Chelidonium majus L. and Berberis thunbergii DC. IC10, IC50, and IC90 doses were established toward hematopoietic cancer cell lines using trypan blue staining. Alterations in the expression of 18 apoptosis-related genes in cells exposed to IC10, IC50, and IC90 were evaluated using real-time PCR. Sanguinarine and Chelidonium majus L. extract exhibit significant cytotoxicity against all studied cell lines. Lower cytotoxic activity was demonstrated for berberine. Berberis thunbergii DC. extract had no influence on cell viability. Berberine, sanguinarine, and Chelidonium majus L. extract altered the expression of apoptosis-related genes in all tested cell lines, indicating the induction of apoptosis. The presented study confirmed the substantial cytotoxicity and proapoptotic activity of sanguinarine, berberine, and Chelidonium majus L. extract toward the studied hematopoietic cell lines, which indicates the utility of these substances in anticancer therapy.Isoquinoline alkaloids belong to the toxic secondary metabolites occurring in plants of many families. The high biological activity makes these compounds promising agents for use in medicine, particularly as anticancer drugs. The aim of our study was to evaluate the cytotoxicity and proapoptotic activity of sanguinarine, berberine, and extracts of  Papaveraceae, Ranunculaceae, and Berberidaceae families. Berberine is a tertiary, protoberberine-derived alkaloid occurring in Berberidaceae, Papaveraceae, Menispermaceae, Ranunculaceae, and Rutaceae families. Due to their toxicity, sanguinarine and berberine play a defending role against viruses and fungi that are pathogenic towards plants [Sanguinarine and berberine are two of the most intensively investigated isoquinoline alkaloids in terms of their use in medicine. Sanguinarine is a benzophenantridine-type alkaloid occurring in s plants ,2.Sanguinarine exhibits various pharmacological activities, including antibacterial , anti-inThe most intensively investigated of these is the anticancer activity of sanguinarine and berberine. Many pieces of evidence indicate that sanguinarine inhibits cell cycle and induce apoptosis in various types of cancer cells ,20. SangNumerous studies have evidenced the anticancer activity of berberine ,30. BerbClinical applications of sanguinarine and berberine are limited by their toxic effects . In IndiDespite the toxicity and mutagenicity of sanguinarine and berberine, the compounds are still extensively studied due to the possibility of synthesis of derivatives with reduced toxicity. In optimized doses, these alkaloids could exhibit potential therapeutic effects with limited side effects. A particularly important direction of research for this aspect is to determine the effect of the compound and mechanisms of action towards different types of cancer cells. Therefore, in this study we performed an assessment of cytotoxic and proapoptotic activities of sanguinarine and berberine on selected hematopoietic cell lines derived from various types of leukemia, including HL-60, HL-60/MX1, HL-60/MX2 (acute promyelocytic leukemia), J45.01 (acute T cell leukemia), U266B1 (myeloma), CCRF/CEM, and CEM/C1 (acute lymphoblastic leukemia). HL-60/MX1 and HL-60/MX2 cell lines are the multidrug-resistant derivatives of the HL-60 cells, therefore these cell lines were used to evaluate the effects of studied samples on cells resistant to anticancer treatment.Chelidonium majus L., Macleaya cordata Willd., Lamprocapnos spectabilis (L.) Fukuhara, Fumaria officinalis L., Glaucium flavum Crantz, Corydalis cava (L.) Schweigg and Körte, Berberis thunbergii DC., Meconopsis cambrica (L.) Vig., and Mahonia aquifolium (Pursh) Nutt. [Chelidonium majus L. and the highest amount of berberine was found in Berberis thunbergii DC. [Previously a screening of alkaloid composition was performed for nine species: h) Nutt. . The higrgii DC. . TherefoChelidonium majus L. and Berberis thunbergii DC., respectively.Plant extracts are very complex mixtures of various compounds, which could exhibit antagonism or synergy of biological activity. The application of plant extracts in pharmacotherapy has a higher risk of side effects than pure compounds, the advantage of which is a more predictable therapeutic effect. In this study, we evaluated the cytotoxicity and proapoptotic activity of both pure alkaloids, sanguinarine, and berberine, as well as the extracts prepared from plants containing high amounts of these alkaloids, Chelidonium majus L. and Berberis thunbergii DC. toward HL-60, HL-60/MX1, HL-60/MX2, CCRF/CEM, CEM/C1, J45.01, and U266B1 cell lines was evaluated by determination of IC10, IC50, and IC90 doses in trypan blue staining test  was marked as IC50 with an asterisk (IC50*). The IC90 values of berberine were determined only for the CCRF/CEM cells at130 μM and J45.01 cells at200μM. For the remaining cell lines, the IC90 dose could not be determined due to insufficient cytotoxicity of berberine even at the maximum possible dose (250 μM) when the viability of the U266B1, CEM/C1, HL-60, and HL-60/MX1 cell lines did not fall below 90%. For this reason, the maximum dose to which the cells were exposed was marked as IC90*. The most potent cytotoxicity of berberine was found toward CCRF/CEM, J45.01, and HL-60/MX1 cells. The HL-60/MX2 line was the least sensitive to the compound .Chelidonium majus L. extract showed differentiated cytotoxic effect depending on the study cell line. The strongest cytotoxic effect of Chelidonium majus L. extract was exerted on the J45.01, CCRF/CEM, and CEM/C1 cells, and the cells of the HL-60 and HL-60/MX2 lines were the least sensitive to this extract   . StandarBAK1, BNIP3, and CASP9 was observed in U266B1 cells after exposure to sanguinarine, berberine, and Chelidonium majus L. extract. For IC10 doses, the highest gene expression modulatory activity was exhibited by sanguinarine, but in higher doses of tested samples, the most affecting expression of studied genes was berberine. A higher dose of berberine and a lower expression of BCL2, BIK, BNIP2, and CASP3 were observed  were upregulated in cells exposed to sanguinarine in IC10 dose, but in cells exposed to IC90 dose of this alkaloid these genes (excluding BAK1 and MCL1) were downregulated  was observed in CCRF/CEM cells compared to other cell lines. In turn, the highest expression of MCL1 and TP53 was demonstrated in CEM/C1 and HL-60 cells, respectively. The highest induction of genes associated with apoptosis by IC90 dose of sanguinarine was also observed in the CCRF/CEM cell line. In HL60/MX1 and HL60/MX2 cells, sanguinarine caused the lowest alterations in the expression of the studied genes. In these cell lines, the highest increase in expression of studied genes was observed after exposure to IC10 of berberine. Expression of examined genes in cells exposed to a higher concentration of berberine and extracts of Chelidonium majus L. and Berberis thunbergii DC. was differentiated across studied cell lines. Exposure to Berberis thunbergii DC. extract (145.5 µg/mL) caused upregulation of B2M and downregulation of BAD and BNIP2 in all studied cell lines . The PARAFAC univariate analysis explained only 28.3% of the variance, so bivariate analysis was performed, explaining 50.4% of the variance. The obtained results allowed the isolation of IC10 concentration of sanguinarine as an outlier  was similar to the IC50 dose of the sanguinarine solution (0.1–1.05 μM) [Chelidonium majus L. extract, but sanguinarine does not occur in the highest amount in this plant [Inhibitory concentrations received for 1.05 μM) . These fis plant ,54.Chelidonium majus L. extract, as previously evidenced in HL60 cells [Chelidonium majus L. [Our research confirmed the cytotoxic activity of 60 cells . The resmajus L. .Berberine also exhibited cytotoxic activity against all examined cell lines, however it was weaker than sanguinarine. IC10, IC50, and IC90 doses of berberine were significantly higher than determined for sanguinarine and was characterized by a high variation between individual cell lines. Due to low cytotoxicity, we were not able to determine the IC50 dose of berberine for HL-60/MX2 cells and IC90 doses for HL-60, HL-60/MX1, HL-60/MX2, CEM/C1, and U266B1 cell lines. Cytotoxic activity of berberine toward U266B2 cell line was previously evaluated by Hu and collaborators using Cell Counting Kit-8 . The autIn our study, the cytotoxic activity of berberine was evaluated for the first time in relation to J45.01, HL60/MX1, and HL60/MX2 cell lines.Berberis thunbergii DC. extract, which contained a relatively high amount of berberine [Berberis thunbergii DC. extract is insufficient to induce a cytotoxic effect, which is supported by relatively high values of IC10, IC50, and IC90 concentrations (25.15 μM–250.45 μM) determined for berberine.The demonstrated cytotoxicity of berberine toward tested cells suggests the potential activity of erberine . HoweverChelidonium majus L. and Berberis thunbergii DC. on the expression of 18 genes associated with apoptosis was tested in hematopoietic cell lines. The similarities between administered doses and the response of the cells after 24 h exposure to the tested samples were also investigated.In the next step of the study, the influence of sanguinarine, berberine, and extracts from BAK1 and BNIP3 was the most frequently observed. In CCRF/CEM cell line, all studied genes were upregulated with logRQ > 0.5 after exposure to sanguinarine. Berberine also exhibited an ability to raise expression of studied genes, but to a lower extent than sanguinarine. All studied genes were upregulated in HL-60/MX2 cells after IC10 berberine treatment. The expression of TP53 was differentiated across studied cell lines exposed to IC50 of berberine. In CEM/C1, CCRF/CEM, and HL-60/MX2 cells TP53 was upregulated, but in other cell lines the downregulation of this gene was demonstrated. This indicates diverse TP53-mediated responses of studied cells after treatment with berberine. Upregulation of BAK1 and BCL2L2 was the most often observed effect in studied cell lines after treatment with Chelidonium majus L. extract.Exposure to sanguinarine, especially in IC10 dose, caused the strongest upregulation of studied genes associated with apoptosis. Upregulation of TP53 gene expression. This is a beneficial effect because the promotion of neoplastic transformation cancer is associated with a reduced expression of the TP53. These findings support evidence that sanguinarine has a strong antiproliferative effect on cells with TP53 gene dysfunction [TP53 was observed.PARAFAC analysis shows that IC10 dose of sanguinarine was an outlier among other doses of tested samples. Exposure to sanguinarine at this dose induced interesting changes in gene expression, which seem to be more unambiguous in interpretation