influenza HA-specific antibodies FI6g for 2 min at 4 °C, and resuspended in staining buffer . After incubation with antibodies at 4 °C in the dark for 30 min, cells were washed with staining buffer and resuspended in staining buffer for either secondary staining following the same procedure above or stored on ice for flow cytometry analysis or single-cell sorting. For cell counting, CountBright™ Absolute Counting Beads (Invitrogen) were added into cell suspensions prior to flow cytometry analysis.Cultures of K562 and derivative cells were collected, centrifuged at 300 × Flow cytometry analysis was carried out using either BD FACSCanto II cytometer (Duke Cancer Institute Flow Cytometry Shared Resource) or BD LSR II cytometer (The Duke Human Vaccine Institute (DHVI) Research Flow Cytometry Facility). Single-cell sorting was performed with BD Aria II (The DHVI Research Flow Cytometry Facility). The bulk cell line after transfection or transduction were stained with corresponding antibodies and single cells expressing FP or antigen of interest were sorted into 96-well flat-bottom plates containing 100 µl/well of the complete RPMI medium above supplemented with 20% heat-inactivated FBS. Nine days after sorting, healthily proliferating cell clones were transferred into 24-well plates for further expansion. Three days later, individual monoclonal cell lines were validated for the expression of FP or antigen of interest and a single clone with a uniform expression level was selected for further engineering or used as a reporter cell line in immunoassays.FlowJO (Version 10.7.2), Chromas (Version 2.6.6) and Microsoft Office (Version 1808) were used to analyze data and prepare figures for publication.The reproducibility of flow cytometry detections of the expression of FPs and cell surface molecules was confirmed by two or three independently repeated experiments as indicated in figure legends and shown as Further information on research design is available in the Supplementary InformationReporting Summary


The beneficial effects of both combinations are worth further investigation.The isoquinolinamine FX-9 is a novel potential chemotherapeutic agent showing antiproliferative effects against hematologic and prostate cancer cell lines such as B- and T-acute lymphoblastic leukemia and prostate cancer (PC) of different species. Interestingly, FX-9 shows no hemolytic activity and low toxicity in benign adherent cells. The detailed FX-9 molecular mode of action is currently not fully understood. But application on neoplastic cells induces pro-apoptotic and antimitotic effects. Canine prostate cancer (cPC) represents a unique spontaneous occurring animal model for human androgen-independent PC. Human androgen-independent PC as well as cPC are currently not satisfactorily treatable with chemotherapeutic protocols. Accordingly, the evaluation of novel agent combinations bears significant potential for identifying novel treatment strategies. In this study, we combined FX-9 with the currently approved therapeutic agents doxorubicin, carboplatin, the demethylating substance azacitidine as well as further potentially antitumorigenic agents such as dichloroacetic acid (DCA) in order to evaluate the respective synergistic potential. The combinations with 1–5 μM FX-9 were evaluated regarding the effect after 72 hours on cell viability, cell count and apoptotic/necrotic cells in two human prostate cancer cell lines  and a canine prostate cancer cell line (Adcarc1258) representing androgen-dependent and -independent PC/cPC forms. FX-9 in combination with azacitidine decreases cell viability and increases cell death with positive Bliss values. Furthermore, this decreases the cell count with neutral Bliss values on PC-3. Carboplatin in combination with FX-9 reduces cell viability with a neutral Bliss value and increases cell death on LNCaP with calculated positive Bliss values. DCA or doxorubicin in combination with FX-9 do not show synergistic or additive effects on the cell viability. Based on these results, azacitidine or carboplatin in combination with FX-9 offers synergistic/additive efficacy against prostate adenocarcinoma cell lines  FX-9 (3-(p-Tolyl)isoquinolin-1-amine) is a synthesized amino-substituted isoquinoline , antifunin vitro studies on canine mammary cancer cell lines showed therapeutic potential of this agent [Therapeutic approaches using compound combinations have been effectively introduced in several treatment protocols in order to increase therapeutic efficacy. Accordingly, combined applications allow resistance mechanisms to be addressed and the enhancement of drug sensitivity against individual agents . Moreoveis agent , 16. Theis agent . Therapyis agent , 19. Caris agent . It is uis agent  and in dis agent . Dichloris agent . In humais agent . Current®) were developed to lower hypersensitivity reactions but also side effects such as leucopenia and neutropenia [® was withdrawn by the pharmaceutical company [Prostate cancer occurs spontaneously in humans and dogs. A total of 10–20% of human patients develop castration-resistant prostate cancer (CRPC) within five years of diagnosis, and over 84% of CRPC patients have bone metastases . CRPC istropenia . Paccal  company , 31 due  company . Therefo company . In the in vitro on two human prostate carcinoma cell lines and one canine prostate carcinoma cell line at which FX-9 dosage a combination with azacitidine, DCA, doxorubicin or carboplatin could provide synergistic or additive effects.The aim of the study was to investigate FX-9  10 mM stock solution was dissolved in dimethylsulfoxide .Azacitidine  10 mM stock solution was dissolved in DMSO (Merck KGaA).Dichloroacetic acid  was used in ≥ 99% purity and a lot-specific concentration of 1.547 g/mL. A 1 M stock solution was prepared and stored at -4°C for up to four weeks. For the working solution, the DCA stock solution was dissolved in distilled water and adjusted to pH 7 with a sodium hydroxide solution. The final solution was filtered through a 0.22 μm filter.™, Thermo Fisher Scientific GmbH, Darmstadt, Germany), 10% FBS superior , 2% penicillin-streptomycin (Biochrom GmbH)) and divided into aliquots.Doxorubicin  100 μM stock solution and carboplatin  10 mM stock solution were prepared with culture medium  was prepared in a 1 mM stock solution by dissolving 5 mg solid DHT in 17.21 mL ethanol absolute (EtOH).2 cell culture flasks in medium 199 (Gibco™) with 10% FBS superior (Biochrom GmbH) and 2% penicillin-streptomycin (Biochrom GmbH). The cell cultures were cultivated at 37°C and 5% CO2 in a humidified atmosphere.TiHoDProAdcarc1258 (Adcarc1258) is a canine cell line, derived from a prostate adenocarcinoma of a ten-year-old male Briard , 34. LNC® AQueous One Solution Cell Proliferation Assay . Cells were seeded in 96 well plates at a density of 7,500 cells per well and allowed to adhere overnight. Cells were exposed to 1 μM, 2 μM, 3 μM, 4 μM and 5 μM FX-9 single application and in combination with 3 μM azacitidine, 3 mM DCA, as well as cell line specific dosages of 75 nM (Adcarc1258)/100 nM doxorubicin (PC-3 and LNCaP) or 20 μM (Adcarc1258)/60 μM (PC-3)/80 μM (LNCaP) carboplatin. Single applications of Azacitidine, DCA, doxorubicin and carboplatin were tested at the concentrations outlined above. After 72 hours, 100 μL of MTS -5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) dissolved in culture medium were added to each well. After two hours of incubation, the absorbance at 490 nm was measured with the Multi-Mode Reader Synergy 2 . The amount of formazan produced by the cells by reducing MTS is proportional to the viability of the cells. The mean value of four wells was used per concentration and per experiment. The experiment was performed in triplicates.Metabolic activity was measured using the CellTiter 96P) with the observed effect (EO). EP is calculated according to the formula: EP = Ea + Eb—EaEb. Ea and Eb represent the effects of the single application of the agents. The difference between the predicted and observed effect is the Bliss value, which determines the degree of synergy. Bliss values greater than zero indicate a synergistic effect, equal to zero an additive effect and less than zero an antagonistic effect [Synergistic effects of drug combinations were calculated using the Bliss independence model. This mathematical method is used to compare the predicted effect  and counted with an automatic cell counter . The mean value of three wells was used per concentration and per experiment. The experiment was performed in biological triplicate.The three cell lines were seeded in 6-well plates at a density of 100,000 cells per well in culture medium and allowed to adhere overnight. The cells were exposed to 1 μM, 2 μM and 3 μM FX-9 single application and in combination with 3 μM azacitidine or 20 μM (Adcarc1258)/80 μM (LNCaP) carboplatin. Single applications of azacitidine and carboplatin were tested in aforementioned concentrations. The cells were incubated at 37°C and 5% CO® Analyzer 10  was used for the flow cytometric measurements. Data analysis was performed with the software FlowJo 7.6.5 . The mean value of three wells was used per concentration and per experiment. The experiment was performed in biological triplicate.The apoptosis rate was analyzed by flow cytometry in all three cell lines after 72 hours of compound exposure. Adherent cells and culture medium were collected to analyze the non-attached cells as well. Samples were pelleted and resuspended in 250 μL binding buffer  before being filtered through a 70 μm filter. Subsequently, 2.5 μL Annexin V-FITC (PromoCell GmbH) and 0.5 μL TO-PRO-3 iodide  were pipetted onto each sample. MACSQuant2. After incubation, the cells were harvested by TrypLE™ Express Enzyme  and counted with an automatic cell counter . The mean value of three wells per concentration and per experiment was used. The experiment was performed in biological