Targeted inhibition of aberrant signaling is an important treatment strategy in cancer, but responses are often short-lived. Multi-drug combinations have the potential to mitigate this, but to avoid toxicity such combinations must be selective and given at low dosages. Here, we present a pipeline to identify promising multi-drug combinations. We perturbed an isogenic PI3K mutant and wild-type cell line pair with a limited set of drugs and recorded their signaling state and cell viability. We then reconstructed their signaling networks and mapped the signaling response to changes in cell viability. The resulting models, which allowed us to predict the effect of unseen combinations, indicated that no combination selectively reduces the viability of the PI3K mutant cells. However, we were able to validate 25 of the 30 combinations that we predicted to be anti-selective. Our pipeline enables efficient prioritization of multi-drug combinations from the enormous search space of possible combinations.  •Systematics drug perturbations measurements in isogenic PI3K wild-type and mutant cells•Mutant-specific reconstruction and quantification of signal transduction networks•Short-term signaling response is predictive for longer term cell viability•25 out of 30 model predictions of mutant-specific multi-drug combinations validated Bioinformatics; Pharmacoinformatics; Systems biology; In silico biology The dependency of tumors on activated signaling pathways results in therapeutic responses to inhibitors that block pathway activity . Howeverin vitro testing of all combinations is infeasible. Computational approaches are required to prioritize promising combinations.These findings warrant further exploration of multi-drug combination strategies. This will require a systematic way to explore promising drug combination treatments, including optimizing the dosing of the different drugs. The combinatorial explosion of the search space — there are more than two million possible 4-way combinations of the 89 (as of 2020 ) FDA-appRecently, Nowak-Sliwinska and collaborators presented a “Feedback Systems Control” approach to explore the search space of possible multi-drug combinations . While tH1047R mutation knocked in under its endogenous promoter  and drug combinations were tested with both drugs at their IC50 values, to obtain a total of 34 different perturbations. We measured the response after 2 h of drug treatment (log2 fold change relative to DMSO control) of nine main nodes in the PI3K and MAPK signaling pathway using a multiplexed luminex assay to obtain more than 600 signaling drug-response measurements  (To establish how the PI3Kon (CNR)  of the MWe used the canonical MAPK and PI3K pathway interactions as prior information, and added four edges that were proposed by the CNR algorithm based on hyperparameters selected in a leave-one-out cross-validation loop A and S2AH1047R cells. These differences are highlighted in blue in H1047R (green) cells.  and phospho-ERK (pERK), which had a Pearson correlation with cell viability of 0.36 and 0.42, respectively A. The reWe therefore investigated whether a model combining the response of multiple nodes described the cell viability data better. To this end, we first fitted a linear model using all nodes as predictors We noticed that for both linear models there is a clear structure in the relation between the residuals and the fitted values A and S4B2 fold changes of pERK and pAKT upon drug treatment k. The parameters 2 fold changes of pERK and pAKT that cause 50% inhibition of cell viability. They differ slightly between the two cell lines, but the bootstrapped 95% confidence intervals strongly overlap  show no anti-selectivity, yet this combination is predicted to be one of the most anti-selective ones.  three-drug combinations are likely to have a mutant-specific impact based on a combination of single and two-drug high-dose drug-response measurements and mathematical modeling. We have used drug perturbation experiments to reconstruct, quantify and compare signal transduction networks of an isogenic cell line pair, and linked the responses of these networks to cell viability. No single signaling readout is highly predictive, but a non-linear model combining the response of pERK and pAKT gave a good fit. Importantly, this showed that the short-term signaling response is predictive for cell viability, which is measured in longer term experiments. Based on the so-obtained models, we were able to predict combinations that are specifically effective in one cell line but not another. 25 of the 30 of the combinations that we predicted to be anti-selective indeed were so in validation experiments, despite the fact that the differences between the cell lines in the training data were very subtle. It is to be expected that it will be easier to find mutation-specific drug combinations when the effect of a mutation on the signaling networks and on cell viability is stronger.50) individually show no selectivity toward the parental cells, the combination IGF1Ri + PI3Ki + GSK3i is one of the most anti-selective drug combinations, both as predicted by our model and as measured by validation experiments. This underscores the need for mathematical modeling in prioritizing promising combinations.All of the most strongly anti-selective drug combinations we identified contained the IGF1R inhibitor, but as monotherapy low-dose IGF1Ri is only mildly anti-selective. More generally, which multi-drug combinations are most selective or anti-selective is often far from obvious. For instance, while in the training data PI3Ki and GSK3i at their lower dose -proteins to hundreds of drug perturbations  in largeIn conclusion, here we have shown that it is feasible to make accurate, non-trivial predictions about (anti-)selectivity of multi-drug combinations based on mathematical models of signaling transduction networks. In combination with suitable model systems, this framework makes it possible to rationally design biomarker-selective low-dose multi-drug combinations.H1047R mutation more strongly than their parental counterparts, at least in this particular model system The absence of oncogene-specific sensitivities is presumably due to an absence of “oncogene addiction” .Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Evert Bosdriesz (This study did not generate new unique reagents.H1047R/+ MCF10A cell lines were obtained from Horizon discovery (HD PAR-003 and HD 101-011). Cells were cultured in DMEM/F-12 including 2.5 mM L-glutamine and 15 mM HEPES, supplemented with 5% horse serum, 10 μg/mL insulin, 0.5 μg/mL hydrocortisone and 0.1 μg/mL cholera toxin. Mycoplasma tests were performed every 2 months.Human parental and PI3KS133, EGFRY1068, ERK1T202/Y204, GSK3S21/S9, MEK1S217/S221, p70RSKT389, PRAS40T246 and RPS6S235 were purchased from ProtATonce Ltd. The luminex antibody against AKT1T473 was purchased from BioRad.The following inhibitors were used in this study: EGFRi (Gefitinib), IGF1Ri (OSI-906), RAFi (LY3009120), MEKi (Trametinib), ERKi (SCH772984), GSK3i (3F8), PI3Ki (BKM120), AKTi (MK-2206), mTORi (AZD8055). All inhibitors were purchased from MedKoo Biosciences. The luminex antibodies against CREB150 and IC90 values were fitted using the R-package MixedIC50 (https://github.com/NKI-CCB/MixedIC50).All the cell-viability measurements were performed in biological triplicates, each with 2 technical replicates, using black-walled 384-well plates (Greiner 781091). Cells were plated at the optimal seeding density (200 cells per well) and incubated for approximately 24 hours to allow attachment to the plate. Drugs were then added to the plates using the Tecan D300e digital dispenser. 10 μM phenylarsine oxide was used as positive control (0% cell viability) and DMSO was used as negative control (100% cell viability). Three days later, culture medium was removed and CellTiter-Blue (Promega G8081) was added to the plates. After 2 hours incubation, measurements were performed according to manufacturer’s instructions using the EnVision (PerkinElmer). Viabilities were normalized per cell line according to ixedIC50  . 300K cells per well were plated and incubated for approximately 24 hours to allow attachment to the plate. Drugs were then added to the plates and protein was harvested after 2 hours using the Bio-Plex Pro Cell Signaling Reagent Kit (BioRad 171304006M) according to the manufacturer’s instructions. Protein concentration of the samples was normalized after performing a Bicinchoninic Acid (BCA) assay , according to the manufacturer’s instructions. Cell lysates were analyzed using the Bio-Plex Protein Array system  according to the suppliers protocol as described previously . IntensiH1047R mutant cell lines were reconstructed based on the Luminex drug-response data using Comparative Network Reconstruction (CNR)(2 fold change of node i in response to perturbation k in cell line x) to the matrix of unobserved interaction strengths i with respect to node j in cell line x) and the direct perturbation effects k on node i in cell line x). These matrices are related throughMAPK and AKT signaling networks of the parental and PI3Kion (CNR). Brieflyion (CNR). It linkIn principle, ••Each inhibitor-target pair has a single indicator for the difference in perturbations strengths for both inhibitor concentrations, i.e. if •Most inhibitors are modelled as a perturbation to their direct target, i.e. EGFRi, MEKi, ERKi, GSK3i and AKTi are modelled as perturbations to EGFR, MEK1, ERK1, GSK3 and AKT1 respectively.•The MEK inhibitor interferes not only with MEK phosphorylation, but also with its catalytic efficiency. Hence, MEK inhibition was additionally modelled as a perturbation to its downstream proteins c.f. .•Some inhibitors target kinases that were not measured in our assay. The effect of these inhibitors was modelled as a perturbation to the  downstream nodes of the kinases being inhibited. Specifically, IGF1R inhibition was modelled as a perturbation to MEK1 and AKT1, PI3K inhibition as a perturbation to AKT1, RAF inhibition as a perturbation to MEK1, and mTOR inhibition as a perturbation to AKT1 and p70S6K.Additional constraints reflecting the experimental design were added to the CNR problem.Prior information about network topology was included by setting the indicators of a set of canonical MAPK and PI3K pathway interactions https://github.com/evertbosdriesz/cnr-selective-combos/blob/master/python/cnr-mcf10a-pi3k.ipynb.The full Comparative Network Analysis can be found in the Jupyter notebook under the following link: To obtain the distribution of residuals shown in Similarly, the distribution of residuals shown in The relation between signaling output and cell viabilityFinally, to obtain the distribution of residuals for random differences between the cell lines shown in k, 2-fold changes of pAKT and pERK relative to DMSO control upon perturbation k, respectively. The viability (relative to DMSO control) upon perturbation Equation vk=21+2−Rhttps://github.com/evertbosdriesz/cnr-selective-combos/blob/master/R/02-perturbations/mapping-signaling-drugresponse.Rmd.All code and details for this analysis can be found in the RMarkdown-file under the following link: k, i in response to this, CNR gives an estimate of the direct target inhibition of each drug only for the concentrations at which the drug was applied. To be able to simulate the effect of unseen drug concentrations, the relations between the applied concentration of drug The parameters  package . For con2 fold changes in response to a perturbation with drugs A, B and C, at concentration x relative to y was optimized by solving the following optimization problem:y should have under the 3-drug treatment, and that we (somewhat arbitrarily) set to 0.8. Specifically, to optimize selectivity we use For each possible 3-drug combination, the (anti)-selectivity for cell line Similarly, unselective control combinations were obtained by solving the optimization problem:A power analysis of the predictions was done by performing 1000 simulations with addition gaussian noise with a mean 0 and a standard deviation 0.25  to the results, and counting in what fraction there was an observable difference between the two groups.https://github.com/evertbosdriesz/cnr-selective-combos/blob/master/mathematica/optimize-combinations.nb.The optimizations were performed in Wolfram Mathematica  (version