and more beneficial than those induced after exposure to higher doses of the compound. Sanguinarine in IC10 doses induced an increase in function . In the Chelidonium majus L. extract on the expression of genes related to the apoptosis process is very similar to the effect of sanguinarine in the IC10 dose. This suggests similarities in the mechanism of apoptosis induction between sanguinarine and Chelidonium majus L. extract. Upregulation of CASP8 and CASP3 observed in cells exposed to IC10 of sanguinarine and IC10, IC50, and IC90 of Chelidonium majus L. extract indicated induction of apoptosis in the extrinsic pathway. The intrinsic apoptosis is probably prevented due to the accumulation of sanguinarine close to the outer side of the inner mitochondrial membrane during its stimulation. Sanguinarine neutralizes the effects of stimulation of the mitochondrial membrane, inhibits the synthesis of ATP, and breaks the oxidative phosphorylation [CASP9 expression observed in cells exposed to Chelidonium majus L. extract suggests the intrinsic course of apoptosis, probably conjugated with the extrinsic apoptosis pathway.The presented results show that the effect of rylation . An incrChelidonium majus L. extract and low sanguinarine doses is the increase in the expressions of BAK1, BAD, and BNIP3. BAK1 gene encodes the Bak protein, which participates in the formation of mitochondrial transmembrane channels and mediates the release of proapoptotic factors from the intra-mitochondrial space, including cytochrome c [BAD gene, has the ability to form heterodimers with antiapoptotic Bcl-2 and Bcl-xL proteins, enhancing the proapoptotic activity of the BAX and Bak proteins [BCL2 family. This protein is responsible for the neutralization of anti-apoptotic proteins after the onset of the mitochondrial permeabilizing membrane [BCL2 family is regulated by sanguinarine and Chelidonium majus L. extract via the BNIP3 protein. Sanguinarine seems to be the main factor inducing apoptosis of cells exposed to Chelidonium majus L., despite a lower amount of this compound compared to the amounts of other alkaloids in this plant [Another effect that clearly indicates promotion of the process of apoptosis in cells exposed to both chrome c ,23. The proteins . The BNImembrane ,61. It cis plant ,54.BAK1, BAD, and BNIP3 was lower, suggesting the lower proapoptotic potential of sanguinarine in higher doses.It should be noted that in the cells exposed to the higher doses of sanguinarine, the expression of TP53 gene, whose expression was strongly elevated in the CCRF/CEM cells (for IC50 by 303% and for IC90 by 342%), but in other cell lines after exposure to the IC50 and IC90 doses of sanguinarine was reduced.Both the PARAFAC analysis and the clustering with the Euclidean distances indicated that CCFR/CEM cell line clearly differed from other cell lines after exposure to all doses of sanguinarine. In this cell line, all doses of sanguinarine increased the expression of almost all analyzed genes. This is particularly important in the case of the Chelidonium majus L. extract. In these cells, as well as in the HL-60/MX1 cells, the anti-apoptotic BCL2 expression was lowered. The remaining cell lines responded with an increase in BCL2 gene expression after exposure to all doses of Chelidonium majus L. extract. This result indicates that the sensitivity of HL60 and HL60/MX1 cells to the proapoptotic action of Chelidonium majus L. extract is mediated by the downregulation of BCL2.PARAFAC analysis and Euclidean clustering showed that the HL60 line is an outlier after exposure to all doses of Berberis thunbergii DC. extract for the transcription of 18 apoptosis-related genes in hematopoietic cancer cell lines. Berberine is a particularly interesting compound because it is characterized by a lack of toxicity in relation to normal cells, while the cytotoxicity towards cancer cells were reported [In this research, we evaluated the effect of berberine and reported . Despitereported ,32, the CASP3 expression was observed in the CEM/C1, CCRF/CEM, and HL-60/MX2 cells after exposure to all doses of berberine. This result clearly indicates that the induction of cell death of these lines was in the apoptosis way. Moreover, an increase in CASP9 expression, observed in the case of all cell lines exposed to berberine in the IC10, IC50, and IC90 doses, suggests the induction of programmed cell death with the intrinsic pathway.In the current study, a high increase in The multidrug resistant HL-60/MX2 cell line was the most sensitivity to sanguinarine and was characterized by higher upregulation of proapoptotic genes compared to HL-60 and HL-60/MX1 cells, which exhibit weaker multidrug resistance potential , Table 2Chelidonium majus L. extract , which may explain a lower increase in the expression of the same genes. It may also suggest the possibility of proapoptotic activity of other compounds present in the extract, whose identification requires further investigation. Our results confirmed that berberine and the Berberis thunbergii DC. extract have an activity promoting the apoptosis with minimal cytotoxicity in studied concentrations. Exposure to the The limitation of this study is assessment of cytotoxicity and gene expression in exposed cells independently from cell population growth parameters, like doubling time or maximum growth rate. Gene expression was evaluated after 24 h of exposure, however, mRNA levels may change in shorter time  and be a result of complex interaction with other RNAs. The influx and efflux time in relation to concentration of study samples could also affect the outcome. The influence of these variables on the sensitivity of the cells to cytotoxic activity of studied compounds should be elucidated in further investigations with optimized time points of the exposure.Chelidonium majus L. and Berberis thunbergii DC. Understanding the mechanisms that accompany the change in the expression of the studied genes is substantial when trying to determine the effects associated with the interaction of sanguinarine, berberine and the extracts studied for their anticancer activity. The obtained results significantly enrich the knowledge of the antineoplastic activity of substances and may accelerate their introduction into the clinical trial phase in the treatment of various types of leukemia.Observations presented in current study may have a significant impact on the understanding of the regulation of apoptosis at the transcriptome level after exposure to sanguinarine, berberine and extracts from The reference compounds sanguinarine and berberine were of analytical grade from Sigma-Aldrich Company . The purity of each compound was more than 98%, according to the manufacturer. 10 mM stock solution of sanguinarine in DMSO was prepared. For the cytotoxic study, a series of dilutions was prepared with concentrations 0.8, 0.6, 0.5, 0.4, 0.2, 0.1 and 0.01 mM. The final concentration of sanguinarine in cell suspensions were 1000-fold lower. Due to the limited solubility of berberine in DMSO (maximum 50 mM), berberine dilutions were prepared in 25 mM, 10 mM and 5 mM concentrations.Chelidonium majus L. extract and 29.1 mg/mL concentration of Berberis thunbergii DC. extract. Due to a harmful effect of DMSO to cells [Plant material selection and procedure of extraction were carried out as previously described . For cytto cells , to the 2 per well) in standard conditions  for 24 h using Galaxy B incubator .HL-60 (CCL-240), HL-60/MX1 (CRL-2258), HL-60/MX2 (CRL-2257), CCRF/CEM (CCL-119) and CEM/C1 (CRL-2265) cell lines were from ATCC collection and were purchased from LGC Standards, UK. J45.01 (ECACC 93031145-1VL) and U266B1 (ECACC 85051003-1VL) cell lines were from ECACC collection and were purchased from Sigma-Aldrich Co. . Before experiments, 1 mL of cell suspension, containing 5–9 × 105 cells, was cultured in sterile 12-wells plates . The supernatant was discarded, to the cells were added 1 mL PBS  and centrifugation was repeated. The supernatant was removed and the cells were resuspended in 50 µL of PBS. Subsequently, 10 µL of suspension was mixed with 10 µL of trypan blue solution  and incubated for 1 min in 37 °C. The total number of cells, as well as the percentages of normal and death cells, were counted on Counting Slides using Automated Cell Counter (Bio-Rad). The experiments were performed in triplicates on independent cell lines passages and the mean values were calculated. The curves presenting relationships between cell viability and the concentration of the studied samples were prepared in order to determine IC10, IC50 and IC90 doses for each of the studied cell lines.After 24 h exposure to IC10, IC50 and IC90 doses of studied samples, cells were centrifuged at 800 rpm for 10 min and the supernatant was discarded. Total RNA was isolated from the cells using Chomczyński and Sacchi method  and TRI The quality and quantity of isolated RNA was assessed by NanoDrop2000 UV-VIS spectrophotometer with NanoDrop2000 Operating Software . RNA samples with A260/A280 ratio ranged between 1.8 and 2.0 were intended to further investigations.Synthesis of cDNA was performed using High Capacity cDNA Reverse Transcription Kit , according to the manufacturer procedure. Briefly, the following reaction mixture was assembled on ice: 2 µL of 10X RT Buffer, 0.8 µL of 25X dNTP Mix (100 mM), 2 µL of 10X RT Random Primers, 1 µL of MultiScribe Reverse Transcriptase (50 U/µL), 0.5 µL of RNase Inhibitor (40 U/µL), 1 µL of isolated RNA and 1 µL of DEPC-treated nuclease-free water. The reaction mixture was incubated in thermocycler Mastercycler Personal  in the following conditions: 25 °C for 10 min, 37 °C for 120 min and 85 °C for 5 s. Obtained cDNA samples were stored in −20 °C. B2M, BAD, BAK1, BAX, BCL2, BCL2L1, BCL2L2, BID, BIK, BNIP1, BNIP2, BNIP3, CASP3, CASP8, CASP9, MCL1, PMAIP1, and TP53) using real-time PCR method. cDNA samples were amplified using a 7900HT Fast Real-Time PCR System . Reaction mixtures contained 1.25 µL of gene-specific TaqMan probe  described in −ΔΔCt) [Gene expression analysis was carried out for 18 genes related to apoptosis (n −ΔΔCt) ,65. The Berberis thunbergii DC. extract due to the lack of cytotoxicity in relation to the tested cell lines.Clustering analysis with Euclidean distance and parallel factor analysis (PARAFAC) were applied to reduce the large dimensionality of the data and to assess the similarities between the doses of the tested compounds, between the analyzed cell lines, and between changes in the expression of examined genes after exposure to the samples. The analysis was performed using R programming software. The analysis did not include data obtained after exposure to the