quadruplicate.The cell lines Adcarc1258, PC-3 and LNCaP were seeded in 6-well plates at different seeding densities /15,000 cells (PC-3)/100,000 cells (LNCaP) per well) due to different growth behavior and exclusion of growth inhibition by cell contacts. Cells adhered over night and were exposed to 10 nM DHT  or with Statistical analysis was performed with SAS Enterprise Guide 7.1 . The values of the measurements were tested for normal distribution. The student’s t-test was used for LNCaP and Adcarc1258, and the Wilcoxon test for PC-3 to calculate p-values in the androgen sensitivity experiment. Dunnett’s t-test was used to calculate p-values in the remaining experiments. p < 0.05 was considered statistically significant.A dose-dependent decrease in viability was observed in all cell lines by FX-9 single application starting at 1 μM/2 μM  and 4 μM (PC-3). The combinations with azacitidine, DCA, doxorubicin and carboplatin resulted in a dose-dependent and cell line-specific significant decrease in cell viability compared to the single application of the two combined agents .In detail, the combination of all tested FX-9 concentrations with azacitidine decreased cell viability significant to the respective DMSO-controls in PC-3, LNCaP and Adcarc1258 . CompareAll tested FX-9 concentrations with DCA reduced cell viability in LNCaP and Adcarc1258 compared to the DMSO control, whereas in PC-3, the reduction started at 3 μM FX-9. The combination with DCA decreased the cell viability significantly only with 2 μM FX-9 compared to FX-9 single application in PC-3. FX-9 combinations 3–5 μM (PC-3)/2-5 μM (LNCaP)/1-5 μM (Adcarc1258) with DCA resulted in a reduction in cell viability compared to the single application of DCA.The combination of all tested FX-9 concentrations with doxorubicin decreased the cell viability to the respective DMSO-controls in PC-3, LNCaP and Adcarc1258 significantly. Doxorubicin with 1–2 μM /1 μM (Adcarc1258) FX-9 caused a lower cell viability than FX-9 single applications. FX-9 combinations starting at 4 μM (PC-3) or 3 μM  resulted in reduced cell viabilities compared to a single application of doxorubicin.The combination of all tested FX-9 concentrations with carboplatin decreased cell viability significant to the respective DMSO-controls in PC-3, LNCaP and Adcarc1258. Carboplatin with 1–4 μM (PC-3)/1-3 μM (LNCaP)/1 μM (Adcarc1258) FX-9 decreased cell viability compared to single application of FX-9. In comparison with the single application of carboplatin, no combination with FX-9 in PC-3 and combination with 2–5 μM FX-9 in LNCaP and Adcarc1258 caused a reduction in cell viability.Bliss values for the combination of FX-9 with azacitidine gave positive values for 1 μM FX-9 on Adcarc1258 and 2 μM, 3 μM, 4 μM FX-9 on PC-3 . For 1 μThe concentrations of FX-9 with azacitidine or carboplatin that significantly decreased cell viability with positive or neutral Bliss values in the human cell lines  were further tested using cell count. As an interspecific comparison, the canine cell line Adcarc1258 was also tested. A dose-dependent reduction in cell count after single application of FX-9 was observed starting at 1 μM FX-9 in Adcarc1258, and at 2 μM FX-9 in PC-3 and LNCaP . The comThe concentrations of FX-9 with azacitidine or carboplatin that significantly decreased cell viability with positive or neutral Bliss values in the human cell lines  were further tested using flow cytometry with annexin and TO-PRO-3 iodide staining. As an interspecific comparison, the canine cell line Adcarc1258 was also tested. FX-9 in combination with azacitidine or carboplatin decreased the fraction of vital cells, and increased the fraction of apoptotic cells in all tested cell lines and concentrations significantly compared to the DMSO-control . The fraIn the human cell line PC-3 and the canine cell line Adcarc1258 there was no significant difference in cell growth between the control and presence of DHT . With LNThe aim of this study was to evaluate at which FX-9 dosage a combination with azacitidine, DCA, doxorubicin or carboplatin could provide synergistic or additive effects on human and canine prostate cancer cell lines. Azacitidine, DCA, doxorubicin or carboplatin were tested in concentrations achievable in plasma –41 and sAzacitidine is an FDA-approved  cytidineCarboplatin, which was approved by the FDA in 1989 , is usedWhen comparing the cell viability and cell counting methods, differences are evident. Looking at the results of the cell count assay, the compounds appear to be significantly more effective at many concentrations than the cell viability results suggest. This effect was distinct in PC-3, as the combination of FX-9 with azacitidine showed 20–50 percentage points higher efficacy in the cell number compared to cell viability. This phenomenon is well known for the response of cancer cells to DNA synthesis-targeting agents . ResponsThe measured synergism was selective regarding concentration and cell