Genomic instability is a hallmark of breast cancer, contributes to tumor heterogeneity, and influences chemotherapy resistance. Although Gap 2 and mitotic checkpoints are thought to prevent genomic instability, the role of these checkpoints in breast cancer is poorly understood. Here, we assess the Gap 2 and mitotic checkpoint functions of 24 breast cancer and immortalized mammary epithelial cell lines representing four of the six intrinsic molecular subtypes of breast cancer. We found that patterns of cell cycle checkpoint deregulation were associated with the intrinsic molecular subtype of breast cancer cell lines. Specifically, the luminal B and basal-like cell lines harbored two molecularly distinct Gap 2/mitosis checkpoint defects . All subtypes of breast cancer cell lines examined displayed aberrant DNA synthesis/Gap 2/mitosis progression and the basal-like and claudin-low cell lines exhibited increased percentages of chromatid cohesion defects. Furthermore, a decatenation Gap 2 checkpoint gene expression signature identified in the cell line panel correlated with clinical outcomes in breast cancer patients, suggesting that breast tumors may also harbor defects in decatenation Gap 2 checkpoint function. Taken together, these data imply that pharmacological targeting of signaling pathways driving these phenotypes may lead to the development of novel personalized treatment strategies for the latter two subtypes which currently lack targeted therapeutic options because of their triple negative breast cancer status. Unique subtypes of breast cancer display distinctive patterns of genomic instability that affect cell division. Jacquelyn J. Bower and colleagues at the University of North Carolina at Chapel Hill, USA, assessed the molecular function of several G2-M cell cycle checkpoints—which ensure that cells don’t begin mitotic cell division until DNA replication is complete and they have had a chance to repair any damaged DNA—in 24 breast cancer and mammary epithelial cell lines that collectively represent four of the six intrinsic molecular subtypes of breast cancer. The distinct patterns of cell cycle checkpoint deregulation that the researchers found suggest that pharmacological targeting of the dysfunction in each type of breast cancer could lead to personalized treatment strategies. The findings also point to diagnostic biomarkers that could help predict patient responses to novel drugs or existing chemotherapies. The G0/G1 restriction point is the point in G1 at which the withdrawal of growth factors no longer induces reversion to a quiescent state; thus, it controls the cell’s commitment to division.4 The restriction checkpoint is largely controlled by the Rb/E2F signaling pathway: release of E2F transcription factors from Rb allows E2F to transcriptionally activate genes that promote the initiation of DNA replication and S phase entry. Both overexpression of upstream regulators of Rb (such as Cyclin D1/cdk4/6) or inactivating mutations in the RB1 gene can degrade this checkpoint and lead to early activation of DNA replication.6 The G1 DNA damage checkpoint delays the initiation of DNA replication in the presence of DNA damage and is largely controlled by the p53/p21/Mdm2 pathway.8 Loss of this checkpoint can often occur via TP53 mutations or inactivation of wild-type p53 by viral proteins and lead to error-prone DNA synthesis due to the diminished amount of time allotted for DNA repair.10At least four cell cycle checkpoints may be deregulated in cancer cells: the restriction point  without overt DNA damage.17 Major players in both the DNA damage and decatenation G2 checkpoint include the ATM/Chk2/p53 pathway and attenuation of either G2 checkpoint leads to chromosomal instability.18 Finally, the SAC acts in mitosis to delay the onset of anaphase until all chromosomes exhibit bipolar attachment to the mitotic spindle.20 Major effectors of SAC function include APC/C, BubR1, and Mad2, and impaired SAC signaling often leads to the formation of multipolar spindles and/or the unequal partitioning of sister chromatids into daughter cells.21 Defects in all of these cell cycle checkpoints have been shown to play an integral role in tumor initiation and/or progression and affect the sensitivity of tumors to both cytotoxic and endocrine drugs.24Two types of G1 checkpoints in breast cancer signaling, genome maintenance, and chemosensitivity has been previously characterized;25 however, the relationship between G2 or SAC checkpoint function, genomic instability, and chemoresistance is poorly understood. Six intrinsic molecular subtypes of breast tumors have been proposed,27 and several studies imply that these subtypes contain similar molecular defects that contribute to disease progression and variability in chemotherapeutic response,30 which may confer synthetic lethality to subsets of cancer cells.32 Although these studies imply that genomic instability plays a role in these processes, the nature of the specific molecular defects contributing to breast cancer outcomes remains elusive. To explore the underlying mechanisms of genomic instability in breast cancer cells, G2 and M checkpoint functions were assessed in a panel of 24 cell lines using a flow cytometry-based mitotic entry rate (MER) assay that allows for discrimination among three molecularly distinct G2/M checkpoints: the DNA damage G2 checkpoint, the decatenation G2 checkpoint, and the SAC. The panel is comprised of cell lines representing four of the six intrinsic molecular subtypes of breast tumors: six luminal B (LumB), four basal-like (BL), six claudin-low (CL), and four Her2-enriched (Her2E) cell lines. Luminal A and normal-like breast cancer cell lines are unavailable, and four immortalized non-tumorigenic human mammary epithelial cell lines (HMECs) were employed as positive controls. These cell lines recapitulate the gene expression profiles, abnormal genomic features, and phenotypes of in vivo breast tumors,35 and exhibit subtype-specific responses to several chemotherapeutic agents.32The role of the restriction and G2/M checkpoints has been described for breast cancer cell lines to date and existing reports almost exclusively utilize three breast cancer cell lines  to investigate the accumulation of cells in G2/M (but not checkpoint function), the data generated from this study provides a valuable resource for delineating the role of G2/M checkpoints in breast cancer cells. Furthermore, the findings of this study indicate that deregulation of G2 and M checkpoint functions, aberrant cell cycle regulation patterns, and chromatid cohesion defects coincide with breast cancer intrinsic molecular subtypes and that a gene signature for decatenation G2 checkpoint function identified in the cell line panel correlates with clinical outcomes in breast cancer patients. These data collectively suggest that pharmacological targeting of pathways driving