Tamoxifen treatment of estrogen receptor (ER)-positive breast cancer reduces mortality by 31%. However, over half of advanced ER-positive breast cancers are intrinsically resistant to tamoxifen and about 40% will acquire the resistance during the treatment.In order to explore mechanisms underlying endocrine therapy resistance in breast cancer and to identify new therapeutic opportunities, we created tamoxifen-resistant breast cancer cell lines that represent the luminal A or the luminal B. Gene expression patterns revealed by RNA-sequencing in seven tamoxifen-resistant variants were compared with their isogenic parental cells. We further examined those transcriptomic alterations in a publicly available patient cohort.We show that tamoxifen resistance cannot simply be explained by altered expression of individual genes, common mechanism across all resistant variants, or the appearance of new fusion genes. Instead, the resistant cell lines shared altered gene expression patterns associated with cell cycle, protein modification and metabolism, especially with the cholesterol pathway. In the tamoxifen-resistant T-47D cell variants we observed a striking increase of neutral lipids in lipid droplets as well as an accumulation of free cholesterol in the lysosomes. Tamoxifen-resistant cells were also less prone to lysosomal membrane permeabilization (LMP) and not vulnerable to compounds targeting the lipid metabolism. However, the cells were sensitive to disulfiram, LCS-1, and dasatinib.Altogether, our findings highlight a major role of LMP prevention in tamoxifen resistance, and suggest novel drug vulnerabilities associated with this phenotype.The online version of this article (10.1186/s12885-018-4757-z) contains supplementary material, which is available to authorized users. Approximately two thirds of breast cancers are estrogen receptor (ER) positive. As the receptor stimulates proliferation of mammary epithelial cells, it is also an important target in anti-hormonal cancer therapy. One of the most prescribed ER antagonists for first line therapy is tamoxifen that has helped millions of women since its discovery 50 years ago . HoweverIn addition to its intended anti-cancer effects, tamoxifen is known to have both direct and indirect effects on the cellular lipid metabolism. It has been shown to reduce blood cholesterol levels  and to bOn the other hand, reprogrammed metabolism is one hallmark of cancer cells  and has In the present study, we have delineated mechanisms underlying tamoxifen resistance by extending our drug screening and exome sequencing analyses on tamoxifen-resistant cell lines  by perfo2.The development, characterization and culturing of the tamoxifen-resistant and their isogenic parental cell lines has been published previously . In brieRNA isolation, library preparation, sequencing, and data-analysis were done as explained in Kumar A et al. . BrieflyRNA-sequencing data analysis was performed as described in detail in Kumar et al.  and inclp-value less than 0.001 (1E-3) were accepted as significantly altered. Log2 ratios of differentially expressed genes of the cell lines were visualized using heat maps. In the heat map analysis, genes and samples were ordered using unsupervised complete linkage clustering with Euclidean distance measure [Public patient transcriptome data  was down measure . PatientParental and resistant T-47D cells were plated on 6-well plates and grown in their default media +/− 1 μM 4-OH-tamoxifen for 72 h in triplicates. The cells were scraped into 2% NaCl and the subsequent lipid extraction was based on the Bligh and Dyer method . Cell lyParental and resistant T-47D cells were seeded on coverslips and grown in the default media +/− tamoxifen for 72 h. Cells were fixed with 4% paraformaldehyde for 20 min at room temperature, and permeabilized with 0.3% Triton X-100 for 5 min, followed by 30 min blocking with 3% BSA-PBS at 37 °C. The primary and secondary antibodies were diluted in 1% BSA-PBS and consecutively incubated for 60 and 30 min at 37 °C. For detection of free cholesterol, cells were stained with 0.05% filipin (Sigma)  in 10% F2 to remove the detection of debris in the image background. Starting from the detected nuclei, the segmented regions were propagated to cover cell cytoplasm stained with ceruloplasmin. The cells that touched the image border were discarded. Finally, spot detection was used to segment galectin-3 stained spots. The maximum radius of the spots was set to 1 μm. We defined cells with more than 1 spot as galectin-3 positive to exclude false positive detection. Further, we calculated the percentage of galectin-3 positive cells and the average spots per cell.In order to measure the integrity of lysosomal membranes, we performed the detection of damaged lysosomes by galectin-1 and -3 translocation according to the previously published protocol . We firsThirty-three compounds that target lipid and cholesterol metabolism, or induce LMP, were selected for the DSRT by literature and vendor research  diluted 1:200 in PBS and Hoechst for nuclei detection. Twenty-five fields-of-view per well were acquired with the PE Opera Phenix HCS system (PerkinElmer) in a confocal mode with the 40× water immersion objective (NA 1.1). Exposure time and laser power were kept constant for each individual staining across different cell lines and conditions. Images were analyzed using a custom pipeline to measure LipidTOXGreen signal and image-based DSS based on cell counts. Images were preprocessed by applying flatfield correction using CIDRE method , and theer 2.2.0 . First, p < 0.05. All comparisons of the measurement of triglycerides, free cholesterol and cholesterol esters, LMP assay, and LipidToxGreen staining can be found in Additional file For all experiment that were at least done in triplicates the values were expressed as mean ± SD. One way ANOVA was performed on the mean of each measurement followed by Tukey test to enable multiple comparisons between groups. Statistically significance was accepted as SERPINA1, PLXDC2, NAV1, DOCK10 and LRP1) with altered expression were detected in the luminal A cell lines , also changed its expression in the patient samples . CP (Ceruloplasmin), a gene that was highly upregulated in the resistant T-47D cell lines (240–290 fold-increase) as well as MCF-7 Tam1 (26 fold-increase), was also found to be overexpressed in all the 3 metastatic patient samples ranging from 12-fold increase (patient 2) to 50–57 fold increase , which is known to regulate several lipid droplet proteins, was upregulated in resistant cells. In addition, the ATP Binding Cassette Subfamily A Member 1 (ABCA1), which functions as a cholesterol efflux pump was downregulated  with antibodies detecting the lysosomal-associated-membrane-proteins 1 and 2 (Lamp1 and Lamp2). Based on this analysis, we observed that most of the free cholesterol accumulated into lysosomes. We also discovered an increase in the amount and size of lysosomes as well as divergences in shape compared to their typical round form seen in the parental cell lines Fig.  and b. AAs lysosomal integrity, with cholesterol content of lysosomal membranes and cathepsin D among its regulators, plays an important role in the induction of cell death , we moniGuided by our RNA-sequencing results we selected 33 drugs, known to affect the genes or pathways involved in lysosomal alterations and lipid metabolism as well as some drugs identified in our previous screen . All the clones responded to AZD8055, a dual mTOR inhibitor, although T-47D Tam1 and Tam2 showed reduced sensitivity. Atorvastatin, which inhibits HMGCoA reductase, did not affect the CTG DSS levels Fig.  but was To see whether any of the compounds are able to revert the lipid phenotype prior to reducing the cell viability (ATP-measurement) or induction of cell death (cell count), we specifically monitored the changes in neutral lipids by quantifying the average LipidToxGreen intensity per well  - independent manner, which leads to an increase in its expression [SERPINA1, which is accompanied by a slight increase of ESR1 transcription  is another mechanism by which cells avoid lysosomal induced cell death . We specALDH2, another target of disulfiram, is upregulated in T-47D Tam1 but not in Tam2 , encoding for a rate-limiting enzyme in the biosynthesis of monounsaturated fatty acids, in the tamoxifen-resistant T-47Ds  was observed in tamoxifen-resistant cells Figs.  and 4c. ynthesis . The comTaken together, our results highlight that tamoxifen resistant cell lines can potentially be used as a representative model for studies of tamoxifen-resistant patients. We propose that the breast cancer cells can acquire tamoxifen resistance by dysregulation of different cellular pathways, dependent on their individual molecular phenotypes. Here, we highlight the inhibition of lysosomal membrane permeabilization as one of the mechanisms to avoid cell death, whereas an increase in neutral lipids may enable the further survival of these cells. We further propose that drugs targeting cellular antioxidation machinery may be able to overcome tamoxifen resistance. However, investigating the relevance of the proposed mechanism of acquired resistance in patients remains a challenge. Given the vulnerability of tamoxifen resistant cells to approved drugs such as disulfiram and dasatinib, it would be interesting to investigate whether these compounds could also be effective in clinical trials in tamoxifen-resistant breast cancer patients.Additional file 1:Antibodies used in this study. (XLSX 10 kb)Additional file 2:Drugs used in this study. (XLSX 11 kb)Additional file 3:Statistical analysis of triglycerides, free cholesterol and cholesterol esters, LMP assay, and LipidToxGreen staining. (XLSX 38 kb)Additional file 4:RNA-sequencing statistics. (XLSX 10 kb)Additional file 5:Figure S1. Tamoxifen-resistant cell lines display distinct expression changes. Tamoxifen-resistant clones derived from same parental cells (A) and of the luminal A subtype (B) differ in their expression changes. Venn diagrams show overlap in numbers and percentage of genes that are differentially expressed. (PDF 363 kb)Additional file 6:Detected fusion genes of parental and tamoxifen-resistant cell lines. (XLSX 40 kb)Additional file 7:Batch corrected CPM counts of cell lines and patients. (TXT 28313 kb)Additional file 8:p < 0.001. (XLSX 22 kb)Enriched pathways with adjusted Additional file 9:Drug Sensitivity Scores  of CTG mAdditional file 10:Mean of the mean LipidToxGreen intensity values. (XLSX 59 kb)