line. Synergistic or additive effects occurred dose-dependent at low concentrations of FX-9. It is possible that FX-9 exhibits a dose-dependent mechanism of action, leading to synergism or additivity with azacitidine or carboplatin at low concentrations. There is evidence that antimitotic agents cause drug- and concentration-dependent variation in cell response . PaclitaProstate cancer in humans is usually dependent on androgen and is treated by hormone deprivation therapy. CRPC develops from this disease despite therapy . The twoIn conclusion, FX-9 provides a dose-dependent selective synergistic potential with azacitidine and carboplatin. The cause of this selective potential should be further investigated regarding possible dose-dependent mechanisms of action. The variance between cell lines after exposure to FX-9 supports the tumour-specific use of chemotherapeutic agents for optimal treatment of prostate cancer . FurtherS1 FigPC-3 was stained with Annexin V-FITC and TO-PRO-3 iodide after exposure to FX-9 and azacitidine. Cells in area Q4 were counted as vital, cells in Q3 as apoptotic, and cells in Q1 and Q2 as necrotic.(TIF)Click here for additional data file.S2 FigAdcarc1258 was stained with Annexin V-FITC and TO-PRO-3 iodide after exposure to FX-9 and azacitidine. Cells in area Q4 were counted as vital, cells in Q3 as apoptotic, and cells in Q1 and Q2 as necrotic.(TIF)Click here for additional data file.S3 FigLNCaP was stained with Annexin V-FITC and TO-PRO-3 iodide after exposure to FX-9 and carboplatin. Cells in area Q4 were counted as vital, cells in Q3 as apoptotic, and cells in Q1 and Q2 as necrotic.(TIF)Click here for additional data file.S4 FigAdcarc1258 was stained with Annexin V-FITC and TO-PRO-3 iodide after exposure to FX-9 and carboplatin. Cells in area Q4 were counted as vital, cells in Q3 as apoptotic, and cells in Q1 and Q2 as necrotic.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.


Chronic neutrophilic leukemia (CNL) is a rare myeloproliferative neoplasm that is genetically characterized by the absence of both the Philadelphia chromosome and BCR-ABL1 fusion gene and the high prevalence of mutations in the colony-stimulating factor 3 receptor (CSF3R). Additional disease-modifying mutations have been recognized in CNL samples, portraying a distinct mutational landscape. Despite the growing knowledge base on genomic aberrations, further progress could be gained from the availability of representative models of CNL. To address this gap, we screened a large panel of available leukemia cell lines, followed by a detailed mutational investigation with focus on the CNL-associated candidate driver genes. The sister cell lines CNLBC-1 and MOLM-20 were derived from a patient with CNL and carry CNL-typical molecular hallmarks, namely mutations in several genes, such as CSF3R, ASXL1, EZH2, NRAS, and SETBP1. The use of these validated and comprehensively characterized models will benefit the understanding of the pathobiology of CNL and help inform therapeutic strategies. Chronic neutrophilic leukemia (CNL) is a distinct but rare myeloproliferative neoplasm that is BCR-ABL1 negative. CNL is diagnosed on the basis of neoplastic expansion of granulocytic cells and is also included in the WHO classification of hematological malignancies . EpidemiCSF3R is the receptor for colony-stimulating factor 3 (previously referred to as granulocyte colony-stimulating factor) which is known to play a key role in the growth and differentiation of granulocytes . MutatioThe CSF3R mutations fall into two classes: the so-called truncation mutations (a premature truncation of the cytoplasmic tail of the CSF3R) and membrane proximal mutations (point mutations in the extracellular domain), most commonly T618I ,16. MiceThese data suggest that high-frequency oncogenic mutations in the CSF3R are a defining molecular abnormality of CNL and thus clearly represent a major diagnostic criterion ,3. RecenThe survival of CNL patients is dismal and the therapeutic options are limited and do not exhibit proven disease-modifying benefits . This unPreviously, a pair of cell lines was established from a patient with CNL, albeit at different time points during disease progression ,23. ThesMost importantly, both cell lines carry the telltale CSF3R mutation, specifically the T618I variant B. In addIn summary, a major impediment to further investigation of CNL is the lack of informative and faithful models that allow functional interrogation of driver genes and the impact of the acquisition of additional mutations. To address this knowledge gap, we have highlighted here the existence of such a valuable CNL model in the form of two sister cell lines and have undertaken a global characterization of these cells. The presence of typical molecular hallmarks indicates the use of these cell lines as vital preclinical models in the analysis of CNL pathogenesis and in the search for therapeutics.www.dsmz.de, accessed on 1 January 2021).Sister cell lines CNLBC-1 and MOLM-20 have been established from a patient with CNL. Both cell lines are characterized by a distinct mutation landscape which corresponds to that of primary CNL samples, in particular, they carry the CNL-typical mutations CSF3R, ASXL1, and SETBP1. The cell line MOLM-20 is available from the public cell line repository DSMZ (