these genomic instability phenotypes may lead to the development of new personalized treatment strategies for breast cancer subtypes that currently lack targeted therapies.Because no comprehensive functional analysis of G2/M checkpoint function, a MER assay utilizing two topo II inhibitors exhibiting distinct mechanisms of action was employed to monitor the G2-M transition rate. Cells were incubated with colcemid (to prevent mitotic exit) for 2–6 h in the presence or absence of the topo II catalytic inhibitor ICRF-193 (which does not overtly damage DNA) to measure decatenation G2 checkpoint function or the topo II poison etoposide (which induces DNA damage) to measure DNA damage G2 checkpoint function at concentrations that arrest 98–100% of normal human diploid fibroblasts (NHF1-hTERTs) and immortalized lymphoblasts in G2.36 The use of colcemid in the MER assay blocks cells from exiting mitosis, allowing for a strict examination of the G2 to M transition, thus minimizing any confounding effects related to the rate of mitotic exit. The MER was calculated from the linear portion of the resulting line (2–6 h time points) and is expressed as the percentage of cells entering mitosis per hour.36To assess G2 to M; however, in the presence of the catalytic inhibitor ICRF-193 (open circles), mitotic accumulation of R-HMEC-E cells was severely inhibited suggesting that the majority of these cells activate a decatenation G2 checkpoint response. In the presence of the topo II poison etoposide, the mitotic accumulation of R-HMEC-E cells was also severely inhibited (closed triangles), indicating that this cell line exhibits an effective DNA damage G2 checkpoint. Conversely, the LumB breast cancer cell line MDA-MB-453 continued to accumulate in mitosis in the presence of ICRF-193, but not etoposide , suggesting that a majority of these cells evaded the decatenation G2 checkpoint but arrested in G2 upon DNA damage.MER examples are shown for the HMEC cell line R-HMEC-E in the left panel of Fig 2 checkpoint assay and the averages of three independent experiments for each cell line (grouped according to molecular subtype) are shown in Fig. 2 checkpoint. In contrast, checkpoint functions of the breast cancer cell lines were highly variable  was employed to compare G2 checkpoint functions among the different subtypes ). These analyses established that the LumB class (but not the BL class) of breast cancer cell lines exhibited defects in decatenation G2 checkpoint function . Although the distribution of % inhibition of MER in the BL cell lines initially appeared similar to that of the LumB group, closer inspection of the MER data revealed that the BL group exhibited very low MERs in the absence of a topo II catalytic inhibitor, indicating that this group was not responding to colcemid treatment; thus, the percent inhibition of MER appears artificially low in the BL group.All 24 cell lines were subjected to the MER-based Gble Fig. . Upon gr2 checkpoint using a concentration of the topo II poison etoposide equivalent to that of the catalytic inhibitor ICRF-193 (4 µM). All four HMEC cell lines displayed an effective DNA damage G2 checkpoint in response to etoposide . At least two of these cell lines (HCC1428 and MDA-MB-453) appeared to express low or undetectable levels of p53 despite harboring wildtype TP53 genes.38 Only the MDA-MB-415 cell line exhibited a mutant form (Y236C) and high basal levels of p53, suggesting that p53 activation was attenuated by a mechanism other than mutation in most of the LumB lines. The increment of p-Ser15 p53 upon etoposide treatment for the LumB class was not statistically different from the HMEC class  occurring during continuous culture was also determined by counting the total number of cells during each passage and calculating the doubling time of each cell line (PDL/week). In Fig. Three measures of cell proliferation were compared via flow cytometry including the S phase fraction as determined by EdU incorporation (S), the mitotic index (MI) as measured by MPM22 checkpoint function in LumB cell lines. In Fig. 2/M cell cycle progression to preserve a diploid genome.40 Conversely, this relationship would be disrupted if cell cycle phase transitions were deregulated. Thus, two adjacent markers of cell cycle phase were assessed for the presence of a correlative relationship,41 and an absence of correlation between those markers would imply that the transition between those two cell cycle phases was delayed and/or aberrantly regulated.Because the completion of DNA replication is coupled to the onset of mitosis, concomitant increases in multiple measures of cell proliferation Fig.  are expep < 0.0001 and 0.0041, respectively, Table 2/M phases follows an ordered progression. However, the BL, CL, and Her2E classes exhibited no correlative relationship between S phase fraction and MI suggesting that cell cycle progression is delayed or aberrantly regulated at some point between the initiation of DNA replication and mitotic exit. To further refine the deregulated point(s) in the cell cycle, S phase fraction was compared to the MER measurement to determine if S or G2 progression was delayed. Only the HMEC class (p = 0.0006) exhibited a correlation between the S and MER; all of the breast cancer cell line classes displayed decoupling of the S phase fraction and MER suggesting that these classes may spend longer periods of time in the S or G2 phases, or exhibit aberrant cell cycle control during S/G2. Finally, the HMEC, LumB, and CL classes all exhibited a correlative relationship between the time-dependent MER and MI  suggesting that these classes maintain ordered mitotic progression. In contrast, the BL and Her2E classes showed no significant correlation between MER and MI, indicating that these classes may not respond to the microtubule polymerization inhibitor colcemid and/or exhibit difficulty completing mitosis. The HMEC and LumB classes exhibited a highly correlative relationship between S phase fraction and MI . In contrast, the CL and BL classes appeared to exhibit an increased probability of harboring cohesion defects that ranged from mild to severe . In addition, both the BL and CL classes exhibited significantly higher percentages of cells with aneuploidy when compared to HMECs (>4 CEP9 foci per metaphase), suggesting that cohesion defects may lead to inappropriate segregation of duplicate chromosomes during cytokinesis and confirming the presence of genomic instability in these classes  assay utilizing a probe targeting the centromere of chromosome 9 (CEP9) to confirm the presence of cohesion defects and evaluate severity in the BL and CL classes. In diploid metaphase cells with properly cohered sister chromatids, the centromeres of the sister chromatids are so close together that they appear to be a single CEP9 locus upon FISH staining. However, in cells exhibiting cohesion defects, the centromeric