Thymoquinone has been proved to be effective against neoplasms, including skin cancer. Its high lipophilicity, however, may limit its potential use as a drug. Melanoma remains the deadliest of all skin cancers worldwide, due to its high heterogeneity, depending on the stage of the disease. Our goal was to compare the anti-cancer activity of free thymoquinone and thymoquinone-loaded liposomes on two melanoma cell lines that originated from different stages of this cancer: skin-derived A375 and metastatic WM9. We evaluated the proapoptotic effects of free thymoquinone by flow cytometry and Western blot, and its mitotoxicity by means of JC-1 assay. Additionally, we compared the cytotoxicity of free thymoquinone and thymoquinone in liposomes by WST-1 assay. Our results revealed a higher antiproliferative effect of TQ in WM9 cells, whereas its higher proapoptotic activity was observed in the A375 cell line. Moreover, the thymoquinone-loaded liposome was proved to exert stronger cytotoxic effect on both cell lines studied than free thymoquinone. Differences in the response of melanoma cells derived from different stages of the disease to thymoquinone, as well as their different responses to free and carrier-delivered thymoquinone, are essential for the development of new anti-melanoma therapies. However, further research is required to fully understand them. Melanoma is a malignant neoplasm that arises from melanocytes, the cells that are responsible for the production of melanin, which absorb UV radiation . CutaneoThe achievements that have been made in the systematic treatment of melanoma provide the decrease of the death rates of patients in the USA in recent years. Nevertheless, melanoma is responsible for 0.6% of deaths caused by cancer worldwide, emphasizing the need for investigation of novel therapies . The choThymoquinone (TQ) is a member of monoterpenes, a group of chemical compounds containing one isoprenoid unit, and it is the main component of the essential oil of Nigella sativa (black seed), which for years has been known to display various beneficial properties. In the human body, it is metabolized to thymohydroquinone or to semiquinone, and the latter presents anti-cancer activities, increasing ROS in cancer cells ,9. AlthoIn recent years, even more studies highlight the possible effects of TQ in melanoma. The anti-cancer activity of TQ has been proved to be caused by various mechanisms, including a direct cytotoxic effect, enhancing apoptosis, DNA damage, and ROS formation. These effects have been attributed to the inhibition of Janus kinase 2 (JAK2) and the activator of transcription 3 (STAT3) signaling pathways ,16, as wDespite the advances in research into the effects of TQ on malignant melanoma, there have been few reports showing the effect of this monoterpenoid in human melanoma cells. Additionally, there have been no reports comparing its effects in cells derived from the neoplasms coming from different stages of the disease. Therefore, we evaluated the efficacy of TQ on two human melanoma models: cell culture derived from skin neoplasm and cell culture originated from melanoma nodular metastasis. Additionally, we compared the effectiveness of free TQ with the effectiveness of liposome-encapsulated TQ in the cell lines used. The presented study points out the differences and similarities in response to free TQ and TQ-loaded liposome treatment between the two melanoma cell lines.2, in a humidified atmosphere.A375  and WM9  human malignant melanoma cell lines were cultured in a high-glucose Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum , 2 mM L-glutamine , 1 mM sodium pyruvate , and antibiotics . HEM   were grown in Melanocyte Growth Medium . All the cell lines were incubated at 37 °C, 5% CO4 cells/culture dish) were cultured for 24 h and then treated with free TQ (20 and 40 μM) for 48 h. Alternatively, the cells were pretreated with 10 μM Z-VAD-FMK general caspase inhibitor  for 1 h, before TQ treatment. Cells treated with DMSO were used as a negative control and those incubated with 1 μM staurosporine , as recommended for melanoma cells  × 100%. The readings were acquired from at least 4 independent experiments (each conducted in quadruplicate).The antiproliferative/cytotoxicity activity of free TQ and TQ in liposomes was evaluated using the Cell Proliferation Reagent WST-1 assay . In the present study, A375 and WM9 cells were seeded in 96-well plates .Data are presented as individual samples or mean ± standard deviation. Spearman’s rank correlation coefficient was used to evaluate the associations between TQ concentration and its effects. Differences between the TQ-treated and control groups, as well as differences between the two cell lines studied, were assessed by Mann–Whitney U test. A To assess the response of the different types of melanoma cells to free TQ, A375 and WM9 cell lines were treated with 20 and 40 μM TQ in DMSO for 48 h. The concentrations were chosen based on previously published reports , from whIn order to compare the sensitivity of both studied cell lines to TQ with their sensitivity to a known apoptosis stimulator, the cells were treated with 1 μM staurosporine, an inhibitor of protein kinases, for 48 h. A significantly higher percentage of apoptotic cells was shown for the population of the cells treated with 40 μM TQ than for staurosporine-treated cells, in the A375 cell line, but not in WM9 cells A,B.As far as the apoptotic stages are concerned, the late/early apoptosis ratio was significantly higher only in the A375 cell line treated with 40 μM TQ when compared with the DMSO control, whether or not the all-caspase inhibitor was used A. RegardTo compare the two tested melanoma cell lines in terms of the presence of apoptosis-associated proteins, we examined the expression of the cleaved PARP, an enzyme characteristic of the late stage of apoptosis, in the cells incubated with 5, 10, 20, and 40 μM TQ, by using Western blot A,C and sm) indicator. In healthy mitochondria, with normal ΔΨm, JC-1 is present as J-aggregates showing red fluorescence; whereas in unhealthy or apoptotic cells with low ΔΨm, the dye remains in monomeric form (J-monomers) that fluoresces in green. Consequently, the drop in the red/green fluorescence ratio indicates mitochondrial depolarization [m in both studied cell lines  for 48 h, or with 100 μM FCCP, an oxidative phosphorylation uncoupler, for 4 h (as recommended by the FCCP producer), as a positive control. Mitochondrial function was assessed by using JC-1 dye, a mitochondrial membrane potential . Both samples were characterized by homogeneous size distribution with a PDI of ca. 0.1. The determined zeta potential was −18.4 and −11.4 mV for non-loaded liposomes and TQ-LP, respectively. The negative charge of the liposomal membrane results from the presence of POPG, an anionic phospholipid. The determined values of the zeta potential point out the incipient instability of the system (Kumar and Dixit 2017); however, the measurements repeated 30 days after preparation did not show any particular changes in the liposome size or zeta potential during storage  by a thin lipid film hydration method. The parameters describing liposomal formulations obtained for the study are presented in  storage .The concentration of TQ in the TQ-LP measured by the gas chromatography/mass spectrometry method reached 1100 μg/L. The A375 and WM9 cells were treated with TQ-LP with the final concentration of TQ reaching 1.25 and 2.5 μM after 48 h. For a comparison, both cell lines were also incubated with free TQ (1.25 and 2.5 μM), as well as with a vehicle control (liposome) for the same period of time. A very strong negative relationship between the TQ-LP dose and cell viability was shown in both studied melanoma cell lines  with free TQ  for 24 h. For a comparison, A375 cells were treated analogously. TQ showed a selective antiproliferative effect on A375 cells, with a strong relationship between its dose and cell viability A. No staIn the last few years, there has been a renewed interest in compounds of plant origin as potential anti-cancer drugs, or as components of adjuvant therapy ,25,26,27To complete our knowledge of the effects of thymoquinone on human melanoma cells, we chose two different cell models for our experiments: an A375 cell line, originally isolated from the skin of a patient with malignant melanoma, and WM9 cells, derived from a metastatic site (an axillary node). Our results revealed that free TQ induced apoptosis in a dose-dependent manner, in both analyzed cell lines. Surprisingly, no difference in the percentage of apoptotic cells was observed in the two cell lines treated with TQ following the administration of an all-caspase inhibitor when compared to the cells that had not been pretreated with the Z-VAD-FMK. This, however, might be explained by a possible insufficient caspase blockage by the Z-VAD-FMK, which led to the execution of apoptotic cell death by the low level of active caspases . AlthougAs reported by Hatiboglu et al., TQ induced apoptosis by the blocking of the Janus kinase 2 (JAK2)/signal transducer and the activator of the transcription 3 (STAT3) pathway and, as a consequence, decreased the expression of anti-apoptotic proteins (Bcl-2 and survivin), and increased the expression of pro-apoptotic proteins, such as BAX and caspase 3, in B16F10 murine melanoma cells, within 24 h . Our stuAn important part of the middle apoptosis is mitochondrial outer membrane permeability (MOMP) that leads to membrane depolarization . In thisRegarding general cytotoxicity, TQ was proved to decrease cell viability in a dose-dependent manner in many cancer cell lines, including melanoma ,47,48. J50 value between 2.5 and 5 mg/mL, in A375 cells. The TQ-loaded liposomes used in our study proved to have much more cytotoxic properties than free TQ, with the lowest significant cytotoxicity observed at the concentration of 1.25 μM compared to 40 μM for free TQ. Notably, the cytotoxic effect of TQ-LP was shown for both A375 and WM9 cells, with no significant differences between the two cell lines. The results are consistent with previous reports on the use of TQ-loaded nanoparticles proving the lower toxicity of free thymoquinone in relation to the TQ packed in nanocarriers [The great pharmacokinetic advantage of TQ is its ability to penetrate the blood–brain barrier, due to a low molecular weight. However, to reach the adequate bioavailability of TQ, the usage of nanocarriers might be needed, as the molecule has poor water solubility and high lipophilicity ,51. Althcarriers . The lacTreatment recommendations for melanoma depend on many factors, including surgery, radiotherapy, and chemotherapy , and, usually, assume a systemic drug administration . RecentlIn conclusion, TQ proved to be toxic to both melanoma cell lines studied, with no apparent toxicity to normal melanocytes. Although the proapoptotic effect of free TQ was more evident in the A375 cells when compared to WM9, its antiproliferative activity proved to be stronger in the metastatic cell line. The incubation of A375 and WM9 cells with TQ-loaded liposomes resulted in much higher reduction in cell viability compared to free TQ, with no significant differences observed between the responses of the two cell lines. The knowledge of the differences in the response to free TQ and TQ-loaded nanocarriers of melanoma cells derived from different stages of the disease is crucial for the development of effective anti-cancer therapies. Further research using more in vitro melanoma models is needed to explore this topic.