Mycoplasmas spp. is among major contaminants of eukaryotic cell cultures. They cause a wide range of problemsassociated with cell culture in biology research centers or biotechnological companies. Mycoplasmas are also resistant toseveral antibiotics. Plasmocin™ has been used to treat cell lines but Plasmocin™-resistant strains have been reported.InvivoGen has developed a new anti-Mycoplasma agent called Plasmocure™ in order to eliminate resistant Mycoplasmacontamination. The aim of this study was the selection of the best antibiotics for treatment of mycoplasma in cell cultures.Mycoplasma species were evaluated by microbiological culture (as the gold standard method), indirect DNA fluorochromestaining, enzymatic (MycoAlert™), and universal or species-specific polymerase chain reaction (PCR) detection methods.In this study, animal and human cell lines available in National Cell Bank of Iran, were treated with Plasmocure™. Thetreatment efficacy and cytotoxicity of Plasmocure™ were compared with those of commonly used antibiotics such as BM-cyclin, Plasmocin™, MycoRAZOR™, sparfloxacin and enrofloxacin. In this experimental study, a total of 100 different mammalian cell lines contaminated with differentMycoplasma spp. A moderatetoxicity was observed during Mycoplasma treatment with plasmocure™; But, after elimination of Mycoplasma, cells werefully recovered. Mycoplasma infections were eliminated by Plasmocure™, BM-cyclin, Plasmocin™, MycoRAZOR™,sparfloxacin and enrofloxacin. However, the outcome of the treatment process  varied among different antibiotics. Plasmocure™ is comprised of two antibiotics that act through various mechanisms of action than those inPlasmocin™. Two-week treatment with Plasmocure™ was enough to completely eliminate Mycoplasma infections in mammalian cell cultures especially for precious or vulnerable cells.The highest number of cured cell lines was achieved by using Plasmocure™ which also had the lowestregrowth rate after a period of four months. As a conclusion; Plasmocure™ might be considered an effective antibiotic to treat Mycoplasma spp. contaminations cause a wide range of economical and biotechnical troubles in cell cultures in biological research laboratories as well as biotechnology companies  is used to treat cell linesinfected by Mycoplasmas and related cell wall-lessbacteria. Plasmocin™ can also be used as prophylaxisfor Mycoplasma and other bacterial contaminations. However, some Mycoplasmas have been reported to be resistant to Plasmocin™ . Plasmocure™is comprised of two antibiotics that act through mechanisms different from those of Plasmocin™ . Two-week treatment with Plasmocure™ is enough to completely eradicate Mycoplasmas .Methods for eliminating c cells , 8. Threlemented , 10. Thasmocin™ . To eradoplasmas . In the www.celljournal.org). All cell lines were analyzed by indirect DNA fluorochrome staining , mycoplasma enzymatic detection kit , universal or species-specific polymerase chain reaction (PCR) detection technique and microbiological culture as the reference method. During the experiments, the cells were incubated at 37°C in 88% humidified air containing 5% CO2 and cultured in medium including 10-20% fetal bovine serum    , 14. In Mycoplasma contamination by above-mentioned methods. For every harvested cell line, Mycoplasma contamination was evaluated after 3-5 days of culturein an antibiotic-free medium. In order to confirm theabsence of contamination with other microorganisms, cell lines were examined through the quality control of microbiological culture , Roswell Park Memorial Institute medium 1640 , F12 nutrient mixture , McCoy’s 5A medium , eagle’s minimum essential medium , Leibovitz’s L-15 medium , earle’s balanced salt solution , horse serum , Trypsin-EDTA , fischer’s medium , penicillin/streptomycin , non-essential amino acid , oxalate, pyruvate, and insulin , human insulin (Sis), bovine insulin , human endothelial cell growth factor , MEBM/MEGM   medium, fibroblast growth factor-basic from bovine pituitary , 200 mM L-glutamine , 100 mM sodium pyruvate , oxalate, sodium bicarbonate , 2-mercaptoethanol , hypoxanthine , thymidine , epidermal growth factor ,granulocyte macrophage colony-stimulating factor (GM-CSF) recombinant human protein . At the beginning, culture media, FBS, trypsin and phosphate-buffered saline  were analyzed and checked for  culture , 15. Celibiotics .Mycoplasma contamination. All the cured cultures were re-examined for regrowth of Mycoplasmas for 4 months following the treatment , Plasmocin™ and MycoRAZOR™ were chosen according to the manufacturer’s instructions. Furthermore, sparfloxacin and enrofloxacin working concentrations were determined according to previously published reports , 16-18. reatment .Mycoplasma colonies, every 3-4 days (The suspended cells 1 ml) were added to 10 ml of Pleuropneumonia-Like Organisms (PPLO) broth medium  supplemented with 10% horse serum , 1% yeast extract agar , L-arginine , D-glucose  and cultured at 37°C for 48-72 hours. In the next step, PPLO medium was vigorously stirred to observe monotonous turbidity. After centrifugation at 1500 rpm for 15 minutes, the precipitate (100 µl) was transferred to a solid PPLO agar  culture plate and incubated at 37°C for 4-6 weeks. Microscopy observation was used to investigate the formation of non-typical colonies or egg form of  ml were Mycoplasma bodies were detected as polymorphous particles with bluefluorescence. For indirect staining, the supernatants of cell cultures that were suspected to be contaminated with Mycoplasma, were added to the Mycoplasma-free Vero cell line   , 20. Mycoplasma detection kit was used according to the manufacturer’s instruction. Briefly, the ratio of the ATPs level in each sample before (Reading A) and after (Reading B) the addition of MycoAlert™ substrate, was considered an indicator for the presence of Mycoplasma contamination. The presenceof contamination was proved if the Reading B/Reading A ratio was greater than 1  was also determined using PCR-based method as described previously , indirect DNA DAPI staining, enzymatic MycoAlert™ and PCR detection  methods. Vero cell line (NCBI C101a) contaminated with several Mycoplasma species, andMycoplasma-free Vero cell line (NCBI C101b) distinct from different sources, were prepared and Mycoplasma-free NSO (NCBI C142) cell line were evaluated by above-mentioned methods and confirmed as positive and negative controls, respectively. Three different Mycoplasma strains including M.hyorhinis, M.arginini and M.fermentans were detectedand identifiedin the positive control cells (Vero cell line contaminated with Mycoplasma (NCBI C101a) by species-specific PCR primers) (The control cell lines of the study were assessed for primers) , 21.2) was used for comparisons of two-bytwo in six groups. In Chi-square tests, the difference among the antibiotics for the treatment of Mycoplasma-infected cell lines was analyzed and interpreted. Differences with a P<0.05 were considered statistically significant.Statistical analysis was performed using SPSS 24.0 software . Non-parametric Chi-square test (χMycoplasmacontamination. The type of mollicutes in each cell line determined by PCR-based method indicating that 65/100 (65%) of the infected cell cultures was contaminated by one Mycoplasma species. Moreover, 19/100 (19%) samples were contaminated with two species and 16/100 (16%) were contaminated with three different species (Table S1) . M.hyorhinis was detected in 46/100 (46%) of the studied samples, M.arginini in 40/100 (40%), M.fermentans in 32/100 (32%), M.orale in 12/100 (12%), A.laidlawii in 6/100 (6%), M.salivarium in 4/100 (4%), M.pirum in 3/100 (3%), and M.hominis, M.genitalium, U.urealyticum and M.pneumoniae in 2/100 (2%). In this study, 100 different human and animal cell lines were randomly selected and assessed for Mycoplasma treatment process are summarized in Table S1  and Figure 2. Mycoplasma infections were eliminated by Plasmocure™, BM-cyclin (Roche), Plasmocin™, MycoRAZOR™,sparfloxacin and enrofloxacin in 91, 70, 66, 55, 33 and 15% of the contaminated cell cultures, respectively. Furthermore, decontamination was confirmed by PCR, as no Mycoplasma was detected in cured cell cultures 14 days after the completion of the treatment period. Mycoplasma regrowth (reinfection or recurrent infection) was observed in 3, 12, 17, 42, 62 and 83% of the cured cell lines four months after treatment with Plasmocure™, Plasmocin™, BMcyclin (Roche), MycoRAZOR™, sparfloxacin and enrofloxacin, respectively. According to the obtained results, the highest level (22%) of cell cytotoxicity (culture death) was observed among Plasmocin-treated cell lines. While, BM-cyclin (Roche), Plasmocure™, sparfloxacin, MycoRAZOR™andenrofloxacin were cytotoxic to up to 13, 6, 5, 3 and 2% of the studied cell lines, respectively . Overall, results reported on antibiotic treatments of cell cultures by different studies are summarized in Table 3. Mycoplasma contamination remains one of the majorproblems in cell culture laboratories. Mycoplasmascan cause significant biological changes in culturedmammalian cells. In fact, consequences of Mycoplasmacontamination are unpredictable and may affect molecularand cellular properties of the infected cells  and Plasmocin™ were used successfully. In case of mild contaminations, particularly for vulnerable or precious cells such as myeloma, lymphoma, hybridoma or primary cultures, MycoRAZOR™ along with fluoroquinolones (sparfloxacin and enrofloxacin) can be used as alternative antibiotics. Plasmocin™ (comprised of a macrolide and a quinolone) acts on the protein machinery and DNA replication by interfering with ribosomal translation and replication fork, respectively. BM-cyclin (Roche) binds to the 30S and 50S ribosomal subunits and inhibits protein synthesis. According to the manufacturer’s information, the bacteriostatic components of BM-cyclin (Roche) are pleuromutilin and tetracycline whereas Plasmocin™ is composed of a macrolide and a quinolone , and vice versa. In case of cytotoxicity and cell death, especially in severe and intensive contaminations with multiple uinolone , 31, 32lication .Mycoplasma infections. Similarly, Molla Kazemiha et al. , ciprofloxacin and MRA , in 65, 66.25, 20 and 31.25% of the cell lines, respectively. In addition, cytotoxicity was reported in 0, 10, 17.5 and 25% of the cell lines treated with ciprofloxacin, MRA, BM-cyclin (Roche) and Plasmocin™, respectively. Nevertheless, recurrent Mycoplasmas infection was observed in 10 to 80% of thestudied cell lines after four months. In another study done by Molla Kazemiha et al. , MRA and ciprofloxacin, respectively. It is noteworthy that, the risk of cell culture loss was 0, 12.5 and 17.5% for ciprofloxacin, MRA and BM-cyclin (Roche), respectively. However, 82.5 (for ciprofloxacin), 62.5 (for MRA) and 12% (BM-cyclin) of the treated cell lines showed Mycoplasma regrowth , sparfloxacin (62%) or MycoRAZOR™ (42%). Plasmocure™, BM-cyclin (Roche) and Plasmocin™ were effective especially in eliminationof M.hyorhinis, M.arginini, M.fermentans and M.orale which were resistant to the other antibiotics used in