The phosphoproteome was profiled to a depth of 2003 phosphopeptides, of which 207, 854, and 1335 were identified in MCF10A, MCF7, and MDA-MB-231 cell lines, respectively. Furthermore, 2450 phosphorylation sites were mapped to 855 distinct proteins, covering a wide range of functions. The identified proteins are associated with several diseases, mostly related to cancer. Among the phosphoproteins, we validated four enzymes associated with cancer and present only in sEVs isolated from MCF7 and MDA-MB-231 cell lines: ATP citrate lyase (ACLY), phosphofructokinase-M (PFKM), sirtuin-1 (SIRT1), and sirtuin-6 (SIRT6). With the exception of PFKM, the specific activity of these enzymes was significantly higher in MDA-MB-231 when compared with MCF10A-derived sEVs. This study demonstrates that sEVs contain functional metabolic enzymes that could be further explored for their potential use in early BC diagnostic and therapeutic applications.Small membrane-derived extracellular vesicles have been proposed as participating in several cancer diseases, including breast cancer (BC). We performed a phosphoproteomic analysis of breast cancer-derived small extracellular vesicles (sEVs) to provide insight into the molecular and cellular regulatory mechanisms important for breast cancer tumor progression and metastasis. We examined three cell line models for breast cancer: MCF10A , MCF7 , and MDA-MB-231 . To obtain a comprehensive overview of the sEV phosphoproteome derived from each cell line, effective phosphopeptide enrichment techniques IMAC and TiO Extracellular vesicles (EVs) derived from human cancer cell lines are involved in multiple biological processes in tumor biology, such as modulation of the microenvironment, angiogenesis, sustained growth, and tissue invasion ,4,5,6,7.In order to elucidate the molecular changes that coincide with cancer or discover new biomarkers, it is important to measure protein expression in EVs, followed by the identification of post-translationally modified proteins such as phosphorylated proteins. Protein phosphorylation is the most important post-translational modification that dominates signaling transduction, which plays an essential role for almost all cellular functions and is involved in major regulatory mechanisms of cell signaling networks . Recent Some studies reported the detection of phosphoproteins in biofluids for disease diagnosis ,19,20. FCell lines are commonly used as models in cancer biology since they are easily grown and relatively inexpensive, making them suitable for high-throughput testing and omics studies . Blood iThe aim of this study was to gain insight into the phosphoproteome of sEVs derived from the metastatic BC cell lines MDA-MB-231 and MCF7 and from the non-cancerous cell line MCF10A. Differences in phosphoproteomes were observed between the control cell line, MCF10A, and BC cell lines, MDA-MB-231 and MCF7. We selected and validated four phosphoenzymes: ATP citrate lyase (ACLY), phosphofructokinase-M (PFKM), sirtuin-1 (SIRT1), and sirtuin-6 (SIRT6) identified in both MDA-MB-231 and MCF7 that might be potentially relevant to BC diagnosis.8 cells.Breast cancer epithelial cell lines MDA-MB-231 (ATCC HTB-26) and MCF7 (ATCC HTB-22) and the non-tumorigenic breast epithelial cell line MCF10A (ATCC CRL-10317) were obtained from the American Type Culture Collection (ATCC) and used in this study. MDA-MB-231 and MCF10A cells were cultured as described in our previous work [g for 10 min. Apoptotic bodies were removed by centrifugation at 2000× g using a Sigma13190 rotor (MBI) for 20 min. Samples were then spun at 16,500× g for 1 h using an SW28 Ti rotor  to deplete microvesicles. To pellet sEVs, the same rotor was used to centrifuge samples for 3 h at 100,000× g. Collected sEVs were washed with PBS and centrifuged at 100,000× g for 1 h and finally resuspended in 200 μL PBS and stored at −80 °C. A Bradford protein assay kit  was used to assess sample protein concentrations. The average amount of EV protein from three replicate samples was roughly 22 ± 5, 27 ± 6 and 34 ± 6 μg for MCF10A, MCF7 and MDA-MB-231 cell lines, respectively. These samples were used for proteomic analysis.Small EVs were isolated from cell culture supernatant by UC, as described previously . ConditiThe average particle diameter and concentration of isolated EV samples were measured by the ZetaView PMX-110 . The camera shutter speeds were adjusted to 85 and 40. The instrument was calibrated and focused with 102 nm polystyrene beads .g, and the supernatant was then collected.Isolated sEVs were resuspended in the lysis buffer with a volume ratio of 4:1 (fraction/buffer) with a final concentration of 20 mM HEPES, pH 8.0, 5% glycerol, 0.1% n-Dodecyl-β-D-Maltoside, 0.2 mM DTT, 1.6 M urea, and 1/250 (v:v) phosphatase inhibitors  and gently vortexed for 3 min. The suspension was centrifuged for 1 min at 10,000× 2-phosphopeptide enrichment was carried out according to the manufacturer’s protocol . All enriched phosphopeptide samples were acidified by adding TFA , subsequently desalted , and dried in a vacuum evaporator.Protein samples obtained from the isolated sEVs were reduced, alkylated, digested, and desalted, as described previously . Phospho2O/1% formic acid (v/v), three microliters of sample was injected onto an in-house packed column and eluted for 65 min at a flow rate of 300 nL/min . The ESI+ parameters were set as follows: top speed mode, ion source temperature 250 °C, ion spray voltage 2.1 kV, and a full-scan MS (m/z 350–2000) resolution of 60,000. For collision-induced dissociation (CID), the automatic gain control (AGC) target was set to 5 × 105 for full scans and 1 × 104 for MS/MS scans. Precursor ions were filtered from +2 to +7 charge states within 2 m/z isolation windows. CID in the linear ion trap was performed at a normalized collision energy of 35%. For the higher-energy collision dissociation (HCD) method, the AGC target for precursor ions was set to 5 × 105 with an ion filling time of 150 ms. The highest intensity ions were isolated and fragmented with a normalized collision energy of 32% and detected at a mass resolution of 15,000. The AGC target for MS/MS was set to 5 × 104 with a maximum injection time of 150 ms and a dynamic exclusion of 30 s.To process samples, the Orbitrap Fusion mass spectrometer  coupled to an UltiMate 3000 nanoRSLC  was utilized, as previously described with modified instrument parameters ,7. FolloMS raw files were analyzed with MaxQuant (version 2.0.1.0)  and the MaxQuant output tables for protein groups, peptides, and phosphosites were used for all analyses in R . We onlyDisGeNET protein–disease annotations were retrieved with the disgenet2r R package . Cancer-All MS raw data were submitted to the PRIDE repository (Accession: PXD030424) at the European Bioinformatics Institute.Cells and sEVs were lysed by gentle vortexing in extraction buffer provided by the ACLY Activity Assay Kit . Supernatants were collected and used for the analyses of enzymatic activities. ACLY Activity Assay Kit was used according to the manufacturer’s instructions. The assays were performed at room temperature for 60 min.The phosphofructokinase activity was measured using kits from Sigma-Aldrich (Phosphofructokinase Activity Colorimetric Assay Kit MAK093) according to manufacturer’s instructions. The assays were performed at room temperature in 100 μL of reaction mixture. The phosphofructokinase activity was calculated via the standard curve of the NADH standard at known concentrations. The samples were mixed and incubated for 30 min, and the absorbance was monitored at 450 nm.+. Fluorescence intensities of SIRT1  and SIRT6  were measured using a microplate reader (FilterMax F3 and F5 Multi-Mode Microplate Readers from Molecular Devices). The activity of enzymes was calculated from the assay time between 5 and 10 min.The enzymatic activity of SITR1 and SIRT6 was assessed by using the Fluorescent Screening Assay Kit  following the manufacturer’s instructions. The protein extract from cells and EVs was obtained by treatment with Triton X-100 . The assay was performed in 96-well black microplate  with a reaction volume of 50 μL per well. Briefly, the reaction was started by incubating the protein extract with the reaction mixture containing an acetylated peptide substrate. Samples were incubated for 30 min at 23 °C. Control samples were prepared in absence of NADSDS electrophoresis and Western blot experiments were carried out as previously described .In this work, we carried out a systematic analysis of the phosphoproteome of sEVs derived from MCF10A, MCF7, and MDA-MB-231 cell lines to provide insight into the molecular mechanism of breast cancer. To evaluate the range of measured diameters of sEVs isolated by differential ultracentrifugation, nanoparticle tracking analysis was performed. Isolated vesicles, from the three cell lines, measured 50 to 300 nm in diameter, with an average size of about 125 nm A. Additi2 affinity chromatography. An overview of our experimental strategy is presented in 2 affinity chromatography from all sEVs , the probability of correct site analysis identified a total of 1613 class I (>75% confidence), 774 class II and III (25–75% confidence), and 63 class IV (<25% confidence) phosphosites, respectively [A total of three biological replicates were prepared from MCF10A-, MCF7-, and MDA-MB-231-derived sEVs and examined independently. EVs were buffered with phosphatase inhibitors, digested, and the phosphopeptides were enriched to enhance the identification of low-abundance phosphorylated proteins. In addition, we