Aspergillus, Penicillium and Fusarium. They can be synthesized on the field, during harvest as well as during storage. They are fairly stable compounds and difficult to remove. Among several hundreds of mycotoxins, according to the WHO, ochratoxin A, aflatoxins, zearalenone, deoxynivalenol, patulin, fumonisins as well as T-2 and HT-2 toxins deserve special attention. Cytotoxicity is one of the most important adverse properties of mycotoxins and is generally assessed via the MTT assay, the neutral red assay, the LDH assay, the CCK-8 assay and the ATP test in different cell lines. The apoptotic cell ratio is mainly assessed via flow cytometry. Aside from the assessment of the toxicity of individual mycotoxins, it is important to determine the cytotoxicity of mycotoxin combinations. Such combinations often exhibit stronger cytotoxicity than individual mycotoxins. The cytotoxicity of different mycotoxins often depends on the cell line used in the experiment and is frequently time- and dose-dependent. A major drawback of assessing mycotoxin cytotoxicity in cell lines is the lack of interaction typical for complex organisms .Mycotoxins are secondary metabolites of molds and mainly produced by species of the genera  Aspergillus, Fusarium and Penicillium. They can be detected in numerous foodstuffs, including nuts, spices, cereals and fruits, both pre- and post-harvest. Mold growth and, consequently, mycotoxin production can be affected by numerous factors such as weather conditions. The thermal stability and ability of mycotoxins to withstand food processing is one of the main reasons for concerns. Out of several hundreds of mycotoxins most commonly occurring and threatening human and animal health, we highlight patulin, aflatoxin, ochratoxin A, fumonisin, deoxynivalenol, T-2 and HT-2 toxins, zearalenone, citrinin and enniatin [Mycotoxins are toxic compounds mainly produced by species of the genera enniatin .Aspergillus and Penicillium, mainly by Penicillium expansum in rotten fruits and fruit juices. The temperatures at which P. expansum can produce patulin range from 0 to 24 °C [Patulin is a mycotoxin produced by several species of the genera Aspergillus species, mainly by Aspergillus parasiticus and Aspergillus flavus. They can be divided into Aflatoxin B1, B2, G1 and G2. These mycotoxins can be produced during storage as well as on the field, typically in warmer and more humid climates. Intoxication by aflatoxins can lead to immune system suppression, child development impairment, cancer or, in severe cases, death [Aflatoxins are produced by s, death .Penicillium verrucosum, Aspergillus ochraceus and Aspergillus carbonarius [Ochratoxin is a mycotoxin that occurs during storage under inadequate conditions. This mycotoxin is mainly produced by bonarius .Fusarium species, including Fusarium proliferatum and Fusarium verticilioides. Among several fumonisins, fumonisin B1 is the most common one [Fumonisins most commonly occur in dry and warm regions and often contaminate maize. They are mainly produced by mmon one .Fusarium culmorum, Fusarium graminearum and Fusarium nivale. It is generated during growth and harvest periods in relatively humid and cool weather conditions [This mycotoxin is one of the trichothecenes, produced mainly by nditions .Fusarium species. High levels of these toxins are found mostly in developing countries as they require high temperatures and relatively high humidity  to produce mycotoxins [The T-2 toxin and its derivative HT-2 toxin are further examples of trichothecenes produced mainly by cotoxins .Fusarium moniliforme, F. culmorum, F. graminearum, F. oxysporum, F. sporotrichides and F. crookwellence [Zearalenone is largely produced during the growth period in moderate climates with relatively high humidity. The main producers are wellence ,9.Penicillium, Aspergillus and Monascus. It most frequently occurs in rice (where it is responsible for yellow rice disease in Japan), cereals, fruits and cheese [Citrinin is a mycotoxin produced by several species of d cheese .Fusarium and comprised of Enniatin A, Enniatin A1, Enniatin B and Enniatin B1. They can be found in nuts, spices, fruits, cocoa, coffee, several grains as well as their products. Enniatins are considered as emerging mycotoxins [Enniatins are secondary metabolites mainly produced by the genera cotoxins .Cytotoxicity is defined as the ability to harm living cells, causing, among others, protein synthesis disruption or weakening of the cell membrane, ultimately leading to cell death (both necrotic and apoptotic) ,13. CytoSince only living cells can reduce MTT to formazan, the amount of formazan is used to assess the number of living cells . The CCKThe neutral red uptake  assay relies on the ability of living cells to absorb neutral red dye. The dye penetrates cells via non-ionic diffusion and is accumulated in the lysosomes. After washing, the dye is extracted from cells using acidified ethanol and measured spectrophotometrically. This assay relies on the capability of cells to retain a pH gradient which allows the dye to penetrate the cell membrane and retain in the lysosomes. Since only living cells have this ability, the amount of measured dye is directly correlated with the number of living cells . The AlaVarious different cell lines are used for cytotoxicity assessment. The cell lines used in the reviewed literature are summarized in All reviewed articles are summarized in In the case of patulin, MTT was the most frequently used test, and HepG-2 was the most frequently used cell line. Results showed a decrease in cells’ viability, as well as ROS formation increase, cell cycle arrest and p53 gene expression. IC50 values for patulin range from µM to mM, depending on experiment conditions, time of incubation and cell line used x is combination index for n components at x% of cell proliferation inhibitionD)j is the dose of n mycotoxins that causes x% cell proliferation inhibition in the combination(xD)j is the dose of each n mycotoxin individually that causes x% cell proliferation inhibition, a method derived from the median effect principle ,99. The CI < 0.9, CI = 0.9–1.1, CI > 1.1 generally indicate synergistic, additive and antagonistic effects, respectively. Additionally, when the CI indicates a synergistic effect, dose reduction indices (DRI) can be calculated. DRI indicate a fold of reduction of dose of each component at a given effect level compared to a dose of each component individually and can be calculated from equation [DRI is dose reduction indicesmD is median effect doseequation :(2)CIxn=In the case of combinations of two mycotoxins, MTT was the most frequently used test, while HepG-2 was the most frequently used cell line. Results showed that cytotoxicity of binary combinations strongly depend on components . Moreover, a concentration of individual components seems to have an effect on mixture cytotoxicity . Moreover, a concentration of individual components seems to have effect on mixture cytotoxicity . This problem could be partially solved by using primary cell lines instead of continuous cell lines  or by using 3D printed models of tissues to emulate the natural environment better.


B‐cell progenitor fate determinant interferon regulatory factor 4 (IRF4) exerts key roles in the pathogenesis and progression of multiple myeloma (MM), a currently incurable plasma cell malignancy. Aberrant expression of IRF4 and the establishment of a positive auto‐regulatory loop with oncogene MYC, drives a MM specific gene‐expression program leading to the abnormal expansion of malignant immature plasma cells. Targeting the IRF4‐MYC oncogenic loop has the potential to provide a selective and effective therapy for MM. Here we evaluate the use of bromodomain inhibitors to target the IRF4‐MYC axis through combined inhibition of their known epigenetic regulators, BRD4 and CBP/EP300. Although all inhibitors induced cell death, we found no synergistic effect of targeting both of these regulators on the viability of MM cell‐lines. Importantly, for all inhibitors over a time period up to 72 h, we detected reduced IRF4 mRNA, but a limited decrease in IRF4 protein expression or mRNA levels of downstream target genes. This indicates that inhibitor‐induced loss of cell viability is not mediated through reduced IRF4 protein expression, as previously proposed. Further analysis revealed a long half‐life of IRF4 protein in MM cells. In support of our experimental observations, gene network modeling of MM suggests that bromodomain inhibition is exerted primarily through MYC and not IRF4. These findings suggest that despite the autofeedback positive regulatory loop between IRF4 and MYC, bromodomain inhibitors are not effective at targeting IRF4 in MM and that novel therapeutic strategies should focus on the direct inhibition or degradation of IRF4. Constitutive activation of MYC signaling is detected in more than 60% of patient‐derived cells and one of the most common somatic genomic aberrations in MM is rearrangement or translocation of MYC.MYC locus, causing their aberrant proliferation.MYC transcription.One way to target the IRF4‐MYC axis is through upstream epigenetic regulators. Bromodomain and extra‐terminal (BET) proteins inhibitors have emerged as potential therapeutic agents for the treatment of hematologic malignancies.CREB binding protein (CBP) and EP300 are bromodomain‐containing histone acetyltransferases.Given the positive auto regulation loop between MYC and IRF4 in MM, we hypothesized that combining the two classes of inhibitors with distinct transcriptional effects would have a synergistic impact on MM cells. To confirm this, we explored the effect of combinations of BET and CBP/EP300 inhibitors on the viability of a panel of MM cell‐lines. To assess whether the protein and mRNA levels for MYC, IRF4 and their downstream targets following drug exposure were consistent with those expected from the IRF4‐MYC auto‐regulatory loop model, we compared their experimentally measured with their simulated expression in a network model of MM molecular interactions. We found that within the time frames used there is no synergistic effect on the viability of MM cell‐lines. For all inhibitors we experimentally measured largely unaffected levels of IRF4 protein and downstream target protein mRNA levels. These results are consistent with the continued presence of IRF4 protein in MM cells due to its long half‐life. Our network modeling of MM therefore suggests that cell death induced by CBP/EP300 bromodomain inhibition is not exerted directly through IRF4 but indirectly through MYC.22.1® Cell Viability Assay. Each experiment was reproduced 3 times per cell line.Cell viability assay and statistical analysis were performed as described in the supplemental methods. In brief, cell viability after inhibitors treatment was assessed using CellTiter‐Blue2.2β‐actin . HRP‐conjugated secondary antibodies: anti‐rabbit  anti‐mouse .Detailed protocols for western blotting are available in the supplemental methods. Primary antibodies: IRF4 , MYC  and 2.3RNA extraction, cDNA synthesis, and quantitative real time PCR was performed as in the supplemental methods.2.4To measure protein half‐life, cells were treated with 10 μg/ml cycloheximide for up to 72 h followed by western blotting. Detailed protocols are available in the supplemental methods.2.5https://github.com/SiFTW/MMModel/). Regulated reactions were modeled as described previously.Computational models were constructed using Ordinary Differential Equations and solved using MATLAB 2020a and ode15 s. All code, equations and parameters used in modeling are available on Github  and JQ1+SGC‐CBP30 (0.28–0.67 μM). However, treatment with SGC‐CBP30 alone (IC50 1.58–5 μM) and ISOX‐DUAL (2.15–7.70 μM) showed reduced efficacy. The poor inhibitory activity of ISOX‐DUAL could be explained by its reduced affinity for BRD4 and CPB/EP300 (IC50 1.5 and 0.65 μM) when compared to JQ1 and SGC‐CBP30.50 comparable with that of JQ1 alone. Similar results were obtained when treating the cells for 72 h  and one acute leukemia (OCI‐AML3) cells lines were treated for 48 h with different concentrations of these compounds. As shown in Figure h Figure . Taken t3.250 value  and MYC (CDK4 and hTERT) downstream targets. We treated the cells with a concentration of drugs corresponding to their IC50 value for 4, 8, 24, 48 and 72 h  but consistent reduction of IRF4 protein levels in response to treatment with the JQ1+ SGC‐CBP30 combination we then asked whether bromodomain inhibitors work through repression of MYC, IRF4 or both. To do so, we used gene and protein network modeling to simulate the effect of a drug acting on the transcription of MYC, IRF4 or both Figure  using th4In this work we studied the effects on MM cell‐lines of two classes of bromodomain (BET and CBP/Ep300) inhibitors, with putatively distinct transcriptional effects, with the aim to disrupt the oncogenic feedback loop between MYC and IRF4. Specifically, we wanted to evaluate the possibility that the combination of these bromodomain inhibitors would have synergistic impact on the viability of MM cells and on the transcription and protein levels of IRF4 and MYC.Our data showed that while the two BET inhibitors JQ1 and OTX015 showed the most effective inhibition on cell viability, the CBP/Ep300 inhibitor SGC‐CBP/Ep300 and the dual BET‐CBP/Ep300 inhibitor ISOX‐DUAL caused the least effect. Since the combination JQ1+SGC‐CBP30 has a stronger inhibitory effect on cell viability compared to the dual inhibitor alone this suggests that the limited effect of ISOX‐DUAL is caused by its reduced affinity for BRD4 and CPB/EP300. Our data also indicate that combining JQ1 and SGC‐CBP30 does not lead to synergistic or antagonistic cytotoxic effects on MM cell‐lines. In line with previous studies,Our data shows that IRF4 is characterized by a long half‐life in a panel of MM cell‐lines. Previous studies have shown a variability in the half‐life's values for IRF proteins .A growing body of preclinical and clinical evidence suggests that bromodomain inhibition could be an important therapeutic approach in a number of hematologic malignancies.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.https://publons.com/publon/10.1002/hon.3016.The peer review history for this article is available at No ethical approvals were required for the studies conducted in this article.Supporting Information S1Click here for additional data file.Figure S1Click here for additional data file.Figure S2Click here for additional data file.Figure S3Click here for additional data file.Figure S4Click here for additional data file.Figure S5Click here for additional data file.