thisstudy. Plasmocure™ showed the lowest frequency (3%) of regrowth in our experiments while regrowth was observed in 17 and 12% of cell lines treated with BM-cyclin (Roche) and Plasmocin™, respectively. Plasmocure™, BM-cyclin (Roche), Plasmocin™ and MycoRAZOR™ effectively eradicated mollicutes and cured 91, 70, 66 and 55% of the cell lines, respectively. However, sparfloxacin and enrofloxacin were considerably less efficient as they cured only 33 and 15% of the cell lines, respectively. In this study, we observed high frequency ofPlasmocin™ caused the highest rate of culture death (22%), although, it targets the prokaryotic DNA replication and protein synthesis machineries which are different from those of eukaryotic cells. In addition, MycoRAZOR™ showed lower cytotoxicity on the studied cell lines (culture death of 3%), which might reduce the risk of culture loss. Therefore, it may be recommended as the first-line alternative especially in case of expensive or hard-to-obtain cell lines , 10, 34.Mycoplasmas. Thus, we suggest using two or more antibiotics in alternating periods for a successful treatment or eradication of Mycoplasma contamination. For example, BM-cyclin (Roche) or MycoRAZOR™ (inhibitors of protein synthesis) can be used alternately along with ciprofoxacin, enrofloxacin or sparfloxacin (inhibitors of DNA gyrase activity and DNA replication) with specified intervals during the treatment period. Finally, we observed that the combination of two or more antibiotic with different mechanisms of action makes the interpretation of the results more complicated. Based on our experiments, this might increase the risk of culture death or antibiotic resistance of Mycoplasma agent in comparison with other antibiotics for elimination of Mycoplasma contamination in cultured cells. As a conclusion, we recommend Plasmocure™ as an effective antibiotic for the treatment of Mycoplasma infections in mammalian cell cultures especially for precious or vulnerable cells. These findings may also help researches at biotechnology laboratories for selection of appropriate antibiotics for treatment of Mycoplasma contamination in cell cultures. This report suggests Plasmocure™ as a reliable anti-


Among the four cell lines, SNU1196 showed the highest mRNA and protein levels of hENT1. Expression of hENT1 showed a linear correlation with the log value of the half-maximal inhibitory concentration of gemcitabine. During incubation with gemcitabine, pretreatment with hENT1-specific small interfering RNA (siRNA) resulted in higher cell viability than that in samples pretreated with control siRNA. In a clinical evaluation, the median progression-free survival was 24 and 11 weeks among patients with strong and weak intratumoral hENT1 immunohistochemical staining (P = 0.05), and the median overall survival was 52 and 26 weeks (P = 0.15), respectively. In conclusion, this study showed that increased hENT1 expression is associated with a stronger toxic effect of gemcitabine on BTC cell lines. The clinical outcomes in this study suggest that increased intratumoral hENT1 immunohistochemical staining is a possible biomarker predicting better therapeutic effects of gemcitabine on patients with advanced BTC. Further studies are needed to determine the precise role of hENT1 in BTC.Gemcitabine is a principal chemotherapeutic agent for biliary tract cancer (BTC). Expression of human equilibrative nucleoside transporter 1 (hENT1) is regarded as a potential predictive biomarker for a gemcitabine response in some cancers. This study was conducted to investigate the association between hENT1 expression and the effects of gemcitabine on BTC cell lines and on patients with advanced BTC receiving gemcitabine-based chemotherapy. A total of four BTC cell lines, HuCCT1, SNU-478, SNU-1079, and SNU-1196, were tested. mRNA and protein expression levels of hENT1 were measured by quantitative reverse-transcription polymerase chain reaction and western blotting, respectively. Cell viability after gemcitabine treatment was measured in a chemosensitivity assay. For clinical assessment, 40 patients with unresectable or recurrent BTC who were treated with gemcitabine (1000 mg/m Biliary tract cancer (BTC) is a relatively uncommon malignant tumor that originates in the biliary tract including the intra- and extrahepatic bile duct and gallbladder. This cancer has a poor prognosis because of the advanced stage at diagnosis in many cases or frequent recurrence even after curative resection , 2. ThusGemcitabine is an analog of cytidine and a commonly used chemotherapeutic agent for various cancers, including BTC. Just as other nucleoside analogs, it is a prodrug that requires cellular uptake and intracellular phosphorylation. Human equilibrative nucleoside transporter 1 (hENT1) and human concentrative nucleoside transporters 1 and 3 (hCNT1 and -3) are important for the transport of gemcitabine into the cell. Inside the cell, gemcitabine is metabolized to active gemcitabine diphosphate and triphosphate by deoxycytidine kinase , 5. DuriRecently, intratumoral hENT1 has been reported as a candidate predictive biomarker for gemcitabine therapy responses in various cancers –11. NeveThe present study was conducted to investigate the association between hENT1 expression and the effects of gemcitabine both on BTC cell lines and on patients with advanced BTC who follow a GP regimen.2. Gemcitabine was provided by Lilly Korea  for in vitro experiments. The company was not involved in anything related to the study.Four BTC cell lines were analyzed in this study. The HuCCT1 cell line (intrahepatic cholangiocarcinoma origin) was purchased from the RIKEN BioResource Center . The SNU-478 (ampulla of Vater adenocarcinoma origin), SNU-1079 (intrahepatic cholangiocarcinoma origin), and SNU-1196 (extrahepatic cholangiocarcinoma origin) cell lines were acquired from the Korean Cell Line Bank . All the cell lines were cultured in the RPMI 1640 medium  supplemented with 10% of fetal bovine serum (Gibco) and 1% of a penicillin/streptomycin solution (Gibco) and were maintained at 37°C and 5% atmospheric COTotal-RNA isolation, cDNA synthesis, and quantitative reverse-transcription polymerase chain reaction (RT-PCR)hENT1 and GAPDH were as follows: hENT1, 5′-CAGGCAAAGAGGAATCTGGA-3′ and 5′-GGCCCAACCAGTCAAAGATA-3′; GAPDH, 5′-TTCACCACCATGGAGAAGGC-3′ and 5′-GGCATGGACTGTGGTCATGA-3′. The primers were designed by means of published sequence data from GenBank (https://www.ncbi.nlm.nih.gov/genbank/) and a BLAST search (https://blast.ncbi.nlm.nih.gov/Blast.cgi/). The Power SYBR Green PCR Master Mix (Applied Biosystems) served as the reaction mixture, in a 20 μL volume in each reaction capillary. The mRNA expression of the gene under study was normalized to the corresponding expression of GAPDH.Total-RNA was isolated from the cells using the RNeasy Plus Mini Kit , and cDNA was synthesized with the High Capacity cDNA Reverse Transcription Kit . The primer sets for RT-PCR analysis of Cells were washed rapidly with ice-cold PBS and lysed in RIPA buffer  containing 1 mM of the protease inhibitor PMSF  and the Protease Inhibitor Cocktail . The cell extracts were centrifuged at 13,000 rpm for 20 min. The total-protein concentration in the supernatant was measured with the BCA reagent . For each sample, equal amounts of total protein were denatured and separated by sodium dodecyl sulfate 10% polyacrylamide gel electrophoresis and then transferred onto a nitrocellulose membrane. The membranes were blocked for 1 h in 5% skimmed milk powder solution composed of Tris-buffered saline (TBS) containing 0.1% Tween (TBST). Membranes were incubated overnight at 4°C with primary antibodies against hENT1  and β-actin . After three washes with TBST, the membranes were incubated with the appropriate horseradish peroxidase–conjugated secondary antibodies for 2 h . Bound antibodies were detected by means of the ECL reagent  and an X-ray film .Cells were cultivated in culture plates to 60–70% confluence. After a wash with PBS, the cells were treated with the Lipofectamine RNAiMAX reagent  mixed with hENT1-targeting small interfering RNA  or nontargeting control siRNA  at a final amount of 10 nmol in Opti-MEM (Gibco). At 72 h after siRNA treatment, cells were harvested to examine silencing efficiency and the effect of the hENT1 knockdown.50) were calculated in the SigmaPlot software.These hENT1 knockdown cells and control cells were seeded at 3,000–5,000 per well in 96-well plates. After overnight incubation, the cells were treated with gemcitabine (0–100 μM) for 72 h. Cell viability was determined using the Cell Titer-Glo Luminescent Cell Viability Assay Kit . Luminescence was measured on a LMAXII 384 microplate reader . Half-maximal inhibitory concentrations (IC2 and cisplatin 25 mg/m2) [Between June 2012 and May 2014, 193 patients with BTC who received gemcitabine-based therapy were identified via electronic medical records at Seoul National University Bundang Hospital. Among them, 153 patients were excluded due to the following: gemcitabine as adjuvant therapy, single gemcitabine or combination with other agents as palliative therapy, a dose different from that in the current first-line regimen  as previously reported .Grading of hENT1 staining was performed by an experienced pancreaticobiliary pathologist (HK) as follows: 2+, membranous staining in more than 50% of tumor cells; 1+, membranous staining in 5–50% of tumor cells; 0, no hENT1 staining or staining in <5% of tumor cells. Grade 0 or 1+ was regarded as low hENT1 expression, and grade 2+ was considered high hENT1 expression according to another study .in vitro experimental results shown are representative of three or more independent experiments. Pearson’s χ2 test or Fisher’s exact test was conducted to determine the differences between categorical variables. Continuous variables were compared by the Mann–Whitney U test. Kaplan–Meier analysis was carried out to generate survival curves and calculate the median survival periods, which were compared by the log rank test. A two-sided P value less than 0.05 indicated statistical significance. All statistical analyses were performed in GraphPad Prism 7.0  and in R software v3.4.3 (The R Development Core Team).The hENT1 in each cell line was measured in comparison to that in HuCCT1 cells by quantitative RT-PCR  that was used in the ABC-2 trial [This study has several strengths. First, we conducted both cell line experiments and clinical analysis. Owing to the limited number of studies on BTC cell lines, our results are important because we demonstrated that the experimental and clinical results were consistent. Second, all the patients in this study received the same chemotherapy regimen Click here for additional data file.S1 Table(XLSX)Click here for additional data file.