regions of the sister chromatids are separated and exhibit a doublet pattern containing two visually distinct CEP9 loci. Degrees of cohesion defects were defined as mild, moderate, or severe,ses Fig. .43 and severe aneuploidy was observed in both classes  and cell lines that lack a functional SAC (no mitotic accumulation due to an inability to arrest in colcemid). Example flow cytometry profiles for MCF10As (HMEC) and MDA-MB-468s (BL) are shown in the left panel of Fig. middle panels). The average increase in mitotic index for each cell line grouped by intrinsic molecular subtype is shown for three independent experiments Because the MERs of the CL and BL classes were low compared to the HMEC class Fig. , the prees Table , Fig. S22 checkpoint function was identified from the cell line panel using quantitative trait analysis (QTA) , but was close to significant.To determine whether the checkpoint defects identified in the cell line panel were associated with clinical outcomes, a gene expression signature of decatenation GA) Table . This si2 checkpoint signature was also predictive of OS outcomes in LumA patients  or EdU+ cells (S phase) were quantified. Results were obtained from at least three independent experiments for each cell line.Cells were plated (day 0), fed (day 2), and treated (day 3) with 10 µM EdU for 2 h and fixed. Samples were stained with: MPM2-Cy5, Click-It EdU-Alexa 488 substrate, and DAPI ]/log2. PDL/week was calculated by determining the number of PDLs occurring over an average 7 day period.36 Results were obtained from at least three independent experiments for each cell line.On day 3, cells were treated with 100 ng/mL colcemid and 0.1% DMSO, 4 µM ICRF-193, or 4 µM etoposide for 0, 2, 4, and 6 h. MPM2-Cy5/DAPI staining of mitotic cells was performed. The rate of change in the percent of cells in mitosis was calculated by regression of the linear portion of the resulting line (2–6 h time points) and the slope of that line was used to quantify MER (% of cells entering mitosis/h) as previously described.1 Total and phosphorylated proteins were semi-quantified using Image J software v. 1.45 (NIH)  for 0 or 24 h and then fixed, stained, and quantified as described for the MER assay. Results were obtained from at least three independent experiments for each cell line.1 and examined using an Olympus BH2 microscope (100X objective) with a SPOT RT camera. At least 100 metaphases were scored for each treatment group/cell line when available—if fewer than 100 metaphase cells were present, all metaphases present were analyzed. All analyses were performed blinded to sample identity.Metaphases were prepared as previously described,1 At least 100 metaphases were scored per cell line when available—if fewer than 100 metaphase cells were present, all metaphases present were analyzed. All analyses were performed blinded to sample identity on a Zeiss LSM 700 Confocal Laser Scanning Microscope (UNC-CH Microscopy Services Laboratory). The severity of cohesion defects was assessed based on a previous study.42 Metaphases containing more than one type of cohesion defect were categorized as exhibiting the most severe cohesion defect.FISH samples were prepared as previously described.34 Array data were Lowess normalized and expressed as log2 ratios. Of the 24 cell lines comprising the panel, 22 were present in the microarray dataset (R-HMEC-E and BT20 were unavailable). QTA was performed using the significance analysis of microarrays (SAM) package in R.58 Genes that significantly correlated with a quantitative trait (such as % Inhibition of MER in the presence of ICRF-193 to represent decatenation G2 checkpoint function) were selected to generate the gene signature using the QTA tool provided within the SAM package at a low FDR (≤0.1).59 These gene lists were subsequently applied to an independent set of publically available clinical data containing overall survival data, clinical annotations, PAM50 subtyping assignments, and gene expression data for approximately 2000 breast cancer patients.45 Clinical outcome association analyses were performed using a Cox proportional hazards model in the R survival package.Cell line gene expression data were obtained from Dr. Katherine Hoadley and Dr. Charles M. Perou at the University of North Carolina—Chapel Hill and is publicly available.p-values, 95% confidence intervals, and details regarding the specific statistical models employed for each individual analysis are included in the supplementary information .A set of LMMs were developed to compare the intrinsic subtype classes with the HMEC class. All of the results presented in this manuscript are supported by sensitivity analyses performed using the same data set after removal of one or more highly influential data points (based on the well-known Cook’s distance metric). If the statistical conclusions were the same in both the primary and sensitivity analyses, it was concluded that the particular observation did not unduly influence the analysis and the primary analysis result was reported as statistically significant. All Supplementary Figure and Table LegendsSupplementary Figure 1A-DSupplementary Figure 1E-GSupplementary Figure 2Supplementary Figure 3Supplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4Supplementary Table 5Supplementary Table 6Supplementary Table 7Supplementary Table 8Supplementary Table 9Supplementary Statistical Information


The utility of cancer cell lines is affected by the similarity to endogenous tumour cells. Here we compare genomic data from 65 kidney-derived cell lines from the Cancer Cell Line Encyclopedia and the COSMIC Cell Lines Project to three renal cancer subtypes from The Cancer Genome Atlas: clear cell renal cell carcinoma , papillary  and chromophobe  renal cell carcinoma. Clustering copy number alterations shows that most cell lines resemble ccRCC, a few (including some often used as models of ccRCC) resemble pRCC, and none resemble chRCC. Human ccRCC tumours clustering with cell lines display clinical and genomic features of more aggressive disease, suggesting that cell lines best represent aggressive tumours. We stratify mutations and copy number alterations for important kidney cancer genes by the consistency between databases, and classify cell lines into established gene expression-based indolent and aggressive subtypes. Our results could aid investigators in analysing appropriate renal cancer cell lines. Cell lines are central to cancer research, but knowing which cell lines are the best representative of actual tumours is a major challenge. Here the authors provide a resource assessment of 65 renal cell lines to assist researchers in selecting suitable lines for studying specific renal carcinoma subtypes. However, studies have identified molecular differences between commonly used cancer cell lines and human tumour samples1234789Over the past six decades, immortalized