Andrographis paniculata (Burm.f.) Wallich ex Nees that has anticancer properties in various cancer cell lines. The present study aimed to evaluate andrographolide’s anticancer effectiveness and potential molecular pathways using a DBTRG-05MG cell line. The antiproliferative activity of andrographolide was determined using the WST-1 assay, while scratch assay and clonogenic assay were used to evaluate andrographolide’s effectiveness against the cancer cell line by examining cell migration and colony formation. Flowcytometry was also used to examine the apoptosis and cell cycle arrest induced by andrographolide. The mRNA and protein expression level involved in the ERK1/2/c-Myc/p53 signaling pathway was then assessed using qRT-PCR and Western blot. The protein–protein interaction between c-Myc and p53 was determined by a reciprocal experiment of the co-immunoprecipitation (co-IP) using DBTRG-05MG total cell lysate. Andrographolide significantly reduced the viability of DBTRG-05MG cell lines in a concentration- and time-dependent manner. In addition, scratch and clonogenic assays confirmed the effectiveness of andrographolide in reducing cell migration and colony formation of DBTRG-05MG, respectively. Andrographolide also promoted cell cycle arrest in the G2/M phase, followed by apoptosis in the DBTRG-05MG cell line, by inducing ERK1/2, c-Myc, and p53 expression at the mRNA level. Western blot results demonstrated that c-Myc overexpression also increased the production of the anti-apoptotic protein p53. Our findings revealed that c-Myc and p53 positively interact in triggering the apoptotic signaling pathway. This study successfully discovered the involvement of ERK1/2/c-Myc/p53 in the suppression of the DBTRG-05MG cell line via cell cycle arrest followed by the apoptosis signaling pathway following andrographolide treatment.Human glioblastoma multiforme (GBM) is one of the most malignant brain tumors, with a high mortality rate worldwide. Conventional GBM treatment is now challenged by the presence of the blood–brain barrier (BBB), drug resistance, and post-treatment adverse effects. Hence, developing bioactive compounds isolated from plant species and identifying molecular pathways in facilitating effective treatment has become crucial in GBM. Based on pharmacodynamic studies, andrographolide has sparked the interest of cancer researchers, who believe it may alleviate difficulties in GBM therapy; however, it still requires further study. Andrographolide is a bicyclic diterpene lactone derived from  Andrographis paniculata and is widely used as a traditional medicine in Malaysia, Indonesia, China, and India to treat diabetes and malaria [20H30O5 and 350.4 g/mol [Glioblastoma multiforme (GBM) is the deadliest and most difficult to treat brain cancer disease. Patients diagnosed with GBM have a one-year survival rate because of the limitations of current treatments, which have unfavorable side effects on patients and make it difficult to remove the tumor from a sensitive area of the brain . In addi malaria . AndrogrMore interestingly, andrographolide is a potential drug candidate for the treatment of any diseases related to the central nervous system (CNS) because it has a neuroprotective effect in the CNS by preserving the neuronal structure and function . It is aAccording to the literature, andrographolide can be developed further as an anticancer drug to treat cancer cells through various mechanisms such as apoptosis, autophagy, cell adhesion control, proliferation, migration, invasion, anti-angiogenic activity, and other miscellaneous acts . FurtherThe C-myelocytomasis (c-Myc) oncogene is a transcriptionally encoded gene that regulates the expression of over 100 target genes involved in apoptosis, cell cycle arrest, cell growth, survival, and differentiation . C-Myc iMoreover, the development of a new drug that can directly target c-Myc still needs to be explored because many researchers have attempted to explain the molecular mechanism of a new drug involved in activating c-Myc in human cancer. After all, information and knowledge regarding the expression and possible interaction of c-Myc in cancer remain insufficient. This review aims to provide information and knowledge related to c-Myc in the development of GBM. This study also aimed to determine the interaction between andrographolide and c-Myc, as well as how andrographolide is involved in upstream and downstream c-Myc signaling pathways in the human brain cell line, as there is no evidence of this having been yet studied. Then, this study aimed to investigate whether c-Myc or c-Myc’s target genes can be targeted by andrographolide for cancer therapy.50 values were determined at 13.95 µM for 72 h, which demonstrated the greatest cytotoxic effects and were chosen as the main active dose and time in the subsequent experiments. The LC50 values for the normal cell line cannot be computed because, as shown in 50 values below 5.8 μM at 24 h of incubation (Human brain cancer cell lines (DBTRG-05MG) and normal brain cell lines (SVGp12) were exposed to nine different concentrations of andrographolide (0.781–200 µM) for 24 h, 48 h, and 72 h A,B. The cubation B. The effect of andrographolide on cell migration changes of the DBTRG-05MG cell line was assessed using the scratch assay. 50 values) and 27.9 µM  of andrographolide at 72 h was observed. To confirm the effect of andrographolide in reducing cell viability of DBTRG-05MG cell lines, the colony formation of cell lines treated with 13.95 µM (LC50 (8.60%), and 2LC50 (8.95%). This study reveals that andrographolide induced cytotoxic effects through G2/M phase arrest in a concentration-dependent manner.Cell cycle arrest was determined by staining the DNA of apoptotic DBTRG-05MG cells with propidium iodide after treatment with different andrographolide concentrations (13.95 μM and 27.9 μM) compared to the control cell. The apoptosis analysis was elucidated through the flow cytometry technique using double staining with annexin V-FITC and PI. The scattered diagram was obtained from flow cytometry analysis of cancer cell lines after exposure to different andrographolide concentrations (13.95 μM and 27.9 μM) compared with the control cell C. There 50: 13.95 μM) and high (2LC50: 27.21 μM) concentrations of andrographolide, the relative expression of c-Myc significantly increased from 1.12-fold to 5.73-fold. The relative expression of ERK1/2 and p53 were also increased from 1.72-fold to 2.87-fold and from 1.48-fold to 2.25-fold, respectively, compared to untreated DBTRG-05MG cells. This finding was further analyzed via Western blot to determine the protein expression level of c-Myc, ERK1/2, and p53. 50: 13.95 μM) and high (2LC50: 27.21 μM) concentrations of andrographolide were increased, respectively. The upregulation of c-Myc markedly increased the expression of p53. Furthermore, c-Myc was implied to interact with p53 constitutively, and this interaction can be evaluated through co-IP.The effect of andrographolide on the relative expression of ERK1/2, p53, and c-Myc in DBTRG-05MG cells was evaluated by Quantitative Real-Time Polymerase Chain Reaction analysis (qRT-PCR). In The study on the interaction or relation between c-Myc and p53 protein, co-IP, and Western blot analysis was performed accordingly. The reciprocal co-IP assay was performed with the total protein lysate from the DBTGRG-05MG after exposure to the andrographolide. The current anticancer drugs in conventional treatments are becoming resistant to cancer, particularly in GBM, which is one of the uncommon brain cancers produced from glial cells of the central nervous system (CNS). GBM is also the world’s leading cause of death due to the limitations of current conventional therapies such as surgery, radiation, and chemotherapy . This ha50 of 13.95 μM at 72 h. This finding can be supported by a previous study that tested andrographolide using various cancer cell lines with LC50 values ranging from 2.25 to 44.25 µM [50 value 13.95 μM, obtained from this study, was in the acceptable range reported by the previous study. This result also indicated that andrographolide showed more cytotoxicity toward cancer cell lines than a normal cell line. However, these studies remain controversial because no previous study was reported on andrographolide’s effectiveness in reducing the cell viability of the DBTRG-05MG cell line. This is because many studies used other types of cell lines to mimic the GBM diseases, such as Uppsala 87 Malignant Glioma (U-87 MG) [In this study, the andrographolide’s effectiveness in DBTRG-05MG cell lines was assessed by a different assay that covers cytotoxicity, proliferation, and the molecular mechanism of cell death. The cell cytotoxicity and proliferation of DBTRG-05MG were successfully determined by WST-1, scratch, and clonogenic assays. This present study found that andrographolide significantly suppressed the cell viability of the DBTRG-05MG cell line in a time- and concentration-dependent manner. The cytotoxicity of DBTRG-05MG cell lines towards andrographolides was higher compared to SVGp12 normal cell lines with an LC44.25 µM . Hence, U-87 MG) , GBM8401U-87 MG) , which sThen, this study was continued with the clonogenic assay, which showed that andrographolide was able to inhibit the cell proliferation of DBTRG-05MG in a concentration-dependent manner by reducing the colony formation of cell lines over time. A few studies were conducted to evaluate the effect of andrographolide on the colony formation of human glioblastoma cell lines that involved a different type of cell line, such as U-87 MG . The preThis study also revealed that andrographolide induced a cytotoxic effect on the DBTRG-05MG cell line through G2/M cell cycle phase arrest followed by apoptosis in a concentration-dependent manner. The percentage of cells arrested at the G2/M phase in DBTRG-05MG cells after treatment of andrographolide at 13.95 μM for 72 h increased significantly compared with the control. Then, the apoptotic cell increased in the DBTRG-05MG cell line after andrographolide of 13.95 μM treatment for 72 h. According to our findings, andrographolide successfully suppressed the cell proliferation of the DBTRG-05MG cell line and was arrested at the G2/M phase, followed by apoptosis. Then, to gain information on the molecular mechanism involved in the cell cycle arrest and cell apoptosis after the DBTRG-05MG cell line was treated with the andrographolide, the ERK1/2/c-Myc/p53 signaling pathway was investigated in this study. This study was successful in detecting the mRNA and protein expression levels of c-Myc, ERK1/2, and p53 genes via qRT-PCR and Western blot analysis. Our finding successfully found that the upstream and downstream regulator genes of the c-Myc signaling pathway involved ERK1/2 and p53. The mRNA and protein expression level of ERK1/2 increased accordingly after andrographolide treatment compared with control. This finding was consistent with the previous finding that reported andrographolide can interact and activates ERK1/2 . The preOur findings showed that the expression of ERK1/2 was upregulated in DBTRG-05MG along with the upregulation of the c-Myc and p53 after andrographolide treatment. However, another study reported that andrographolide treatment suppressed lung cancer cell growth by downregulating c-Myc expression . Our finOur findings were extended with a co-IP analysis to assess the interaction of c-Myc with p53 via this signaling pathway. After being treated with andrographolide, c-Myc and p53 were able to form a protein complex in DBTRG-05MG; both proteins were bound together to initiate an apoptotic signaling pathway. The positive interaction between c-Myc and p53 also was discovered in the nasopharyngeal carcinoma (NPC) cell line for apoptosis . Then, aBased on co-IP results, c-Myc displayed a single band A, whereaOverall, evidence suggests that andrographolide at low concentrations induced cell cycle