MCTP1 gene in all resistant cell lines. We observed overexpression of the S100A3 and HERC5 genes in TOP-resistant cell lines. Increased expression of the S100A3 gene was also observed in CIS-resistant A2780 sublines. Overexpression of the C4orf18 gene was observed in CIS- and TOP-resistant A2780 sublines. A short time of exposure to CIS led to increased expression of the ABCC2 gene in the W1 and A2780 cell lines and increased expression of the C4orf18 gene in the A2780 cell line. A short time of exposure to TOP led to increased expression of the S100A3 and HERC5 genes in both sensitive cell lines, increased expression of the C4orf18 gene in the A2780 cell line and downregulation of the MCTP1 gene in the W1 cell line. Our results suggest that changes in expression of the MCTP1, S100A3 and C4orf18 genes may be related to both CIS and TOP resistance. Increased expression of the HERC5 gene seems to be important only in TOP resistance.Low efficiency of chemotherapy in ovarian cancer results from the development of drug resistance. Cisplatin (CIS) and topotecan (TOP) are drugs used in chemotherapy of this cancer. We analyzed the development of CIS and TOP resistance in ovarian cancer cell lines. Incubation of drug sensitive cell lines (W1 and A2780) with cytostatic drugs was used to determine the primary response to CIS and TOP. Quantitative polymerase chain reaction (Q-PCR) was performed to measure the expression levels of the genes. We observed decreased expression of the  One of the main reasons for the low effectiveness of chemotherapy in neoplastic disease is the development of drug resistance. A very good model to study this phenomenon is epithelial ovarian cancer (EOC), the most lethal gynecological malignancy . Most paCIS is the most frequently used antitumor agent and is used in lung, testis, and ovarian cancer chemotherapies, among others. CIS inhibits DNA replication and RNA transcription by interacting with the nitrogen atoms of the DNA, preferentially with the N-7 atom of deoxyguanylic acid, which results in intrastrand and interstrand DNA cross-linking , decreasABCG2 gene  and TOP [A2780TR1 and A2780TR2 (A2780 topotecan resistant)] were generated by exposing A2780 cells to CIS or TOP at incrementally increasing concentrations. The human primary ovarian cancer cell line W1 was established using ovarian cancer tissue obtained from an untreated patient. The W1 sublines resistant to CIS [W1CR (W1 cisplatin resistant)] and TOP [W1TR (W1 topotecan resistant)] were obtained by exposing W1 cells to CIS or TOP at incrementally increasing concentrations. The final concentrations used for selecting the resistant cells were 1000 ng/mL of CIS and 24 ng/mL of TOP and were twofold higher than the plasma concentrations of CIS and TOP two hours after intravenous administration. The increase in resistance according to parental drug sensitive cell lines were as follows: 8.9-fold for W1CR vs. W1; 20.0-fold for W1TR vs. W1; 4.1-fold for A2780CR1 vs. A2780 and 3.3-fold for A2780CR2 vs. A2780; 59.6-fold for A2780TR1 vs. A2780 and 48.5-fold for A2780T2 vs. A2780 as described previously ,20.v/v) foetal bovine serum, 2 pM l-glutamine, penicillin (100 units/mL), streptomycin (100 units/mL) and amphotericin B (25 μg/mL)] at 37 °C in a 5% CO2 atmosphere.All the cell lines were maintained as monolayers in complete medium . The graphs were made using Sigma Plot 11.0 .Changes in For amplification, 12.5 μL of Maxima SYBR Green/ROX qPCR Master Mix (Fermentas), 1 μL of each primer  , 9.5 μL t-test.The statistical analysis was performed using Microsoft Excel software. The statistical significance of the differences was determined by applying Student’s MCTP1 gene in all resistant cell lines suggests a non-specific role for MCTP1 in drug resistance. Expression of the C4orf18 gene was characteristic for A2780 drug-resistant sublines. The significance of these genes in the resistance to cytotoxic agents was not described previously and should be further investigated. In contrast, increased expression of the S100A3 and HERC5 genes was described in different cancers. Our results suggest that these genes may also be associated with the development of drug resistance in cancer.In summary, our results show the expression of new genes in response to CIS and TOP treatment in ovarian cancer cell lines. Decreased expression of the


Human cell lines represent a valuable resource as in vitro experimental models. A hepatoma cell line, HuH-7 (JCRB0403), has been used extensively in various research fields and a number of studies using this line have been published continuously since it was established in 1982. However, an accurate genome profile, which can be served as a reliable reference, has not been available. In this study, we performed M-FISH, SNP microarray and amplicon sequencing to characterize the cell line. Single cell analysis of metaphases revealed a high level of heterogeneity with a mode of 60 chromosomes. Cytogenetic results demonstrated chromosome abnormalities involving every chromosome in addition to a massive loss of heterozygosity, which accounts for 55.3% of the genome, consistent with the homozygous variants seen in the sequence analysis. We provide empirical data that the HuH-7 cell line is composed of highly heterogeneous cell populations, suggesting that besides cell line authentication, the quality of cell lines needs to be taken into consideration in the future use of tumor cell lines.The online version of this article (10.1007/s13577-018-0212-3) contains supplementary material, which is available to authorized users. Human cancer cell lines have been widely used in in vitro experiments to study tumor biology and to develop new drugs . Cancer The HuH-7 cell line was established in 1982 from a well differentiated human hepatocellular carcinoma . This ceTumor cells acquire genetic alterations during their evolution, which underlie cancer development, progression and drug resistance . It shouAlthough the HuH-7 cell line has been in regular use for over 25 years, an accurate genome profile has not yet been established. In this study, genetic analyses based on karyotyping, SNP microarray and variant calls on major cancer-related genes were performed to provide a reference standard.l-glutamine, low glucose (1 g/L) and 10% non-heat-inactivated fetal bovine serum without antibiotics. Cells were treated with 0.25% trypsin and 0.02% EDTA, and split at 1/4 dilution. Our standard quality control confirmed that samples were free of mycoplasma and major pathogenic human viruses. Genomic DNA was extracted from cultured cells at passage 49 using the AllPrep DNA/RNA Mini Kit (Qiagen).The HuH-7 cell line has been registered at the JCRB cell bank as JCRB0403 and is distributed worldwide upon request. The culture medium was DMEM with The DNA sample was amplified by the PowerPlex 16 STR System (Promega) and repeat numbers were determined by the ABI 3500 Genetic Analyzer.Metaphase chromosomes were prepared from cells at passage 57 using a conventional protocol . ChromosDNA microarray analysis was performed using a high-density chip, CytoScan HD array (Affymetrix). The data analysis was undertaken using the Chromosome Analysis Suite software (Affymetrix).Target regions were amplified using the Ion AmpliSeq Cancer Hotspot Panel v2 (Life Technologies). Template DNA was prepared using the Ion PGM Hi-Q Chef Kit (Life Technologies) and sequencing was run on the Ion PGM using the Ion 314 chip. Reads were aligned to the hg19 reference and the analysis was carried out using the Ion Torrent Variant Caller Plugin and the Ion Reporter (Life Technologies).Expression of cell surface markers was examined at passage 50 by flow cytometry with a standard protocol  using ei4 cells/cm2, and the saturation density was about 6 × 104 cells/cm2. The doubling time was calculated to be 1.5–2.0 days. Low seeding density or insufficient inhibition of trypsin activity during subculture tended to cause growth retardation.HuH-7 cells showed epithelial morphology and some of them contain small droplets in the cytoplasm . The optimal seeding cell density at passage 45–50 was 1–2 × 10An STR profile of 16 loci is shown in Table S2, which indicates the absence of Y chromosome. This consists of one pattern for 11 loci and two patterns for five loci, which is unique in that the number of homozygous loci is more than that of heterozygous loci.The majority of cells showed a chromosome number between 55 and 63 Fig. . The modAbnormalities were detected in all chromosomes except for chromosome 21, which was an apparently normal chromosome pair t,+der(X)t,-Y,del(1),+der(1)t,del(2),der(2)t,+der(2)t,del(3),+del(3),der(4)t,+der(4)t,der(5)t,+der(5)t,+der(6)t,der(7)t,+der(7)t,del(8),+dup(8),der(10)t,der(11)t,der(11)t,+der(11)t,+der(12)t,der(13)t,der(13)t,der(14)t,der(15)t,+der(17)t,del(18),+der(18)t,+20,der(22)t,+der(22)t[cp10]/59,idem,-del(3),+der(4)t[cp4]/57 ~ 59,idem,-del(3),-der(22),+der(4)t,der(5)t,der(9)t,der(15)t,der(22)t[cp8].Major copy number changes showed regional gains except for a complete deletion at 5q21.3and at Xq21.1 , which were consistent with previous studies . CD44 waThe variable chromosome number in the HuH-7 cell line indicates cellular heterogeneity. This is observed in its diverse karyotypes, implying the presence of clonal variants. The cell population could become heterogeneous due to selective pressure during culture . This woKaryotypes including chromosome rearrangements clearly exhibit differences between normal and abnormal homologues . AnalysiWhole genome sequencing provides a picture of nucleotide composition at the highest resolution; however, the data quality largely depends on the techniques and equipment available . SequencLoss or gain of a whole chromosome is due to chromosome mis-segregation arising through defects in the mitotic checkpoint . MutatioExpression of CD133, known as a cancer stem cell marker, has been reported in a sub-population of the HuH-7 cell line . It is sQuality control of cell lines is essential to obtain consistent and reproducible data, which is important when making comparisons with previous studies or databases . AlthougBelow is the link to the electronic supplementary material.Supplementary material 1 (PDF 896 KB)