cancer cell lines have had an increasingly important role in the study of cancer biology and response to therapeutics. Ideally, a cell line should closely resemble the particular cancer type of interest in order to serve as a suitable With such data now publicly accessible, efforts have been initiated to compare the genomic similarity of commonly used cell lines to known tumour samples. Previous work from our laboratory comparing data from TCGA and CCLE for high-grade serous ovarian cancer (HGSOC) revealed differences between some of the most commonly used cell lines and HGSOC tumour profiles. Additionally, we demonstrated that several cell lines initially classified or widely used as HGSOC were probably derived from other ovarian cancer subtypesVHL, PBRM1, SETD2 and BAP1  is the eighth leading cause of cancer-related death in the US and has an annual incidence of more than 270,000 new cases globallyAP1 refs , 16. RecUtilizing these three rich data sets  we characterize commercially available RCC cell lines with respect to genomic resemblance to human RCC. We further classify the cell lines resembling ccRCC into prognostic groups based on the validated ccA and ccB expression-based subtypes25In our comparison of RCC molecular profiles from TCGA, CCLE and CCLP data, we characterize individual commercially available RCC cell lines and help to distinguish their sub-histology as well as their resemblance to human RCC. These findings may help future investigators select the most appropriate cell line tailored to the RCC subtype under examination.We compared the kidney cell lines from CCLE and CCLP using mutation, CNA and gene expression data , after pDue to the distinct copy number profiles of the common subtypes of RCC, we compared 33 kidney-derived cell lines from CCLE and 32 from CCLP to all 728 TCGA kidney cancer tumours  using CNA data. Our analysis reveals that the cell lines cluster according to well-described RCC subtypes . After eIntriguingly, our comprehensive review of the literature in PubMed Central identified ACHN as the third most highly cited RCC cell line despite the fact that it clusters with pRCC . SN12C iP value 0.003, Fisher's exact test), higher grade . These tumours also display a higher extent of copy number aberrations , and more frequent mutations in genes such as BAP1 (12.3% versus 5.4%), SETD2 (12.7% versus 8.1%) and MTOR (6% versus 4.7%), which are associated with more aggressive disease .It is evident that clustering analysis based on CNAs demonstrated that a subset of ccRCC tumours clusters away from the cell lines, while others cluster with cell lines. We compared these subsets in order to determine the properties of tumours that cell lines might best represent. This revealed that the tumours clustering with cell lines tend to be of higher stage  array and targeted exome data for 415 ccRCC tumours. As expected, our analysis reveals that the cell lines tend to have a higher fraction of genes mutated and higher median CNA compared to the tumours . In the TP53, VHL, PBRM1, SETD2, KDM5C, BAP1, NF2, PTEN, ARID1A, MICALCL, STAG2, SLC1A3, CDKN1A, MTOR, MET, SMARCB1, TCEB1, NFE2L2, PIK3CA, MLL3, FH, FLCN, TSC1, TSC2) recurrently mutated in the three TCGA kidney cancer subtypes based on the three TCGA kidney cancer studies1722ACHN, KMRC1, KMRC3, SNU349, SNU1272, RCC10RGB and TUHR4TKB showing no mutations in these key genes. None of the CCLP cell lines had a mutation in any of the genes FLCN, ARID1A, MICALCL, SLC1A3, STAG2 or TCEB1; while none of the CCLE cell lines had a mutation in FLCN, ARID1A, FLCN, NF2 or TSC1. Discrepancies between CCLE and CCLP for matching cell lines were observed, in line with a previous studyVHL is a notoriously challenging gene to sequence, we additionally culled the existing literature for evidence of VHL mutations in the various RCC cell lines  of CCLE and 14 (42%) of the CCLP RCC cell lines.We next investigated the mutation data for 24 important genes  based on the three TCGA kidney cancer studies1722TP53, VHL, BAP1, NF2, PTEN, ARID1A, CDKN1A, MTOR, MET, SMARCB1, PIK3CA, MLL3, FH, FLCN, TSC1 and TSC2). Given a gene and a kidney cancer cell line common to CCLE and CCLP, we defined three tiers of mutations. Tier 1 consists of cases where both databases report identical mutations. Tier 2 consists of cases where both databases report mutations, but they are non-identical, and Tier 3 consists of cases where one database reports a mutation, while the other does not. Similarly, using 5-valued GISTIC scores for CNAs , we defined three tiers of CNAs. Tier 1 consists of cases where the databases agree on both nature and extent of CNA. Tier 2 consists of cases where the databases agree on the nature (gain/loss) but disagree on the extent, that is, one reports a high-level amplification while the other reports a low-level gain, or one reports a shallow loss while the other reports a deep deletion. Tier 3 consists of cases where one database reports a CNA, while the other reports either no CNA, or an alteration of in the ‘opposite' direction (gain versus loss).To address the discrepancies between CCLE and CCLP data, we investigated mutations and CNAs in 24 key kidney cancer genes .Taken together, our analysis reveals CAL54 as the only cell line with perfect agreement on mutation and CNAs in key kidney cancer genes, with 769-P and a few other cell lines also showing a high degree of concordance. These cell lines might be thought of as the most trustworthy kidney cancer cell lines from the point of view of genomics-directed selection.With respect to canonical copy number events , we firs3132Since CCLP provides allele-specific estimates of integral copy number (using PICNIC)We then analysed gene expression data to investigate whether the cell lines could be classified as the aforementioned prognostic expression-based subtypes ccA or ccBOwing to their genomic diversity and frequent use, we chose to perform xenografts on the three most highly cited RCC cell lines  in order to assess their morphologic features. In our cluster analysis, ACHN co-segregates with tumours displaying amplifications in chromosomes 7 and 17, furthering the notion that this appears to derive from papillary origins. In addition, it has been shown that Type 2 pRCC, which is the more aggressive form, frequently exhibits focal losses in chromosome 9p ref. . ACHN shVHL mutations . Previous studies have confirmed that certain RCC cell lines are truly derived from kidney tumours54041SETD2, BAP1 and MTOR, which have been associated with more aggressive disease and poorer outcomes.By comparing tumours which cluster with the cell lines based on CNAs with tumours that cluster away from the cell lines, we found that the tumours likely to be best represented by the cell lines carry hallmarks of aggressive disease, such as higher stage, higher grade, greater extent of CNAs, and more frequent mutations in genes such as et