arrest and apoptosis via the c-Myc signaling pathway. This finding suggested that ERK1/2 acts as an upstream regulator, enhancing the activation of the c-Myc signaling pathway by modulating the activity of apoptotic proteins such as p53. Andrographolide, temozolomide (anticancer drug), and crystal violet were supplied by Sigma–Aldrich (USA). Andrographolide and temozolomide were dissolved in DMSO and kept at −20°C until needed. RPMI 1640, DMEM, trypsin-EDTA, and FBS were purchased from Gibco (USA). Penicillin–streptomycin was purchased from Nacalai Tesque (USA). WST-1 -2-(4-iodophenyl)-3-(4-nitrophenyl)- 2H-tetrazolium inner salt) was purchased from Roche (Germany). The PI/RNase staining buffer and Annexin V-FITC Apoptosis Detection Kit were obtained from BD Pharmingen (USA) and Invitrogen (Austria).v/v) Fetal Bovine Serum  and 1% penicillin–streptomycin . The cells were maintained in standard cell culture conditions, a humidified incubator with 5% CO2 at 37 °C.Human glioblastoma cell lines DBTRG-05MG and normal glial cell lines SVGp12 were supplied by the American Type Culture Collection (ATCC), USA. Both cell lines were used in this study and were cultured in complete RPMI and DMEM supplemented with 10% , with the final concentration of DMSO adjusted to 0.2% for all concentrations of andrographolide. After incubation, the old medium was discarded, and the cell was washed using PBS. Then, 100 µL of the sterile medium of complete RPMI was supplemented with 10% (v/v) Fetal Bovine and 1% penicillin–streptomycin, and each well-received 10 µL of cell proliferation reagent WST-1 . The plates were incubated in 5% CO2 at 37 °C for 1 h, and the absorbance (Abs.) was measured using the ELISA reader (Multiscan Spectrum) at a wavelength of 450 nm with a reference reading of 630 nm. The optical density values from vehicle-control-treated cells with 0.2% DMSO were considered as 100% of cell viability. The experiments were triplicated to check the consistency. The LC50 values were computed using the GraphPad Prism 8.0.1 software by plotting a cell viability versus concentration graph. The percentage of cell viability was calculated using the formula below:The cytotoxic effect of andrographolide was assessed by WST-1 assay in DBTRG-05MG and SVGp12 cell lines. Both cell lines were seeded into 96-well plates at 6 × 105 cells/ml. Cell lines were cultured in 5% CO2 at 37 °C overnight to allow them to adhere to the plate. After cells reached confluency at 60–70%, the plate was scratched accordingly with 1 mL pipet tips, and the cell was washed once with PBS. Then, the cells were given treatment with an LC50 concentration of andrographolide (13.95 µM) and without andrographolide that was only treated with 0.2% of DMSO as vehicle control. The treated cells were incubated in 5% CO2 at 37°C for 24, 48, and 72 h. The following incubation cell was fixed and stained with 0.5% of crystal violet in 25% methanol for 1 h. The crystal violet was rinsed with tap water and dried on plates for a few days at room temperature. The plate was viewed and photographed on an inverted microscope at ×10 magnification. The wound area was evaluated using ImageJ software.The DBTRG-05MG were harvested and seeded in 6-well plates at 4 × 105 cell/mL of cell density for each well. Then, the cells were incubated in 5% CO2 at 37°C overnight to allow them to attach to the plate. The cells were treated with varying concentrations of andrographolide (13.95 µM and 27.21 µM) and without andrographolide that was only treated with 0.2% of DMSO as vehicle control. The treated cells were cultured in 5% CO2 at 37 °C for 72 h. After incubation, the old medium was removed and the cells were rinsed with PBS. The fixation solution (acetic acid/methanol 1:7 (v/v)) was added to the plate and incubated for 15 min at 25 °C (room temperature). Following the removal of the fixation solution, 1 mL of 0.5 percent crystal violet in 25% methanol was applied to the plates. The plates were placed for 1 h at 37 °C in a 5% CO2 incubator. The crystal violet was removed by immersing in tap water to rinse it off. The plates were allowed to air dry on a tablecloth at room temperature for a few days and were further observed, and images were recorded for the stained monolayer. The formation of the colony was counted and analyzed with ImageJ software. The plating efficiency (PE) and surviving fraction (SF) were calculated by using the formula below:The clonogenic assay was performed to evaluate the effectiveness of andrographolide toward DBTRG-05MG by observing colony formation after treatment. The DBTRG-05MG was harvested using trypsin-EDTA and seeded in 6-well plates with 4 × 104 cells/mL. Then, the cells were placed in a 5% CO2 incubator at 37 °C overnight to allow them to attach to the flask. The cells were treated with varying concentrations of andrographolide corresponding to LC50 (13.95 µM)/2LC50 (27.21 µM) and without andrographolide that was only treated with 0.2% of DMSO as vehicle control. The treated cells were cultured in a 5% CO2 incubator at 37 °C for 72 h. Cell cycle arrests were assessed by PI/RNase staining buffer. The treated cells were harvested, fixed, and permeabilized with 70% ice-cold ethanol overnight. Then, the fixed cells were centrifuged to remove ethanol and washed once with ice-cold PBS. The cell pellet was resuspended in 0.5 mL of PI/RNase staining buffer and incubated for 15 min at room temperature. The cell cycle distribution was analyzed using BD FACS CantoTM Flow Cytometer  using MoD fit software , while apoptosis assay was assessed using annexin V-FITC/PI double staining. Cell pellets were obtained after treatment were collected and re-suspended with 0.1 mL of 1× Annexin V Binding Buffer (AVBB), 5 µL of annexin V-FITC, and 5 µL of PI staining added in the tube. The cells were incubated in the dark at room temperature for 30 min. Flowjo software was used to analyze the stained cells using a BD FACS CantoTM Flow Cytometer .The DBTRG-05MG were cultured in a T25 flask at a concentration of 20 × 1050 (13.95 µM)/2LC50 (27.21 µM) and without andrographolide that was only treated with 0.2% of DMSO as vehicle control. According to the official guidelines, the AurumTM Total RNA Mini Kit  was used to isolate total RNA from DBTRG-05MG cells. The NanoDrop 2000/2000c spectrophotometer was used to measure the RNA concentration . Electrophoresis on a 1% agarose gel was used to evaluate the integrity of total RNA. For use in qRT-PCR later, total RNA was kept at 80 °C.Total RNA was extracted from the DBTRG-05MG cell line treated with varying concentrations of andrographolide correspondent to LCThe qRT-PCR was carried out using a Real-Time PCR machine, an iTaq Universal SYBR Green One-Step Kit on Qiagen Rotor-Gene Q-Pure detection . All reactions on each instrument were optimized to obtain the best amplification kinetics. The RNA template of each sample representing one biological triplicate was diluted to a working concentration of 30 ng/µL for the qRT-PCR analysis. The reaction setup and program thermal cycling protocol were followed according to the manufacturer’s protocol. A 5-point 10-fold serial dilution standard curve of RNA was used to generate a standard curve, while PCR efficiency (E) values ranging from 96.67 % to 109.04% were calculated over all primer sets with average correlations (R2) of 0.99. Primers used in this study were designed using NCBI/Primer-Blast and Primer3Plus as follows: GAPDH, FW 5’-ACATCGCTCAGACACCATG-3’ and RW 5’- TGTAGTTGAGGTCAATGAAGGG-3’; ERK1/2, FW 5’-CATTCAGCTAACGTTCTGCAC-3’ and RW 5’-GTGATCATGGTCTGGATCTGC-3’; c-Myc, FW 5’- TCCTCGGATTCTCTGCTCTC-3’ and RW 5’-TCTTCCTCATCTTCTTGTTCCTC-3’; p53, FW 5’- GACACGCTTCCCTGGATTG-3’ and RW 5’-GACGCTAGGATCTGACTGC-3’. The housekeeping glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference gene for comparing the relative expression of cDNA using the 2-△△CT method.50 (13.95 µM)/2LC50 (27.21 µM), along with cells treated with 0.2% of DMSO as vehicle control. The total cell lysate was isolated from DBTRG-05MG cells using the RIPA lysis buffer  according to the manufacturer’s instructions. The cells, both treated and untreated, were collected and washed twice in ice-cold phosphate-buffered saline (PBS). After, 1 mL 1× RIPA lysis buffer containing protease inhibitor cocktail was added to the cell pellet with occasional vortex. The lysed suspension was incubated for 30 min on ice and the following centrifugation at 10,000× g for 10 min at 4 °C was performed. The cell supernatant was transferred into new tubes immediately for further use in Western blot analysis and immunoprecipitation assays.The total cell lysate was extracted from the DBTRG-05MG cell line treated with varying concentrations of andrographolide correspondent to LCg. Total cell lysates obtained from the previous method were pre-cleared with 50 µL protein G Sepharose beads and incubated at 4 °C for 1 h, followed by gentle mixing using a rotating mixer. The pre-cleared lysate was recovered by centrifugation at 1200× g for 5 min and precipitated with the protein of interest antibody (Ab). The 5 µL of c-Myc Ab was added into pre-cleared lysate and incubated overnight at 4°C with moderate mixing on a rotating mixer. The immune protein complex was recovered by centrifugation at 1200× g for 5 min at 4 °C, then gently resuspended with cold lysis solution 3 times. The immune protein complex was recovered by centrifugation at 1200× g for 5 min at 4 °C, and the protein concentration was quantified by Bradford assay using a Bio-Rad protein assay kit. The immune protein complex was probed by Western blot analysis.The protein G Sepharose beads  were activated by rinsing three times with 1000 µL of lysis buffer and centrifuging at 150× TM ECL Western Blotting Substrate (Thermo Fisher Scientific) and were developed in a dark room. The relative density of the interest protein band to the control protein (α-tubulin) was analyzed with ImageJ.The equal amount of denatured protein sample (30 µg) for each well was resolved on the 12% polyacrylamide gels and transferred to Bio Trace™ NT Nitrocellulose Transfer Membrane (PALL) by electroblotting. The membrane was blocked with 5% skim milk (Sun Lac) in 0.05% TBS-T for 1 h and stored at 4 °C. The membranes were then washed three times with TBST for five minutes each time before being incubated overnight at 4 °C with primary antibodies anti-p53 (1:500), anti-c-Myc (1:500), and anti-tubulin (1:500). After that, the membranes were probed with HRP-conjugated secondary antibodies for 2 h at 4 °C. The signal protein bands were detected using a Piercep ≤ 0.05 was accepted as statistically significant.All the experiments were performed in triplicate, and the results were expressed as mean ± standard error of the mean (SEM). Other data were analyzed in GraphPad Prism version 8.0.1 using a two-way analysis of variance (ANOVA), proceeded by the Student’s t-test and Tukey’s test of one-way analysis. The This study successfully demonstrated the role andrographolide in inducing a cytotoxic effect in DBTRG-05MG cell lines in a time- and concentration-dependent manner. Further, andrographolide was found to reduce colony formation and delayed cell migration into the wound area. This study also evidenced that andrographolide induced a cytotoxic effect via G2/M phase arrest followed by apoptosis via the ERK1/2/c-Myc/p53 signaling pathway in a concentration-dependent manner. To our knowledge, this is the first work to show the mechanisms of andrographolide cell death in the DBTRG-05MG cell line. Because of its efficacy in inhibiting the DBTRG-05MG cell line, andrographolide also has the potential to be one of the anticancer treatments for glioblastoma multiforme disease. However, further study needs to be performed to evaluate the cytotoxicity of andrographolide in vivo and in vitro. The function of other regulatory genes that may interact with the c-Myc signaling pathway must also be investigated intensively.