Arabidopsis thaliana, respond to a subsequent exposure to mild, non-damaging freezing temperatures with an additional increase in freezing tolerance referred to as sub-zero acclimation (SZA). There is comparatively little information available about the molecular basis of SZA. However, previous transcriptomic studies indicated that cell wall modification may play an important role during SZA. Here we show that CA and SZA are accompanied by extensive changes in cell wall amount, composition and structure. While CA leads to a significant increase in cell wall amount, the relative proportions of pectin, hemicellulose and cellulose remained unaltered during both CA and SZA. However, both treatments resulted in more subtle changes in structure as determined by infrared spectroscopy and monosaccharide composition as determined by gas chromatography-mass spectrometry. These differences could be related through a proteomic approach to the accumulation of cell wall modifying enzymes such as pectin methylesterases, pectin methylesterase inhibitors and xyloglucan endotransglucosylases/hydrolases in the extracellular matrix.Cold acclimation (CA) leads to increased plant freezing tolerance during exposure to low, non-freezing temperatures as a result of many physiological, biochemical and molecular changes that have been extensively investigated. In addition, many plant species, such as  Therefore, the temperature-dependent growth of extracellular ice crystals leads to cellular dehydration stress. Furthermore, expanding ice crystals may compress cells physically, potentially leading to disruption of the plasma membrane and cell death. Therefore, the extracellular space and the mechanical properties of cell walls can be considered to be important determinants of plant freezing tolerance.In temperate and boreal climates, plants are exposed to low temperatures on a seasonal basis. Temperatures below 0 °C can lead to freezing of plant tissues, in particular in exposed parts such as leaves. Ice crystallization takes place in the intercellular spaces3, followed by accumulation of cold regulated (COR) proteins and compatible solutes such as sugars and proline6. In addition, modifications of membrane lipid composition have been reported8. In Arabidopsis, development of CA reaches a plateau after about seven days, when maximum freezing tolerance is attained7. Some plant species, such as wheat, rye and oat can increase their CA freezing tolerance further by an additional exposure to mild, non-damaging freezing temperatures11. This is referred to as sub-zero acclimation (SZA).Plants can transiently increase their freezing tolerance upon exposure to low, non-freezing temperatures . CA induces massive changes in gene expression that are in part regulated by the cold induced CBF transcription factorsArabidopsis during SZA treatments at temperatures between −1 °C and −4 °C for up to seven days13. The molecular basis of SZA is largely different from CA because CBF and COR genes are up-regulated more moderately during SZA than CA12, while many genes that are not regulated at 4 °C are strongly induced or repressed at −3 °C14. In addition, the concentrations of compatible solutes are not significantly changed during SZA12. Therefore, SZA-induced changes are predicted to constitute novel mechanisms leading to increased plant freezing tolerance.Freezing tolerance is also increased in Arabidopsis leaves under SZA showed that cell wall synthesis and modification related genes are over-represented among up-regulated genes14. This is in agreement with electron microscopy results showing that oat leave cells accumulate xyloglucans in golgi vesicle and the extracellular space during SZA11. These results indicate that changes in the extracellular space including cell wall and apoplastic fluid might be key determinants of enhanced freezing tolerance during SZA.Transcriptome analysis of 21. In addition, modification of pectins and hemicelluloses is associated with CA in oil seed rape and wheat22. Changes of the composition of the apoplastic fluid during CA have also been reported. For instance, the concentration of soluble sugars, such as fructans, in the apoplastic fluid is increased during SZA in cereals, suggesting that these sugars may prevent freezing injury to the plasma membrane25. In addition, the presence of antifreeze and ice binding proteins in the apoplastic fluid has been shown for several plant species after CA27. In general, however, only little is known about changes in the extracellular matrix during CA and SZA.However, only little is known about changes in cell wall and apoplast even during CA. Several studies demonstrated that cold acclimation leads to an increase in cell wall content, thickness and rigidityArabidopsis thaliana. Different accessions showed natural genetic diversity in the magnitude of increase in freezing tolerance during CA and SZA. In addition, we found significant changes in cell wall monosaccharide composition from GC-MS analysis and evidence for structural changes in the cell wall from ATR-FTIR spectroscopy. These results could be linked to the list of proteins that responded to CA and SZA in their abundance that was obtained from a proteomics approach.In the present study, we have investigated the effects of CA and SZA on Arabidopsis accessions that were already known to possess high freezing tolerance after CA28 using a well-established electrolyte leakage assay  to −7.32 °C (Te-0) in the NA state and by 2.75 °C with a range from −7.27 °C (Van-0) to −10.02 °C (N14) after seven days of CA at 4 °C. After an additional SZA for three days at −3 °C LT50 varied by 4.45 °C from −10.00 °C (Ms-0) to −14.45 °C (N14). It is interesting to note that natural variation in freezing tolerance was higher after SZA than after CA. Among the six accessions, Ms-0 increased its freezing tolerance only by 1.22 °C during SZA, while N14 exhibited a more strongly enhanced freezing tolerance by 4.43 °C. For further detailed analysis we selected Col-0 as the standard accession with a good SZA response (increase in freezing tolerance of 3.80 °C) and N14 as the accession with the highest increase in freezing tolerance during SZA. This resulted in strong SZA responses in both investigated accessions, however, since they did not strongly differ in their SZA response, we did not expect major differences between the molecular reactions of the two genotypes. With this strategy, we aimed to identify robust SZA responses in Arabidopsis.We first evaluated the SZA response of six say Fig. . The LT5Arabidopsis rosettes were detached from soil-grown plants and incubated in water at −3 °C in the dark. Therefore, treatment effects other than temperature could influence results obtained under SZA conditions. To control for these factors, we subjected samples to an extended cold acclimation (CA+) treatment under the same conditions as the SZA treatment, but at 4 °C. The corresponding electrolyte leakage assays with Col-0 and N14 reproduced the effects of CA and SZA treatments shown in Fig. For the SZA treatment, Arabidopsis leaves (Col-0) did not respond to SZA with increased freezing tolerance after one day of CA at 4 °C, while after two or three days of CA this effect was clearly visible. This indicates that at least a short CA treatment is required to enable the plants to respond to the sub-zero temperature stimulus.Finally, we wanted to know whether the CA treatment was required prior to SZA to trigger increased freezing tolerance. Figure 2CO3 fractions), hemicellulose (KOH fraction) and an insoluble residue containing mainly cellulose , the proportions of these four fractions were maintained in the rosettes of both accessions under all conditions  hydrolysis and subjected to GC-MS analysis. In the total cell wall material CA resulted in significant decreases in Rha, Fuc, Ara, Xyl and Man and increases in Glc and Gal content in Col-0 Fig. . The sam−1 was used for PCA. Scores plots of spectra obtained from Col-0  and 215 (N14) soluble apoplastic proteins from samples without CaCl2 and 395 (Col-0) and 376 (N14) cell wall associated proteins from samples extracted in the presence of CaCl2. The soluble apoplastic proteins accounted for about 15–30% of all identified proteins extracted without CaCl2, indicating a strong contamination in particular with plastidic proteins, while cell wall associated proteins accounted for around 70–80% of the total identified proteins in the fractions extracted in the presence of CaCl2  showed that the increase in freezing tolerance during SZA was indeed due to the sub-zero temperature and not to extended time or any other experimental parameters. Significantly, we also showed that SZA requires a minimum of two preceding days of CA to result in increased freezing tolerance. This aspect will be discussed in more detail below.Since relatively large amounts of plant material (around 3 g) were needed for each cell wall or apoplastic protein isolation, we modified the previously used SZA protocol, where detached leaves were incubated in glass tubes at −3 °C standing with the petioles in water. In the present study we incubated whole rosettes that were submerged in water. To ascertain that the rosettes still showed the previously observed increase in freezing tolerance, we performed electrolyte leakage assays with six In general, our results indicate that major changes in cell wall amount and composition took place during seven days of CA, while no further changes were observed when rosettes were detached and incubated in water in darkness for an additional three days at 4 °C. Three days of SZA treatment under these conditions resulted in much smaller modifications of cell wall composition than seven days of CA.Nidularium minutum)38. This has been interpreted as an increase in the mechanical strength of cell walls against deformation during extracellular ice crystallization18. Plant primary cell walls are mainly composed of cellulose, hemicellulose and pectin. There were no changes in the cellulose or pectin content relative to total cell wall dry weight after any of the cold treatments. However, pectin accumulation during CA has been reported previously38, while our data showed that the relative amounts of pectin-enriched fractions (CDTA and Na2CO3) and relative uronic acid content in these fractions did not change during any of the treatments. However, the amount of pectin relative to plant dry matter also increased substantially during CA in our study, in parallel with the increase in total cell wall material.Total cell wall content was drastically increased only during CA. Such an increase in response to low temperature has previously been reported for oilseed rape, pea and a bromeliad , rhamnogalacturonan I (RG-I) and smaller amounts of rhamnogalacturonan II (RG-II)42. Both PMEs and PMEIs are encoded by large gene families in Arabidopsis  of which only a small number has been functionally characterized, mainly for their roles in pathogen defence42. In line with the FTIR results, some PMEs significantly increased in abundance during SZA and no significantly decreased PMEs were detected in either Col-0 or N14. In parallel, nine PMEIs decreased in abundance during SZA, while six low-abundant PMEIs increased in Col-0 and/or N14. In agreement with these findings, total PME activity increases in Arabidopsis after exposure to 0 °C43. In addition, overexpression of the PMEI13 (At5g62360) gene in Arabidopsis decreases freezing tolerance of NA plants42.Mechanistically, pectin can be demethylated by PMEs and their activity can be inhibited by PMEIs39. We found a significant increase in arabinose content in the CDTA fraction after SZA, but not after CA or CA+. The arabinose sidechains in RG-I are thought to be important determinants of the mechanical properties of cell walls, for example by preventing Ca2+-mediated HG cross-linking and cell wall rigidification39. The resurrection plant Myrothamnus flabellifolius has a high content of arabinose in its cell walls44 and the same has been found for the desiccation tolerant seeds of Arabidopsis and common bean (Phaseolus vulgaris L.)46. Moore et al.47 hypothesized that arabinose sidechains may inhibit excessive cross-linking of pectin backbones and thereby stabilize cell walls during dehydration. As extracellular ice crystallization also leads to dehydration stress, the increased arabinose