al., which demonstrated that RCC cell lines had a higher mutational burden compared to primary tumours based on SNP profilingIn addition, we demonstrate that commonly used cell lines have higher fractional mutation rates and median CNAs than human tumours. These findings are consistent with a recent report by Beleut VHL mutations. This finding may relate, in part, to selection pressures for growth of aggressive tumours that are subject to passaging effect over time. Other possibilities include known difficulties in sequencing the VHL gene as well as potential inactivation of the gene via promoter methylation. Moreover, our analysis reveals that genomic alterations with potential activating effect on the mTOR signalling pathway are detected in a significant portion of the ccRCC cell lines. Previous work from our group demonstrated that mTOR pathway activating mutations sensitize patients to rapalogs43in vitro work as well.Further characterization of cell lines that cluster with ccRCC reveals considerable genomic variability with regards to copy number profile and mutations. Through integrative genomic analyses, we highlight the ccRCC cell lines that most closely resemble human tumours based on the presence or absence of characteristic features observed in this particular subtype. More specifically, we show that despite clustering with ccRCC, several of these cell lines lack We address the discrepancy in genomic data from CCLE and CCLP via a detailed comparison of mutations in 15 key kidney cancer genes, and of CNAs in 18 key kidney cancer genes . By emplA growing interest in the prognostic ability of the mRNA-based genetic signature ccA/ccB252Finally, we assessed the morphological architecture of the three most highly cited RCC cell lines  in order to further investigate how the genomic landscape of these cell lines translates histologically. Despite ACHN clustering with pRCC based on CNA data as well as previous reports suggesting similar original histology2038We acknowledge the limitations of this study, including the lack of complete mutational and expression data for every cell line despite utilizing several publically available resources and exploring the available literature. In addition, a relatively restricted number of genes were sequenced for CCLE and multiple sequencing platforms were applied in the various analyses used in this study. Furthermore, several discrepancies were found between CCLE and CCLP, especially in mutation data, as previously reported by othersin vitro examination.In summary, we utilize publically available genomic data from TCGA, CCLP and CCLE to compare the molecular profiles of human RCC tumours to those of commercially available cell lines. We show that the vast majority of cell lines resemble ccRCC tumours, but the highly cited ACHN cell line resembles pRCC. We also show that tumours that are most likely to be well represented by cell lines tend to carry hallmarks of aggressive disease, and conversely, most cell lines resemble the expression-based ccRCC subtype associated with more aggressive disease. This study may therefore serve as a guide for future investigators as to the suitability of particular RCC cell lines for Mutation, CNA and gene expression data for CCLE kidney cancer cell lines was obtained from the CCLE websiteTo compare mutation counts, we used the mutation data available from CCLE and TCGA, which excluded various kinds of putative neutral and common variants. We further excluded mutations from intronic, untranslated region, flanking and intergenic regions, as well as silent and RNA mutations. To compare mutations across the same set of genes, we only used TCGA data for the same 1,651 genes for which CCLE provides mutation data. CCLP and CCLE mutation data was compared using the 1543 genes present in both data sets. For CCLE, we used the file listed as ‘preferred data set' by CCLE, that is: CCLE_hybrid_capture1650_hg19_NoCommonSNPs_NoNeutralVariants_CDS_2012.05.07.maf. This dataset filters out variants that are any of the following: common polymorphisms, have an allelic fraction of <10%, are located outside the CDS for all transcripts, or are putative neutral variants based on low conservation in vertebrates. CCLP only provided one dataset, which had been filtered for likely germline variants by comparison with ∼8,000 normal data sets , we converted the CCLP data to log2 ratios by computing the average total copy number per sample, dividing the integral copy number of each segment by the average total copy number, and taking the logarithm to the base 2. Correlating this with CCLE data revealed a high inter-data set similarity among matched cell lines, confirming that the conversion was meaningful.CN, a threshold T and a length L(i) of segment i:Fraction Genome Altered (FGA) was calculated as follows for a given log2 (sample intensity/reference intensity) value In other words, FGA is the ratio of the sum of the lengths of all segments with signal above the threshold, to the sum of all segment lengths. A log2 (sample intensity/reference intensity) threshold of 0.2  was used for both the TCGA tumour samples as well as the CCLE cell lines. The fraction of chromosome 3p lost was similarly calculated using a threshold of −0.2.For clustering CNA data, we used the gene-wise copy number data for KIRC, KIRP, KICH and CCLE and CCLP kidney cell lines, and (1—Spearman's correlation) as the distance. Hierarchical clustering was employed with average inter-cluster distance based agglomeration for combining sub-clusters.To compare the TCGA KIRC tumours which grouped with or away from the majority of kidney cancer cell lines from CCLE and CCLP , we cut To resolve the discrepancies between CCLP and CCLE data, we compared the mutation and CNA data between kidney cancer cell lines in common for 16 out of 24 key kidney cancer genes (since CCLE only includes these 16 genes among the 1651 genes it screened for mutations). For mutations in a given gene and cell line, we defined three ‘tiers' of mutations, depending on the extent of disagreement between the two databases. Tier 1 consists of cases with identical mutations in both CCLE and CCLP. Tier 2 comprises cases with non-identical mutations in the same gene—while these are discrepancies, they are often close to each other and could potentially be the same mutation, with the discrepancy a result of alignment and other technical issues. Tier 3 consists of cases where a mutation is reported in one database, but not in the other.Similarly, for CNAs, we defined three tiers using GISTIC scores  for a given gene and CNA. Tier 1 comprises cases where CCLP and CCLE agree on the nature and amplitude/extent of the CNA. Tier 2 consists of cases where CCLP and CCLE agree on the nature but disagree on the amplitude/extent of the CNA, that is, one database reports a high-level amplification but the other reports a low-level gain, or one reports a