Insulin resistance (IR) plays a key role in gestational diabetes mellitus (GDM) pathogenesis. The antiaging protein klotho has been proven to be closely related to IR. The purpose of this study was to investigate the effect of klotho on IR in GDM trophoblast cells.  The GDM cell model of HTR-8/SVneo cells was induced by high glucose (HG). Plasmid transfection was used to mediate the overexpression or silencing of klotho. The effects of klotho on cell viability, IR, and the IGF-1/PI3K pathways were observed by RT-qPCR, western blot, Cell Counting Kit-8 detection, glucose uptake assay, and immunofluorescence detection. α, INSR-β, IRS1, IRS2, and GLUT4), and glucose uptake in HTR-8/SVneo cells of the HG group. In addition, the overexpression of klotho inhibited the levels of IGF-1, IGF-1R/p-IGF-1R, and the phosphorylation and activation of the signal transduction molecules PI3K/Akt/mTOR. On the contrary, klotho deletions could reverse these changes of HTR-8/SVneo cells induced by HG. Conclusion. In a word, the results of this study showed that the regulation of klotho played an important role in the IR of trophoblast cells induced by HG, which was mediated at least in part by the IGF-1/PI3K/Akt/mTOR pathway. Klotho expression was up-regulated in HG-induced cells. Overexpression of klotho could reduce the cell viability, insulin signaling molecules (INSR- Gestational diabetes mellitus (GDM) is one of the most common complications of pregnancy, which refers to the abnormal glucose tolerance that occurs during pregnancy or is first discovered . GDM oftThe intracellular signal of placental insulin is mediated by stimulating its tyrosine kinase receptor and activating insulin receptor substrate -1 (IRS-1) . Activatβ-klotho increased in the GDM placenta, which could be one of the reasons for IR -2-deoxy-glucose (2-NBDG) . Briefly, the cells were cultured in a medium with a 100 The expression and distribution of p-IGF-1R were detected by immunofluorescence staining. Cells were pre-seeded on the slide and fixed with 4% paraformaldehyde for 15 minutes. After scouring with PBS to remove residual paraformaldehyde, the cells were infiltrated with 0.1% TritonX-100 for 30 minutes to permeate the cell membrane. Then, goat serum was used to block the nonspecific antigen for 15 minutes and spent the night with the anti-p-IGF-1R antibody  at 4°C. After washing with PBS, the cells were incubated with secondary bodies anti-Rabbit IgG (H + L) for 90 minutes. The nucleus was incubated with DAPI. Finally, the cells were sealed with buffered glycerin and stored away from light for observation under a fluorescence microscope .t-tests were used for the statistical analysis between the two groups, and one-way analysis of variance (ANOVA) determined three or more groups. p < 0.05 was considered significant.The statistical analysis was performed by using the GraphPad Prism 8 software. All data were represented as the mean ± SD. 'Student's To study the important influence of klotho on GDM trophoblast cells, we simulated the HTR-8/SVneo cell model by HG induction. RT-qPCR was performed to test klotho levels in HTR-8/SVneo cells . The resα, INSR-β, IRS1, and IRS2. The results showed that GLUT4, INSR-α, INSR-β, IRS1, and IRS2 levels in trophoblast cells under HG significantly decreased than in the control group  were significantly decreased in HG-induced HTR-8/SVneo cells, which is consistent with previous studies [Many studies showed that IR played an important role in GDM , 27. At  studies . Moreove studies , which wStudies have shown that klotho-mediated insulin metabolism and aging are related to IR , 31, 32.β signaling pathway [It was reported that klotho inhibits the IGF signaling pathway, induces SOD expression to reduce oxidative stress, and inhibits the Akt-mTOR signaling pathway to inhibit abnormal growth of the kidney . Klotho  pathway . In addi pathway . Inhibit pathway . The upr pathway .Our study found that the expression of IGF-1R, p-IGF-1R, and IGF-1 was significantly increased in the HG-treated HTR-8/SVneo cells after silencing klotho. Over-expression of klotho significantly reduced the levels of these proteins. It is consistent with previous studies . We furtIn a word, the results of this study showed that silencing of klotho reversed the decrease of cell activity induced by HG and alleviated insulin resistance. In addition, these results were consistent with klotho's view that it could inhibit the activation of IGF-1 and PI3K-Akt-mTOR signals to promote IR. This study proved that klotho might regulate trophoblast cells' IR under HG conditions, which indicated that klotho might be a promising target for treating GDM.


PSEN1, which cause familial Alzheimer’s disease alter the processing of amyloid precursor protein, leading to the generation of various amyloid-β peptide species. These species differ in their potential for aggregation. Mutation-specific amyloid-β peptide profiles may thereby influence pathogenicity and clinical heterogeneity. There is particular interest in comparing mutations with typical and atypical clinical presentations, such as E280G. We generated PSEN1 E280G mutation induced pluripotent stem cells from two patients and differentiated them into cortical neurons, along with previously reported PSEN1 M146I, PSEN1 R278I and two control lines. We assessed both the amyloid-β peptide profiles and presenilin 1 protein maturity. We also compared amyloid-β peptide profiles in human post-mortem brain tissue from cases with matched mutations. Amyloid-β ratios significantly differed compared with controls and between different patients, implicating mutation-specific alterations in amyloid-β ratios. Amyloid-β42:40 was increased in the M146I and both E280G lines compared with controls. Amyloid-β42:40 was not increased in the R278I line compared with controls. The amyloid-β43:40 ratio was increased in R278I and both E280G lines compared with controls, but not in M146I cells. Distinct amyloid-β peptide patterns were also observed in human brain tissue from individuals with these mutations, showing some similar patterns to cell line observations. Reduced presenilin 1 maturation was observed in neurons with the PSEN1 R278I and E280G mutations, but not the M146I mutation. These results suggest that mutation location can differentially alter the presenilin 1 protein and affect its autoendoproteolysis and processivity, contributing to the pathological phenotype. Investigating differences in underlying molecular mechanisms of familial Alzheimer’s disease may inform our understanding of clinical heterogeneity.Mutations in the presenilin 1 gene,  et al., generate patient-derived induced pluripotent stem cells containing the Presenilin 1 E280G mutation. The E280G lines displayed a high relative production of Abeta43, a finding matched in post-mortem tissue with the same mutation. This mutation also disrupts presenilin 1 protein maturity. Together these findings indicate mutation-specific pathogenic mechanisms.Willumsen   However, FAD is caused by rare autosomal dominant mutations in the presenilin 1 (PSEN1), PSEN2 or amyloid precursor protein (APP) genes and APP duplications.4–9Alzheimer’s disease is defined by the cerebral accumulation of pathological misfolded proteins into amyloid beta (Aβ) plaques and neurofibrillary tangles containing tau.11 In the amyloidogenic pathway, APP is cleaved by β-secretase (BACE1) releasing the sAPPβ fragment.12 The remaining membrane-bound C99 fragment undergoes an initial epsilon cleavage, releasing an APP intracellular C domain and a membrane-bound Aβ peptide. Sequential carboxypeptidase-like cleavages of Aβ by the γ-secretase complex releases Aβ peptides of varying length.13 Epsilon Aβ cleavage occurs at residue 48 or 49, giving rise to two cleavage pathways of decreasing peptide length (Aβ49 > 46 > 43 > 40 and Aβ48 > 45 > 42 > 38).14 In PSEN1-FAD, there is a loss of processivity leading to qualitative shifts towards longer Aβ peptides.15The presenilin 1 protein (PSEN1) is the catalytic component of the γ-secretase complex, responsible for the cleavage of APP.16 Studies have revealed that specific FAD mutations produce very distinct Aβ ratios, as shown in both iPSC differentiated into neurons17 and in other cell-based and activity assay models,18–20 recapitulating aspects of disease in vitro. Importantly, these Aβ ratios are likely to reflect early perturbations in Aβ processing, revealing early disease mechanisms.Recently, there have been multiple papers that have generated induced pluripotent stem cells (iPSCs) from FAD and Down’s syndrome patient fibroblasts, reviewed in.PSEN1, most pathogenic PSEN1 mutations cause an increase in the ratio of Aβ42 to total Aβ, and notably an increased Aβ42:40 ratio.21 However, the mechanism by which ratios are altered differs by mutation. Specific PSEN1 mutations affecting γ-secretase activity can influence both absolute amounts of Aβ and the Aβ cleavage pathway selection, leading to altered ratios of the peptides within those pathways.22Compared with wild-type (WT) PSEN1 mutations appear to consistently hinder sequential carboxypeptidase cleavage, leading to the release of longer Aβ peptides, alterations of which can be measured via the Aβ42:38 and Aβ43:40 ratios. This results in a relative increase in, and release of, longer Aβ peptides which are more prone to aggregation,22 in turn leading to more oligomeric Aβ and greater extracellular deposition.23 Mutations that severely compromise γ-secretase carboxypeptidase activity affect residues that map near the proposed γ-secretase active site.24Pathogenic 25 Mutations near the endoproteolytic site are proposed to inhibit endoproteolysis,26 which can lead to altered processivity.27 Interestingly, even within the γ-secretase complex bearing full-length PSEN1, the catalytic pore is still able to be formed,28 which suggests that proteolytic activity can still occur, although it may be reduced.Upon γ-secretase complex assembly full-length PSEN1 undergoes autoendoproteolysis to produce cleaved PSEN1, consisting of the PSEN1 N-terminal fragment (NTF) and C-terminal fragment.PSEN1 R278I mutation has previously been shown to have increased full-length PSEN1 and Aβ43 ratio in patient iPSC-derived neuronal lines by our group.17 Increased Aβ43 and full-length PSEN1, with reduced Aβ40, was also observed in the R278I mutation mouse embryonic fibroblast CF-1 (MEF) cell line30 and mouse models.30 Importantly, alterations in PSEN1 maturity were not caused by differences in PSEN1 expression, and APP expression was not altered,17 implying specific mutations affect disease via influencing PSEN1 structure/function. Along with R278I, other mutations associated with high Aβ43 are located around the intracellular loop. Another important mutation located in this area is the E280G mutation. Clinically, the E280G mutation is associated with atypical clinical presentations, including spastic paraparesis and marked white matter hyperintensities on MRI.32 Pathologically, a brain biopsy from a PSEN1 E280G carrier showed numerous large non-cored Aβ deposits as cotton wool plaques (CWPs) and severe cerebral amyloid angiopathy (CAA).31 An individual with the PSEN1 E280Q mutation has also had a similar CWP pathology observed post-mortem.33 HEK lines expressing the E280G mutation show significantly increased Aβ42 concentration, non-significantly increased Aβ40 concentration and an overall significantly increased Aβ42:40 ratio in conditioned media compared with controls.34 However, as discussed by the authors, these results suffer from the caveat that only mutated PSEN1 is expressed in these lines compared with heterozygous mutation carriers where WT PSEN1 is also expressed. In another study, cloned γ-secretase with a range of PSEN1 mutations was purified, WT or mutant γ-secretase was mixed with APP-C99 in a detergent-based assay and incubated