combined two techniques for the enrichments of phosphopeptides: Fe-IMAC and TiOall sEVs . In addiWe visualized sequence motifs for the phosphorylated amino acids for each cell line fraction . The majIn addition, we used the GPS 5.0 software  to prediTo determine whether identified phosphoproteins are linked to cancer diseases, we annotated proteins using the recent set of disease annotations from the DisGeNET database . PhosphoWe annotated and classified proteins using the KEGG pathway database. Pathways terms such as metabolic pathway, spliceosome, cell cycle, and viral carcinogenesis were highly abundant in MCF7- and MDA-MB-231-derived sEVs, compared with MCF10A-derived sEVs  Table S. InteresACLY, PKFM, SIRT1, and SIRT6 activities were detected in all extracted protein fractions from the three cell lines, as well as their sEVs . The spe2, and fragmentation methods, CID and HCD, were employed to maximize the purification and identification of phosphopeptides. We identified 2450 phosphorylation sites with 60 novel ones. These phosphorylation sites were mapped to 870 distinct proteins, covering a broad range of functions. Many identified phosphoproteins were unique to BC EVs encompassing a variety of signaling, metabolic, and regulatory pathways and cellular processes. The significant difference in the number of phosphoproteins identified from sEVs isolated from MCF10A and either MDA-MB-231 or MCF7 could be due to MCF10A producing a lower number of sEVs. This is supported by studies showing that MCF10A cells produce significantly fewer sEVs and less protein content than MDA-MB-231 [To understand the molecular and cellular regulatory mechanisms important for BC tumor progression and metastasis, we performed a phosphoproteomic analysis of BC-derived sEVs. In our study, we used sEVs from two breast cancer cell lines, MCF7 and MDA-MB-231, and a non-tumorigenic breast cell line, MCF10A. Two enrichment techniques, IMAC and TiOA-MB-231 . MoreoveIn this study, among all groups of kinases, CMGC, CAMKs, STE, AGC, and TKL were predicted to regulate the largest number of phosphorylation events in sEVs C. KinaseIn total, 266 phosphoproteins were associated with cancer in sEVs derived from MCF7 and MDA-MB-231 cell lines. Among them, 78 are related to breast cancer, suggesting that the cargo of phosphoproteins in sEVs may play an important role in cancer progression and metastasis. Moreover, these identified phosphoproteins could serve as biomarkers for BC diagnosis. Therefore, this study identified nine proteins  previousAdvancements in proteomics facilitate discovery of enzymes that can provide an attractive source for cancer biomarker tests. Due to their unique enzyme specificity and selectivity, enzymatic assays are a reliable, simple, and rapid diagnostic method . A phospACLY is a cytoplasmic homologous tetramer composed of four polypeptide chains and acts as a metabolic enzyme involved in fatty acid synthesis in rapidly proliferating cancer cells ,45,46. AExpression levels of phosphofructokinase-M (PFKM) are closely related to the occurrence and development of malignant tumors. To meet the metabolic demands of tumor cells in energy, the activity of PFKM in cancer cells is increased ,54,55,56Sirtuin-1 (SIRT1) is a class-III histone deacetylase (HDAC) enzyme involved in gene regulation, genome stability, apoptosis, autophagy, senescence, proliferation, aging, and tumorigenesis . This enSirtuin-6 (SIRT6) is involved in multiple molecular pathways related to DNA repair, glycolysis, gluconeogenesis, tumorigenesis, neurodegeneration, and cardiac hypertrophic responses . SIRT6 m2 enrichment and CID fragmentation methods were employed for peptide enrichment and fragmentation; the study was based on cell culture lysates and did not identify the four enzymes presented in our work.Previous studies have investigated the phosphoproteome of BC cell lines, as well as their EVs ,71. HoweThe objective of this study, along with previously published works ,7, was tIn this study, we conducted a phosphoproteomic analysis of breast cancer-derived extracellular vesicles from MCF10A, MCF7, and MDA-MB-231 cell lines. In total, 855 distinct phosphoproteins were identified collectively among the cell lines, covering a wide range of functions, most of which are related to cancer. Among these phosphoproteins, we validated four enzymes: ACLY, PFKM, SIRT1, SIRT6. The results demonstrate that the specific activity of PFKM in BC cancer cell lines was not statistically different from the non-cancerous cell line. In contrast, the three phosphorylated enzymes ACLY, SIRT1, and SIRT6 showed a significantly higher specific enzymatic activity in MDA-MB-231 in comparison to MCF10A-derived sEVs. These three enzymes might serve as a potential prognostic biomarker for BC. They have been previously proposed as therapeutic targets for cancer therapy. Thus, our findings justify further investigation of these enzymes as promising drug targets for BC treatment.