shallow loss while the other reports a deep deletion. Tier 3: consists of cases where a CNA is reported in one database, but not the other.For CCLE, we used the file ‘CCLE_Expression_Entrez_2012-09-29.gct' from the CCLE website, and for CCLP, we obtained the data from ArrayExpress54All programming was done in Perl and RThe number of Pubmed Central articles mentioning one of the CCLE kidney cancer cell lines was determined with the Pubmed Central search builder using several punctuation alternatives for the cell line names .3 in volume, mice were euthanized and tumour was collected for histological analysis. For haematoxylin and eosin staining, tissue samples were fixed in 10% formalin and embedded in paraffin. Sections of 5 μm thickness were prepared. haematoxylin and eosin staining was performed as per standard protocol. Each slide was individually reviewed by an experienced genitourinary pathologist (Y.B.C.).All mouse experiments were performed using an approved protocol under Memorial Sloan-Kettering Cancer Center's Institutional Animal Care and Use Committee. For subcutaneous growth, 4 million cells were mixed 2:1 with Matrigel (BD Biosciences) and injected into NSG mice (The Jackson Laboratory). When the tumour reached 300–400 mmDatabases used in this study are the Cancer Cell Line EncyclopediaHow to cite this article: Sinha, R. et al. Analysis of renal cancer cell lines from two major resources enables genomics-guided cell line selection. Nat. Commun.8, 15165 doi: 10.1038/ncomms15165 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figure and Supplementary Tables


Human embryonic stem cells (hESCs) hold tremendous promise for cell replacement therapies for a range of degenerative diseases. In order to provide cost-effective treatments affordable by public health systems, HLA-matched allogeneic tissue banks of the highest quality clinical-grade hESCs will be required. However only a small number of existing hESC lines are suitable for clinical use; they are limited by moral and ethical concerns and none of them apply Good Manufacturing Practice (GMP) standards to the earliest and critical stages of gamete and embryo procurement. We thus aimed to derive new clinical grade hESC lines of highest quality from fresh surplus GMP grade human embryos.A comprehensive screen was performed for suitable combinations of culture media with supporting feeder cells or feeder-free matrix, at different stages, to support expansion of the inner cell mass and to establish new hESC lines.We developed a novel two-step and sequential media system of clinical-grade hESC derivation and successfully generated seven new hESC lines of widely varying HLA type, carefully screened for genetic health, from human embryos donated under the highest ethical and moral standards under an integrated GMP system which extends from hESC banking all the way back to gamete and embryo procurement.The present study, for the first time, reports the successful derivation of highest-quality clinical-grade hESC lines from fresh poor-quality surplus human embryos generated in a GMP-grade IVF laboratory. The availability of hESC lines of this status represents an important step towards more widespread application of regenerative medicine therapies.The online version of this article (doi:10.1186/s13287-017-0561-y) contains supplementary material, which is available to authorized users. The advent of the first derivation of embryonic stem cells in the mouse  and humaab initio were reported in 2007 [Modifications and improvements have been repeatedly achieved for specific components of hESC derivation in order to obtain high-quality hESC lines for clinical use, including inner cell mass (ICM) isolation, development of human feeder cells or feeder-free substrates and fully chemically defined culture media devoid of animal products , etc.  laboratory processes by which the gametes are procured, and the embryo generated and then procured for stem cell derivation. However, previous clinical-grade lines have been derived exclusively from non-GMP-grade embryos and the majority of them were from cryopreserved and well-developed embryos, originally stored for the patients’ own use in fertility treatment and subsequently donated to research. There are two major disadvantages of such lines; the first concerns the source and handling of the embryonic starting material in IVF laboratories operating at non-European Union Tissues and Cells Directive (EUTCD)/GMP standards. Second, there are ethical and moral concerns over the use of cryopreserved embryos which have been stored for the treatment of the parent couple or could be donated to (“adopted by”) another infertile couple. In order to minimise this ethical concern, we consider that it is important to derive hESC lines from oocytes or fresh IVF embryos which cannot be used for clinical treatment –27.Therefore, for both safety and ethical reasons, the next significant advance towards clinical use of hESC lines is to derive them from embryos created in an IVF laboratory designed and operated to GMP standards but which are then surplus to treatment at the point of patient care, under complete xeno-free and GMP conditions. However, as these embryos have very limited developmental potential, this advance also requires the development of a novel and robust protocol in order to derive stem cell lines efficiently from such material.Here we report the derivation and characterisation of the most advanced clinical-grade hESC lines with the highest ethical and moral status to date. We developed a highly efficient clinical-grade hESC derivation culture system without expensive specialist micromanipulation equipment, from fresh, discarded and poor-quality embryos that are not suitable for patient treatment under stringent GMP and xeno-free conditions from the point of gamete procurement in the IVF laboratory onwards. This development brings the execution of pre-clinical and clinical trials using pluripotent stem cells in regenerative medicine an important step closer.All work with human embryos and hESC derivation was carried out with approval of Central Manchester Local Research Ethics Committee (03/CM/684) and the Human Fertilisation and Embryology Authority (HFEA) licence R0067 and R0171. Informed consent was obtained from all couples who donated their embryos for the present study. As standard, all donating couples were screened for transmissible diseases , and staAfter mechanical and manual disruption of the trophoblast cells or as a whole, the embryos were plated on different adhesive matrices and GMP-qualified human dermal fibroblast (hDF) cells inactivated by treatment with mitomycin C in various culture media. The primary colonies of hESCs were passaged by manual dissection up to passage 8, from which point the cells were then robustly passaged with Trypzean to establish the lines. Details regarding manipulation of embryos