at 37°C for 16 h. Aβ peptides produced in the detergent-based assay were measured using an AlphaLISA assay. The assays revealed greater amounts of Aβ42 compared with Aβ40 and a higher Aβ42:40 ratio in association with the PSEN1 E280G mutation, while western blot images also suggested the presence of full-length PSEN1, possibly due to reduced endoproteolytic processing.20 Aβ43 has not, however, been assessed in the PSEN1 E280G mutation.In some mutations, an increase in the ratio of Aβ43 compared with other peptide lengths or increased absolute levels has been linked to an increase in the full-length form of PSEN1. The PSEN1 R278I mutation. Finally, we compared these findings to post-mortem tissue from E280G carriers.In this study, we generated E280G iPSCs from two clinically affected FAD patient donors. We went on to investigate Aβ profiles using ratios measured via an enzyme-linked immune-sorbent assay (ELISA) and an electrochemiluminescence assay, and PSEN1 protein maturation in iPSC-derived neurons, with the hypothesis that the E280G mutation decreases PSEN1 autoendoproteolysis, resulting in the enhanced generation of Aβ43, similar to the proximal 35 Fibroblasts underwent episomal reprogramming according to Okita et al.36 Fibroblasts underwent nucleofection with episomal DNA coding for OCT4 and shRNA against p53, KLF4, SOX2 and c-MYC using plasmids #27077, #27078, and #27080 which were obtained from Addgene. Nucleofection was performed using the Lonza P2 Nucleofection kit (Amaxa PBP2-00675) as per manufacturer’s instructions.Cell lines used in this study are summarized in After nucleofection, cells were grown on geltrex  , maintained in fibroblast media which was changed every three days.TM) 10 cm dishes . On Day 8 after nucleofection, media was switched to human embryonic stem cell media , 20 ng/ml Fibroblast Growth Factor-basic (Peprotech)) and changed daily. Nascent iPSC colonies were picked and transferred into feeder-free conditions . iPSCs were passaged using 0.5 mM ethylenediaminetetraacetic acid.Six days after electroporation, cells were lifted with 1 × trypsin  and resuspended in fibroblast media on cultured on geltrex-coated MEF feeder layer coated  and 1 μM dorsomorphin (Tocris)]. N2B27 media consists of a 1:1 mixture of Dulbecco’s modified eagle medium F12 (DMEM-F12) and Neurobasal supplemented with 0.5X N2, 0.5X B27, 0.5X non-essential amino acids, 1 mM L-glutamine, 25 U/ml pen & 25 µg/ml strep, 50 μM β-mercaptoethanol and 2.5 μg/ml insulin. At Day 12, the precursors were passaged by using a cell scraper  onto laminin-coated wells and at Day 18 passaged using dispase and plated in laminin-coated wells (Sigma) in N2B27 media. The final passage was performed at Day 35 using accutase and maintained in N2B27 media on poly-L-ornithine (Sigma) and laminin (Sigma) coated wells in N2B27 media until the required time point. Neurons were analysed at 100 days post-neural induction. One hundred days were selected as it represents the end of corticogenesis and detectable levels of secreted Aβ.38iPSCs were differentiated into cortical neurons according to the Shi (av), 4°C for 15 mins. After isolating and removing the aqueous phase, the DNA-containing interphase was removed from the organic phase by adding 300 µl of 100% ethanol was added and samples were shaken, incubated for 3 mins and centrifuged at 2000 g(av), 4°C for 5 mins. The DNA was washed twice by the addition of 0.1 M trisodium citrate in 10% ethanol, pH 8.5 followed by incubation for 30 mins with regular inversion followed by centrifugation at 2000g(av), 4°C for 5 mins. The pellet was resuspended in 1.5 ml 75% ethanol and incubated for 20 mins with regular inversion followed by centrifugation at 2000 g(av), 4°C for 5 mins. The pellet was air dried for 10 mins before resuspension in 300 µL 8 mM NAOH, followed by centrifugation at 12 000g(av), 4°C for 10 mins. Genomic DNA from the two patient PSEN1 E280G lines, named E280G.A and E280G.B, were analysed for chromosomal abnormalities using the hPSC Genetic Analysis Kit (Stem Cell Technologies) and the StemCell genetic analysis app. Amplification in a minimal critical region of chromosome 17 of the E280G.A line was observed, with a calculated copy number of 2.44. There were no significant amplifications in the E280G.B line. Additional chromosomal sites  showed no significant abnormalities.DNA was harvested using the TRIzol™  protocol according to manufacturer’s instructions. Briefly, cells were homogenized in 1 ml TRIzol™ followed by the addition of 200 µl Chloroform , shaken vigorously and then incubated for 3 mins followed by centrifugation at 12 000gRNA was harvested from iPSCs 2 days after passage via Monarch total RNA miniprep kit (NEB). 100 ng was used for Nanostring analysis on the nCounter Stem Cell Characterization Panel. The expression of 770 genes was analysed on the nSolver software to compare grouped control (CTL1 and CTL2) and grouped E280G (E280G.A and E280G.B) data. Linear regression analysis was performed using GraphPad Prism.°C. Coverslips were blocked in 5% BSA/0.3% PBS-triton-X-100 followed by primary antibody incubation  and fixed for 15–20 min in 4% formaldehyde followed by excess 1X PBS wash and stored in PBS at 4cubation  in blockantibody  in block17 Conditioned media was centrifuged at 2000 g(av) for 5 min at RT, with 1 ml aliquoted in Sarstedt 2 mL PP tubes (72.694.406) and stored at −80°c.Cortical neurons were incubated in N2B27 media for 48 h prior to collection, as described previously.DCTM Protein Assay, 5 µl of sample and standards (1:1 dilutions of Bovine Serum Albumin in RIPA from 4 mg/ml to 0.125 mg/ml + a RIPA blank) was added to reagent A + S, followed by reagent B, in triplicate. After plate shaking and 15 min development time, absorbance was measured at 750 nm on a SparkTM 10 m plate reader (Tecan). Concentration was calculated from the standard curve using the equation x = (y-m)/b. Lysates of 30 µg loaded with NuPageTM LDS sample buffer and NuPAGETM reducing agent were denatured at 70°c for 10 min followed by brief centrifugation. Electrophoresis was conducted on a NuPageTM 10% Bis-Tris gel in MES SDS running buffer at 150 V for 1 h 30 mins. Transfer to nitrocellulose membrane was conducted at 30 V for 1 h. The membrane was blocked in 5% BSA/0.1% PBS-tween20 (PBS-T) and incubated in anti-PSEN1 overnight at 4°C. The membrane was washed three times with 0.1% PBS-T and incubated in a secondary antibody (Cells that had been used for conditioned media collection were lysed in radioimmunoprecipitation assay buffer (RIPA) containing phosphatase and protease inhibitors (Roche). Using the Bio-Rad antibody  for 1 h,antibody  for 1 h,n = 2).Solid phase sandwich, ELISA for Aβ1-43  was conducted using D100 neuron-conditioned cell media according to manufacturer’s instructions. Briefly, 100 µl of calibrator, control and samples were added in duplicate to individual wells coated with an Aβ1-43 peptide-specific capture antibody. A labelled detection antibody (82E1) was applied followed by chromogen incubation and the eventual addition of the stop solution. The plate was then read at 450 nm, with a reference of 650 nm . Aβ38/40/42 in D100 neuron-conditioned cell media were measured by electrochemiluminescence using the V-PLEX Aβ Peptide Panel 1 (6E10) Kit (1 Plate) , according to manufacturer’s instructions. Briefly, samples were diluted 1:1 in diluent 35 and added in duplicate to individual wells which were coated in mouse monoclonal peptide-specific capture antibodies for human Aβx-38/x-40/x-42. Samples were incubated with anti-Aβ antibody (6E10 clone) as the detection antibody conjugated with an electrically excitable SULFO-TAG. Measurements were made using the MESO QuickPlex SQ 120, MSD. Concentrations were calculated from the ECL signal using a four-parameter logistic curve-fitting method with the MSD Workbench software package. Absolute levels of secreted Aβ43, Aβ42, Aβ40, and Aβ38 were measured and converted to ratios for comparison. Data from the two control lines (CTL1 and CTL2) were pooled and comparisons were made between all cell lines, except for the E280G.B line in Aβ38 analyses due to the low number of inductions  and random hexamers following the manufacturer’s instructions. qPCR for each induction (run in triplicate) was performed using Power SYBR™ Green (Thermo Fisher) on the Mx3000P qPCR system (Aligent). The following primers were used: T-box Brain Transcription Factor 1 , Special AT-rich sequence-binding protein 2 , Class III beta-tubulin  and PSEN1 (F: CAATACTGTACGTAGCCAGA R: AATGGGGTATAGATTAGCTG). Data were normalized to housekeeping gene RPL18a (F: CCCACAACATGTACCGGGAA R: TCTTGGAGTCGTGGAACTGC).RNA was harvested using the TRIzol™  protocol and stored at −8039 For a summary of cases, see 202 in methanol and non-specific binding with 10% dried milk solution. Sections were incubated with the primary antibodies  with the Indica Labs standard macro (Area Quantification v1) optimized for each Aβ antibody.All three FAD cases assessed in this study were obtained through the brain donation programme of the Queen Square Brain Bank for Neurological Disorders, Department of Clinical and Movement Neurosciences, UCL Queen Square Institute of Neurology. The protocols used for brain donation and ethical approval for this study were approved by a London Research Ethics Committee and tissue is stored for research under a license from the Human Tissue Authority. The standard diagnostic criteria for the neuropathological diagnosis of Alzheimer’s disease were used in all cases.tibodies  overnighP < 0.05 considered significant. When ANOVA analysis reached statistical significance, Tukey’s post hoc test was used, adjusting the significant level appropriately for the analysis conducted. All cell data statistics were conducted on GraphPad Prism.Cell data were checked for normality using QQ plots. For statistical analysis, the CTL1 and CTL2 lines have been pooled. When needed for western blot and qPCR analysis, CTL2 was used for normalization. Cell data were assessed using one-way ANOVA with a probability value of less than PSEN1 E280G mutation carriers were reprogrammed to iPSCs via episomal reprogramming. Confirmation of successful reprogramming in the newly generated PSEN1 E280G lines was confirmed by immunocytochemistry (ICC) which showed that all iPSC lines expressed the markers of pluripotency NANOG and SSEA4  with E280G lines (E280G.A and E280G.B) displayed a high correlation (R2 = 0.9932), representing a similar stem cell expression profile in the newly derived lines compared with established iPSCs (Fibroblasts from two nd SSEA4 . Chromos of 2.44 . The exaed iPSCs . Sanger ed iPSCs .PSEN1 M146I and R278I),17 the newly generated lines were subjected to cortical differentiation to generate the cell type affected in FAD. ICC was conducted on all control and FAD cell lines to investigate forebrain patterning at Day 25 via Forkhead Box G1 (FOXG1) and Paired box protein Pax-6 (PAX6) staining  with a significant difference between the control and E280G.B lines . There was no significant difference in SATB2 expression . There was a significant difference in the Aβ42:40 ratio, indicative of both γ-secretase epsilon cleavage efficiency and selection of the Aβ48 or 49 pathway, across the cell lines (P < 0.0001). Compared with controls, there was a significantly increased ratio in the M146I, E280G.A and E280G.B lines . M146I and E280G.A also had significantly increased Aβ42:40 ratios compared with R278I . The M146I line also had a significantly increased Aβ42:40 ratio compared with E280G.B (P = 0.01), To explore Aβ processing down the Aβ48/49 pathways in vitro , we asseP < 0.0001). Compared with controls, there was a significantly higher Aβ43:40 ratio in the R278I, E280G.A and E280G.B lines . The Aβ43:40 ratio in the R278I, E280G.A and E280G.B lines was also significantly higher compared