content of pectin during SZA might prevent the formation of irreversible connections between pectin backbones during extracellular freezing, thereby promoting recovery of cell wall properties after thawing.RG-I is the second major pectin component. It is composed of a rhamnose/galacturonic acid backbone with arabinan, galactan or arabinogalactan sidechains attached to the backbone2), some hemicellulose-associated proteins increased specifically in abundance either during CA or SZA. For example, in both accessions XTH31 and XTH33 significantly increased specifically during CA. XTH31 has xyloglucan endohydrolase (XEH) activity in vivo and may regulate xyloglucan endotransglucosidase (XET) activity together with XTH1750. XTH33 is induced by aphid infestation51. Overexpression of XTH33 causes bigger leaves, wider stems and altered architecture of the adaxial epidermis52, suggesting that this enzyme is a major player in hemicellulose modification. Therefore, alterations of XEH and/or XET activities of XTH31 and XTH33 during CA will affect cell wall properties and contribute to enhanced freezing tolerance. On the other hand, XTH22 specifically accumulated during SZA in both Col-0 and N14. The encoding gene has been found to be cold induced in several studies57. The recombinant enzyme produced in E. coli has higher activity under cold conditions than other XTHs58, while the baculovirus-produced XTH22 behaves similar to other enzymes of this family59. XET activity of XTH22 is regulated by disulfide bond formation and/or N-linked glycosylation60, suggesting that low temperature conditions may cause such modifications. A recent study also revealed the presence of XET activity in XTH2261, suggesting that covalent cross-linking between xyloglucan chains may also play a role during SZA. In addition, the increased abundance of endo-β-mannase 6, together with various glycosyl hydrolases, peroxidases and lipid transfer proteins during SZA indicates additional cell wall modifications that will require further research to elucidate their functional relevance.In the cell wall-bound protein fraction  in several plant species, which inhibit ice crystallization and recrystalizationWhen NA plants were directly exposed to mild freezing temperatures without previous CA, or after only one day of CA, exposure to −3 °C did not result in increased freezing tolerance, suggesting that at least some of the changes induced during CA at 4 °C may be important for the induction of increased freezing tolerance during SZA, even if the corresponding cell wall monosaccharides, proteins and cell wall structures show no further changes during SZA. In line with this argument, a greater number of apoplastic proteins was affected by CA than by SZA. However, it is presently not clear which of the numerous molecular and structural adaptations taking place during CA are functionally relevant for a subsequent SZA treatment. Further mutational studies will be necessary to elucidate such factors.Arabidopsis thaliana plants from the accessions Col-0, Ms-0, N13, N14, Te-0 and Van-0 were grown in soil in a greenhouse at a light intensity of at least 200 μmol m−2 s−1 (8 h day length) and a temperature of 20 °C during the day and 18 °C during the night for three weeks and then at 16 h day length under the same conditions until 35 days after sowing 66. For cold acclimation (CA) plants were transferred to a growth chamber at 4 °C with a 16 h photoperiod (90 μmol m−2 s−1) for an additional seven days as described previously68. For sub-zero acclimation (SZA) and extended cold acclimation (CA+), whole rosettes from CA plants were transferred into 500 mL glass vials containing 20 mL distilled water und placed in a temperature controlled silicon oil bath  at −3 °C (SZA) or 4 °C (CA+) for three days in darkness. During the entire incubation at −3 °C, samples were supercooled under these conditions. Only for the experiment reported in Fig. 12.50 using an electrolyte leakage assay as described in detail recently69.Leaf freezing tolerance was determined as the LTet al.70, Neumetzler et al.36 and Pettolino et al.71. First, approximately 1.5–3.0 g fresh rosette material was dried at 85 °C for 3 d. After weighing, 10 mL 80% (v/v) ethanol were added and samples were homogenized in a ball mill . Subsequently, another 10 mL 80% (v/v) ethanol were added, samples were centrifuged for 5 min at 1,500 g and supernatants were discarded. Pellets were washed at least five times with 10 mL 80% (v/v) ethanol, once with 10 mL acetone and once with 10 mL methanol. Extracted cell wall powders were air-dried. To digest starch in the cell wall preparations, 3 mL 10 mM Tris-maleate buffer (pH 6.9) was added and samples were placed in boiling water for 5 min. Starch was removed by α-amylase digestion (1 U/mg of CWM) for 1 h at 40 °C. This step was repeated with half the amount of α-amylase. Then, 12 mL of cold ethanol was added to the samples and they were incubated at −20 °C for 1 h or overnight. After centrifugation for 5 min at 1,500 g, supernatants were discarded. Pellets were washed three times with ethanol and then air-dried.Cell wall material (CWM) was obtained in accordance with Ruprecht 2CO3 and 4 M KOH (each supplemented with 20 mM NaBH4) in the same way. The final pellets were washed three times with distilled water. Na2CO3 and KOH fractions were acidified to pH 5.0 with acetic acid . All four fractions were transferred into Spectra/Por dialysis tubes  and dialyzed for three days at 4 °C against distilled water, which was exchanged every 12 h. All fractions were dried by lyophilisation .Approximately 30 mg of CWM was transferred into a screw-capped microtube for sequential cell wall fractionation  was transferred into screw-capped microtubes. An aliquot (30 μL) of 1 mg/mL myo-inositol was added to each sample as internal standard for GC-MS analysis. Four biological replicates of each treatment  were prepared.Around 1 mg of all five fractions  for 1 h at 121 °C followed by adding 300 μL 2-propanol and evaporation at 40 °C. This process was repeated twice. Then distilled water (300 μL) was added to each sample and supernatants were collected by centrifugation for 10 min at 14,000 g. Cellulose-derived hexoses were quantified with an anthrone assay2 gas. Subsequently, monosaccharides were converted to alditol acetates as described75 which is modified from Albersheim et al.76. Detection was performed with an Agilent 6890N GC System coupled with an Agilent 5973 N Mass Selective Detector .Neutral monosaccharide composition of all CWM fractions was determined by gas chromatography-mass spectrometry (GC-MS). Aliquots (100 μL) of supernatants after TFA hydrolysis as described above were dried on a heating block under N−1. Spectra were ATR and baseline corrected using the Spectrum 10.4.3 software (PerkinElmer). They were subsequently area-normalized between 1800 and 850 cm−1.The FTIR spectra were obtained from total CWM powder using a PerkinElmer Spectrum GX  spectrometer equipped with a Golden Gate single reflection diamond ATR system . For each sample, 64 scans were accumulated in the range of 400–4000 cmet al.37 with slight modifications. Aerial parts of plants were carefully removed and washed with water. Plant rosettes (Approximately 2–3 g FW) were soaked in 0.3 M sorbitol solution under vacuum. Then rosettes were placed into syringes and centrifuged in a swinging bucket rotor at 1000 × g for 10 min. Exuded sorbitol solution was collected as apoplastic fluid. Subsequently, rosettes were processed with 0.3 M sorbitol solution containing 0.2 M CaCl2 in the same way. To reduce the volume of apoplast fluids, ultrafiltration was performed using an Amicon Ultra 0.5 centrifugal filter system . Protein concentration of the filtrate was measured using the Pierce BCA Protein Assay Kit . Extracted proteins were stored at −80 °C. Four biological replicates of each treatment were prepared.Apoplastic fluids were extracted based on the method of Boudart After the extraction of apoplastic proteins, 50 μg of protein per sample were solubilized with 100 µL 8 M urea in 10 mM Tris-HCl (pH 8) and loaded into Microcon-30 kDa centrifugal filter units (Millipore). Proteins were reduced with 10 mM dithiothreitol and 27 mM iodoacetamide followed by digestion with trypsin . The digested peptides were acidified to pH < 3.0 with 10% TFA. The peptide mixture was purified and desalted on C18 SEP-Pak columns , which were attached to a QIAvac 24 Plus  vacuum manifold. The columns were equilibrated with 1 mL 100% methanol, once with 1 mL 80% acetonitrile (ACN) and twice with 1 mL 0.1% TFA. The peptides were applied to the C18 column and allowed to pass through slowly. The column was washed twice with 1 mL of 0.1% TFA. Peptides were eluted with 800 µL 60% ACN, 0.1% TFA, dried in a vacuum concentrator and stored at −80° prior to mass spectrometry analysis.−1 in a buffer consisting of 3% (v/v) ACN, 0.5% (v/v) acetic acid. Peptide elution was facilitated by increasing the ACN concentration from 3% to 30% (v/v) linearly over 46 min, from 30% to 50% over the next 12 min and from 50% to 80% for the last 10 min. The column was then washed with 80% (v/v) ACN for 7 min at a flow rate of 0.3 μL min−1. Peptide ions were detected in a full scan from mass-to-charge ratio 200 to 1600 at a resolution of 70,000 (AGC target of 3e6 and maximum IT of 100 ms). MS/MS scans were performed for the 15 peptides with the highest MS signal . Peptides for which MS/MS spectra had been recorded were excluded from further MS/MS scans for 15 s.Peptides were resuspended in 30 μL of resuspension buffer . Measurements were performed on a Q Exactive Plus coupled to an Easy nLC1000 HPLC (Thermo Scientific). Eight μL of the samples were loaded onto an Acclaim PepMap RSLC reversed-phase column  at a flow rate of 0.4 μL minQuantitative analysis of MS/MS measurements was performed with Progenesis LC/MS software (Nonlinear Dynamics). Selection of a reference run, alignment and peak picking were performed automatically. The spectra for each MS1 signal peak were exported to Mascot. Mascot search parameters were set as follows: Arabidopsis TAIR10 genome annotation, requirement for tryptic ends, fixed modification: carbamidomethylation (cysteine), variable modification: oxidation (methionine), peptide mass tolerance =±10 ppm, MS/MS tolerance =±0.8 Da, allowed peptide charges of +2 and +3 and maximum of two missed cleavages. Spectra were also searched against a decoy database of the Arabidopsis proteome and results were filtered to ensure a FDR below 1% on the protein level. Additionally, peptide identifications with a Mascot score below 25 were excluded. Mascot results were imported into Progenesis QI. Relative quantitation using Hi-N was selected as a protein quantitation option. The three most abundant peptides were used for this analysis. Lists of peptides and proteins identified in the present study are shown in Supplementary Tables http://suba.plantenergy.uwa.edu.au/) and the total sum of the apoplastic protein abundances was normalized across all samples.Apoplastic proteins were predicted by SUBA3  using the prcomp function in the R software.Statistical significance of differences was determined by Student’s Supplementary Fig. S1, S2, S3, S4; Supplementary Table S1, S2Supplementary Table S3Supplementary Table S4