and primary colonies of hESCs and culture media/matrices are provided in the Additional file All hESC lines were characterised using in-process and final characterisation methods, including immunocytochemistry evaluating the expression of pluripotent stem cells markers, karyotype with G-banding, comparative genomic hybridisation (CGH) arrays, in vitro/vivo differentiation with embryoid body and teratoma formation, and HLA typing, as described in Additional file Detailed procedures are provided in Additional file One of the major challenges in the development of a derivation system for clinical-grade hESC lines has been the lack of a suitable combination of culture medium with supporting feeder cells or feeder-free matrix to support expansion of the inner cell mass (ICM) . We initWe next performed a screen for GMP-compatible culture media suitable for the derivation of hESCs with hDFs as feeders. We compared each culture system (combination of culture medium with hDFs) for long-term expansion of existing hESC lines. The culture media we tested included KO-SR ZF, NutriStem, StemPro, hESF9, TeSR2, Advanced (modified from ). AlthouMedium hESF9 was able to support hESC proliferation for more than four passages on hDFs, although the cells showed some tendency to undergo differentiation. However, we started by attempting derivations in hESF9. The seeded ICM attached on the feeders and initiated growth with proliferation to form a “dome-like” structure, but the growth resulted in rapid differentiation early during derivation. Different culture conditions were therefore employed immediately after the first two passages of the primary ICM-derived cell colony leading to a successful derivation. A part of the colony outgrowth was transferred onto MEFs in standard hESC medium (KO-SR) and gave rise to a hESC line (MAN9 m), while another part of the same outgrowth was transferred onto new hDFs in medium TeSR2 and gave rise to a research-grade line, MAN9. In contrast, the part which remained from this outgrowth continued to be passaged on the hDFs in medium hESF9, but differentiated significantly losing all colony-forming cells after several passages. The above successful procedure coupled with TeSR2 medium for the establishment of a line is entirely GMP-compatible using a two-step culture system on hDFs.To confirm that it was possible to derive GMP-grade hESC lines with this novel two-step medium system using sequentially hESF9 and TeSR2 on hDFs, further derivations were performed in our clean room, resulting in the lines MAN10–14. MAN10 onwards were derived in the GMP-grade clean room, and MAN11-16 under an HTA licence for clinical application (22627). This GMP derivation system for clinical-grade hESC lines employing clinical-grade hDF feeder cells and the sequential culture media hESF9 followed by TeSR2 is highly efficient: lines were derived from blastocysts with almost invisible ICMs e.g. for MAN13  expression of pluripotent markers including OCT-4, SOX2, NANOG, SSEA-4, TRA-1–60 and lack of expression of SSEA1  and (b); while MAN12 has inherited haplotypes (a) and (c). Haplotype (a) is a relatively common HLA haplotype, with a population frequency of 3.9% in northwest England. It is likely, therefore, that MAN10 may also have independently inherited this haplotype from a separate ancestral source, although this cannot be confirmed. Other common haplotypes include the HLA-A*01, B*08, C*07, DRB1*03, DQB1*02 haplotype (approximately 15%), which occurs at increased frequency in several autoimmune disorders and is commonly referred to as “the autoimmune haplotype”. It is likely that MAN15 carries this haplotype.The clinical-grade hESC lines described here, are of the highest quality yet produced for clinical therapeutic use, for the following reasons: (i) they have been derived from sperm and eggs procured and embryos generated in an IVF laboratory specifically designed and operated to GMP standards, as well as derivation and expansion under a GMP Quality Management System, and (ii) they have been sourced from fresh embryos deemed unsuitable for clinical treatment, and therefore have the highest possible ethical and moral status in terms of embryo viability since these embryos were not intended to establish a pregnancy. This status lowers a significant hurdle to the widespread use of hESCs to provide allogeneic tissue banks for regenerative medicine applications, balanced against the opposition to the use of embryo-derived stem cell lines in many parts of the world. Although autologous stem cell therapies using induced pluripotent stem cells (hiPSCs) and/or somatic cell nuclear transfer-derived hESCs are possible avenues for regenerative medicine therapies, a likely route for widespread, cost-effective application of many hPSC-derived cell replacement therapies is via allogeneic tissue banks of high-quality, carefully screened, HLA-matched hESC lines, as reflected by the latest progress in the world’s first clinical trial with hiPSCs . It is During infertility treatment in the UK, one or two embryos are routinely selected as the ‘best’ ones to transfer in each IVF cycle. Any embryos that are not transferred immediately may be cryopreserved for later use. Embryos that score low on developmental and morphological criteria  are clasFor the purposes of deriving clinical-grade hESC lines, which might be used in the treatment of thousands of patients, using embryos generated at GMP standards that are higher than those required for routine IVF treatment provides an important additional aspect of quality. The human embryo undergoes a complete reorganisation of the genome and epigenome immediately following fertilisation and before the point of hESC derivation, and there is evidence that these processes are disrupted by environmental variables in the IVF laboratory (reviewed in ). TherefAlthough studies have shown that even poor-quality or arrested embryos can give rise to hESC lines –47, the Ideally, clinical-grade hESC lines would be derived in a completely defined culture system without feeder cells. A single study by Ludwig et al.  showed tIt is imperative that hESCs used for clinical therapy are of the highest possible genetic quality. Advances in technology and an increasing cytogenetic knowledge base demand high-quality analysis, hence we have carried out high-resolution array CGH on all lines. Reassuringly, no currently plausible pathogenic variants were identified in any of our hESC lines. Minor aberrations were observed in MAN10, MAN11, MAN13 and MAN15 but without any associated evidence of pathogenicity. Moreover, the microarray data did not indicate the presence of mosaic aberrations, suggesting (within the limitations of microarray technology) that these sequence variants were likely to have been present in the donated embryo and were not induced during derivation of hESCs. Indeed variants shown in Fig. Optimally, transplantation