with M146I , The Aβ43:40 ratio, indicative of γ-secretase carboxypeptidase cleavage efficiency in the Aβ49 pathway, was significantly different between cell lines (n number (n = 2). The ratio was significantly different across the cell lines . Compared with controls, M146I, R278I and E280G.A had a significantly increased Aβ42:38 ratio . The M146I line had a significantly increased ratio compared with R278I, and significantly reduced ratio compared with E280G.A (Both P < 0.0001). The R278I line had a significantly reduced ratio compared with E280G.A (P < 0.0001), The ratio of Aβ42:38, indicative of γ-secretase carboxypeptidase cleavage efficiency in the Aβ48 pathway, was compared across all lines, excluding E280G.B due to low P < 0.0001), which indicates γ-secretase epsilon cleavage efficiency in the Aβ48:49 pathways. Controls had a significantly increased Aβ38:40 ratio compared with R278I and E280G.A lines . The M146I line had a significantly increased Aβ38:40 ratio compared with E280G.A (P = 0.0002). R278I also had a significantly increased Aβ38:40 ratio compared with E280G.A (P = 0.02), There was a significant difference across the lines (excluding E280G.B) in the ratio of Aβ38:40 (P = 0.002). Compared with controls, there was a significantly decreased Aβ42:43 ratio in the R278I, E280G.A and E280G.B lines . Additionally, compared with M146I, there was a significantly decreased Aβ42:43 ratio in the R278I, E280G.A and E280G.B lines , Pathway 43, also indicative of γ-secretase epsilon cleavage to the Aβ48 or 49 pathway, significantly differed across cell lines .These data support PSEN1 mutations have been shown to alter γ-secretase stability.41 By using western blots to probe for the PSEN1-NTF, the presence of cleaved and full-length PSEN1 was assessed in the neurons, PSEN1 M146I, PSEN1 R278I and two PSEN1 E280G). Full-length PSEN1 levels statistically differed across lines (P = 0.04), with significantly increased levels in the R278I line compared with the control group (P = 0.03), with a notable trend for increased levels in the E280G lines, see Firstly, the amount of full-length PSEN1 (46 kDa), relative to actin, was normalized to CTL2. Protein expression was then compared between the control lines (pooled data), and the four FAD patient-derived lines (P = 0.0007). Compared with the control group, PSEN1-NTF was significantly higher in the E280G.A and E280G.B lines  and significantly higher in the E280G.A and E280G.B lines compared with the R278I lines , see Secondly, the amount of PSEN1-NTF (25 kDa), relative to actin and normalized to CTL2, was then compared between the control lines (pooled data), and the four FAD patient-derived lines. PSEN1-NTF levels indicate effective autocleavage of PSEN1 into the N and C-terminal fragments. PSEN1-NTF significantly differed across lines (P < 0.0002) with higher full-length PSEN1 levels in the R278I line compared with the control group (P < 0.0001), M146I line (P = 0.0008), E280G.A line (P = 0.002) and E280G.B line (P = 0.002), see Finally, the levels of full-length PSEN1 relative to PSEN1-NTF, normalized to the CTL2 line was compared between the control lines (pooled data), and the four FAD patient-derived lines. This ratio was significantly different across lines , and the four FAD patient-derived lines . TogethePSEN1 mutations that were studied in our cell lines  were available for immunohistochemical investigation. Note, the tissue and iPSC lines are not from the same individuals. The percentage area of Aβ-specific antibody coverage was assessed in the temporal and occipital cortices.Post-mortem cortical brain tissue from three individuals with FAD carrying the same Immunohistochemical analysis displayed intense Aβ42 deposition in all cases in the temporal A–C and oAβ40 pathology was present in all cases, notably as CAA, and cortical deposition . Aβ40 waAβ43 IHC shows mutation-specific differences in Aβ43 deposition . Aβ43 dePSEN1 E280G mutation, which is associated with atypical clinical (spastic paraparesis) and pathological features (amyloid plaques of the cotton wool type). We have shown that in patient-derived iPSC-cortical neurons, the E280G mutation leads to increased ratios of Aβ42 and of Aβ43 compared with other length peptides, which is linked with both reduced PSEN1 autoendoproteolysis and relative abundance of both full-length and PSEN1-NTF.The pathogenic effect of mutations in PSEN1 is mediated through alterations in APP processing with different mutations producing distinct effects on that processing. This may potentially underlie some of the clinical heterogeneity seen in FAD mutation carriers. Here, for the first time, we have defined the consequences of the 43 Mutations which effect the endopeptidase activity could affect both absolute Aβ levels, and which pathway of Aβ production is selected, leading to altered ratios of the peptides within those pathways.22 Whilst PSEN1 mutations vary in whether or not they alter the endopeptidase activity of γ-secretase, they have been found to consistently decrease the efficiency of its subsequent carboxypeptidase activity by destabilizing enzyme–substrate complexes between γ-secretase and the Aβ peptides it is processing.44 The destabilization and compromised carboxypeptidase activity results in the release of longer Aβ peptides which are more prone to aggregation22 This in turn leads to more oligomeric Aβ and greater extracellular deposition.23 We observed a greater Aβ42:38 ratio in the M146I and E280G.A lines, compared with controls, indicative of consistently impaired carboxypeptidase activity in the Aβ48 pathway for these lines. Mutations near the autoendoproteolysis site are proposed to inhibit the cleavage that converts the full-length form into the mature, cleaved product.26 This can then lead to altered processivity by the enzyme.27 Interestingly, even within the full-length PSEN1, the catalytic pore is still able to be formed,28 which suggests that γ-secretase activity can still occur, although it is likely reduced, possibly explaining altered Aβ peptide production in mutations near this location. Such mutations in this region include PSEN1 R278I and E280G. Compared with controls and with M146I, the R278I and E280G lines displayed an increased Aβ43:40 ratio, indicating reduced carboxypeptidase activity in the Aβ49 pathway in a mutation dependant manner. Mutations not within the catalytic domain can likely affect the protein structure/stability and this may also impact interactions with substrates which will contribute to disease.44 For instance, the M146I mutation is not within the catalytic domains, yet displayed altered Aβ production profiles compared with controls, and other mutations.One way in which mutations can affect PSEN1 is via the initial endopeptidase activity of γ-secretase.PSEN1 R278I cell line compared with the control group. This has previously been shown for the R278I line, with a significant increase in full-length PSEN1 observed, and has been linked to Aβ43 production.30 It has also been observed for the E280G mutation,20 with our results suggestive of a trend for increased full-length PSEN1 in the E280G cell lines, although this did not reach significance.Western blot analysis revealed a significantly increased full-length PSEN1 in the The levels of PSEN1-NTF protein were also observed to be higher, and significantly so in the E280G neurons. As PSEN1 expression was not higher in these lines, it suggests that in the E280G lines there is an accumulation of PSEN1-NTF. This increase in PSEN1-NTF may explain the high levels of Aβ42 observed in these lines, in addition to the high Aβ43.Finally, the level of full-length relative to PSEN1-NTF was significantly higher in the R278I line, which reflects the increase in products of the Aβ49 pathway only in this line. This measure also highlights that the R278I mutation specifically affects the processing of full-length to cleaved PSEN1, compared with other lines.PSEN1 R278I and E280G mutation lines. It is likely that the mutation location hinders the processivity of full-length to cleaved PSEN1. The R278I and E280G mutations have a similar impact on Aβ ratios, however, the differential impacts on protein maturity effect may impact the amounts of mutant PSEN1 that are incorporated into the γ-secretase complex. Thus, absolute levels of Aβ could differ. To investigate this, isogenic lines with the homozygous mutations can be generated, as has previously been done for the PSEN1 Intron 4 deletion mutation.45This would allow the impact of these mutations to be assessed on protein maturity, Aβ ratios, and absolute Aβ levels, in the absence of a WT PSEN1 allele. It should be noted there was some variability between the two E280G lines. We cannot rule out that this could be due to the karyotypic abnormality in the E280G.A line in the 17q region. Although we were not able to identify the exact region that is duplicated, it is worth noting that the MAPT gene is located on chromosome 17q, encoding for tau, which forms pathological deposits in multiple dementias including FAD. Reassuringly, analysis of gene expression profiles in this line was normal  ε2/ε3 genotype. Generally, the APOE ε4 allele is linked to sporadic CAA, with a meta-analysis indicating APOE ε4 as a risk variant in grouped dementia patients and controls while APOE ε2 may result in less CAA,58 although a positive APOE ε4 carrier status and CAA severity were not associated in a study of PSEN1 mutation cases.59 Post-mortem analysis in 93 sAD patients revealed a significant association of CAA pathology with the APOE ε4 allele, with increasing APOE ε4 allele number associated with increasing severity of CAA.60 However, the role of the APOE genotype in CAA is not clear cut, as carriers of the APOE ε2 allele have been found to have more severe CAA than those with the APOE ε3/ε3 genotype, despite the APOE ε2 allele conferring lower risk for Alzheimer’s disease.61 Additionally, Alzheimer’s disease patients who were APOE2 carriers had worse small vessel disease 62 and a meta-analysis found APOE ε2 to be associated with certain markers of cerebrovascular disease.63 These factors may contribute to the association between APOE ε2 and CAA. Thus, the APOE ε2 carriership in the M16I mutation case may influence the Aβ40 pathology displayed in this case. However, the R278I case also has an APOE ε2/ε3 genotype, which could also impact the histological observations. In future work, it would be of interest to investigate the Aβ peptides deposited in a larger number of individuals carrying the same FAD mutation but different APOE genotypes. Such larger studies will be needed in order to fully investigate correlations between clinical and pathological features and mutation-associated differences in Aβ processing. While studying pathology and iPSCs from the same individuals would be a gold standard, the altered Aβ ratios observed in iPSC studies are conserved in FAD and also in sAD human tissue, validating the use of iPSCs as models and implying γ-secretase alterations do occur in vivo and can occur in sAD.64 Furthermore, the approach used here has enabled some confirmation of cell line results to human pathological tissue, highlighting the validity of the models to recapitulate FAD mechanisms and their appropriateness to investigate clinical and pathological associations.The mutation-associated differences in PSEN1 and subsequent Aβ production identified in this study are of particular interest in view of the distinct clinical and pathological features associated with the mutations we studied. E280G and R278I mutations both lie within exon 8, near the endoproteolytic cleavage domain. Mutations that are post-codon 200, and particularly within exon 8, have been linked to clinically atypical cognitive presentations and associated with spastic paraparesis.fcac321_Supplementary_DataClick here for additional data file.