Motions of transmembrane receptors on cancer cell surfaces can reveal biophysical features of the cancer cells, thus providing a method for characterizing cancer cell phenotypes. While conventional analysis of receptor motions in the cell membrane mostly relies on the mean-squared displacement plots, much information is lost when producing these plots from the trajectories. Here we employ deep learning to classify breast cancer cell types based on the trajectories of epidermal growth factor receptor (EGFR). Our model is an artificial neural network trained on the EGFR motions acquired from six breast cancer cell lines of varying invasiveness and receptor status: MCF7 (hormone receptor positive), BT474 (HER2-positive), SKBR3 (HER2-positive), MDA-MB-468 , MDA-MB-231 (TN) and BT549 (TN).The model successfully classified the trajectories within individual cell lines with 83% accuracy and predicted receptor status with 85% accuracy. To further validate the method, epithelial–mesenchymal transition (EMT) was induced in benign MCF10A cells, noninvasive MCF7 cancer cells and highly invasive MDA-MB-231 cancer cells, and EGFR trajectories from these cells were tested. As expected, after EMT induction, both MCF10A and MCF7 cells showed higher rates of classification as TN cells, but not the MDA-MB-231 cells. Whereas deep learning-based cancer cell classifications are primarily based on the optical transmission images of cell morphology and the fluorescence images of cell organelles or cytoskeletal structures, here we demonstrated an alternative way to classify cancer cells using a dynamic, biophysical feature that is readily accessible.https://github.com/soonwoohong/Deep-learning-for-EGFR-trajectory-classification.A python implementation of deep learning-based classification can be found at Bioinformatics online. In response to stimuli, cells adapt a number of strategies to regulate the signaling pathways of transmembrane receptors, including changing the composition of the plasma membrane . ReceptoD) and the linear size of the compartment (L) from the mean-squared displacement (MSD) plots. While MSD analysis is widely used in the field, much information is lost when turning the raw trajectories into an MSD plot (We have previously used the single-particle tracking (SPT) techniques to study the movement of epidermal growth factor receptor (EGFR) in the membrane and showed that the aberrant spatial dynamics of EGFR are associated with the upregulated EphB2 and Src pathways in advanced prostate cancer Liu D and theDeep learning approaches, such as convolutional neural network (CNN), and machine learning approaches, such as k-nearest neighbor, have become an increasingly important tool in image-based tumor segmentation and analysis . Here weWe first acquired EGFR motions from six breast cancer cell lines which have different gene expression profiles, hormone receptor status and surface receptor status: MCF7, BT474, SKBR3, MDA-MB-468, MDA-MB-231 and BT549. The methods for EGFR labeling and trajectory acquisition were described in our previous publications  Kao et . TN subtdt)] were acquired from the six cell lines at 37°C on an Olympus IX-71 inverted fluorescence microscope equipped with a Hamamatsu sCMOS camera  reassembled trajectories as inputs and output a vector containing the probability distributions of predicted classes. The prediction results were visualized in three different ways: a confusion matrix  optimized using stochastic gradient descent (SGD) with categorical cross-entropy as the loss function . By obsen matrix  S7, a ren matrix  S8 and ak − 1 times. Eventually, five networks (k = 5) were trained, and a number of evaluation metrics were obtained based on the testing dataset, providing an assessment of the model performance. Each network generated a set of prediction results, and the model metrics were averaged over the k steps.The model was first evaluated using a testing dataset within the StratifiedKFold from the scikit-learn library. The testing dataset was hidden from the network until each fold had completed training. The StratifiedKFold approach ensured each reassembled trajectory to be used in the testing dataset once and used in the training dataset Based on the testing set of 396 trajectories from the six cell lines , our modThe model was also trained and tested based on receptor status of the six cell lines , we obtained two confusion matrices  that werTo further investigate the capabilities of the trained models, we tested EGFR trajectories from MCF10A cells  with and without the induction of epithelial–mesenchymal transition EMT,  S1. MCF1We demonstrated that well-trained deep learning models based on ResNet  and fromGenevestigator , it can be extended to investigate single-cell behaviors in the tumor microenvironment and tumor heterogeneity (This work was supported by the Cancer Prevention & Research Institute of Texas [RR160005 to T.E.Y.]. T.E.Y. is a CPRIT Scholar in Cancer Research. H.-C.Y. acknowledges the support of this work by Texas 4000, the Robert A. Welch Foundation [F-1833], National Institutes of Health [GM129617] and National Science Foundation [2029266]. Y.-L.L. is a recipient of the YingTsai Young Scholar Award of China Medical University [CMU108-YTY-01] and also a recipient of the Young Scholar Fellowship Program from the Ministry of Science and Technology in Taiwan [MOST 108-2636-E-039-001].Conflict of Interest: none declared.https://github.com/soonwoohong/Deep-learning-for-EGFR-trajectory-classification.The dataset and a python implementation of deep learning-based classification can be found at btab581_Supplementary_DataClick here for additional data file.