of embryonic stem-derived cells would require availability of histocompatible hESC lines for every patient. Since significant differences in histocompatibility types exist among various ethnic populations, establishment of a bank of lines representing human genetic diversity will be vital for successful therapeutic purposes. Using a combination of our lines MAN10–16, HLA specificities present in 66.8% of the UK population for HLA-A, 39.9% for HLA-B, 55.0% for HLA-C, 61.2% for HLA-DRB1 and 95% for HLA-DQB1 would be covered. Restricted presentation of HLA class II antigens  would suggest that matching for HLA class I specificities would normally take priority for the use of hESCs. It follows that for maximum histocompatibility, it is useful to have cells which are homozygous for HLA, inheriting two copies of the same haplotype, so that a single specificity is present at each locus, reducing the likelihood of direct allorecognition and rejection. These cells are extremely rare, originating most commonly in consanguineous families, but also arising by chance. MAN15 is homozygous at all loci except HLA-A, and therefore has a restricted capacity to provoke an immune response compared with heterozygous cell lines. This feature is advantageous for use in studies of antigen presentation, where reactivity can be assessed without ambiguity, and in cellular therapies, as the presence of only one specificity at each locus minimises the risk of immunological rejection. Thus, the newly derived MAN10–16 lines, especially MAN15, make a significant contribution to the effort to provide an allogenic tissue bank with wide coverage.To our knowledge, the present study, for the first time, reports the successful derivation of high-quality clinical-grade hESC lines from fresh discarded and poor-quality embryos that were originally generated in a GMP-grade IVF laboratory. As hESCs are natural pluripotent stem cells, they remain the gold standard in this field. hESC-derived cells have been used in clinical trials and the preliminary results are promising , 21. Our


Transplantation of donor-derived allogeneic hematopoietic cells causes increased survival in patients suffering from various blood cancers and other hematologic and immunologic diseases. However, this health benefit is limited to certain patients. One major complication is graft-versus-host disease (GVHD) that occurs when donor-derived immune cells recognize host cells/tissues as foreign and perpetrate subsequent destruction. Cytokines are a major class of effector molecules that are involved in GVHD pathogenesis. Proinflammatory cytokines released by activated immune cells including T cells lead to the onset of GVHD. T cell depletion (TCD) is an effective approach for GVHD prevention. Several immune suppressive drugs are also used to treat GVHD. However, these prophylactic and treatment strategies often lead to an immune compromised state that increases the risk for infection and cancer relapse. Considering the adverse effects of TCD and overall immune suppression, more selective managements such as approaches targeting proinflammatory cytokines have emerged as a promising strategy to control GVHD. Therefore, this work is dedicated to review recent development in the studies of cytokines and their future implication in GVHD therapy. Graft-versus-host disease (GVHD) is serious complication after allogeneic hematopoietic cell transplantation (Allo-HCT). During anallo-HCT procedure, hematopoietic cells harvested from an allogeneic donor are transplanted into a patient who suffers a certain type of hematologic malignancy or other hematologic or immunologic disease. After transplantation, however, donor derived immune cells may recognize normal host cells/tissues as foreign and subsequently cause tissue damages leading to onset of GVHD. The common symptoms of acute GVHD include weight loss, alopecia , skin lesions, gastro-intestinal (GI) tract and liver complications that may lead to death . Among vIn MHC-mismatched models of GVHD, T helper (Th) cells differentiated into Th1 subtypes that mediate tissue damage in GI tract and liver. In the absence of IFN-γ, Th cells differentiated into Th2 and Th17 subsets and caused damage in lung and skin. If there is neither IL-4 nor IFN-γ the dominating subset is Th17 cells that lead to tissue damage in skin. If both the IFN-γ and IL-17 are absent, the dominating subset is Th2 cells that cause idiopathic pneumonia in GVHD patients . Further+Foxp3+Treg homeostasis without causing any adverse effect on the graft-versus-leukemia/lymphoma (GVL) response [Among various Th1 cytokines, IL-2 is one of the most studied cytokines for its role in activation, proliferation and expansion of T cells in GVHD. Several studies indicate a potential role of IL-2 in GVHD –14. The response . On the response . In xenoresponse . Taken tAnother member of Th1 secreted cytokines, IL-10  is a regulatory cytokine that play an important role in GVHD. IL-10 can modulate CD4+ T cells functions by down-regulation of another Th1 cytokine IL-2 . IL-10 dAnother major Th1 cytokine, IL-12, produced mainly by dendritic cells and macrophages, is a heterodimeric cytokine that mediated cellular immunity . The dimPro-inflammatory cytokine IFN-γ is mainly produced by activated T-cells, NKT cells and NK cells. IFN-γ inhibits GVHD in lethally irradiated mice receiving allo-HCT but promotes lethality in un-irradiated and sub-lethally irradiated recipients . The ext+ Foxp3+ regulatory cell suppressive function to attenuates GVHD [Another cytokine from Th1 family that plays important role in the GVHD is TNF-α. It is directly involved in tissue damages by inducing apoptosis or necrosis of target cells and synergizes with cytotoxic T lymphocytes and NK cells . Therefotes GVHD . Also, ates GVHD .In summary, the roles of Th1 cytokines in GVHD pathogenesis are diverse yet not completely defined. Management of Th1 cytokines for GVHD prophylaxis and treatment appears promising, but optimization is still an issue that needs further effort.Th2 cytokines can attenuate Th1 cytokines such as TNF-α and interrupt Th1 cytokine cascade post allo-HCT and therefore was once thought to lead to the suppression of GVHD pathogenesis . RecentlTh17 cells are known to release cytokines including IL-17and IL-22 . Th17 cein vivo and is also known as a thymotropic cytokine along with SCF [+CD26+CD4+ T cells in part induces chronic GVHD of the lungs [Apart from Th1, Th2 and Th17 cytokines, various other cytokines also play important roles in GVHD directly or indirectly. For example, TGF-β-dependent CD103 expression is involved in regulating destruction of gut epithelium by CD8+ T cells during GVHD pathogenesis . The antwith SCF ,59. A rewith SCF . Both ILwith SCF . Considewith SCF . In addiwith SCF . The rolwith SCF . Furtherwith SCF . Anotherhe lungs . On the he lungs .+CD103+Fop3+natural Treg can directly migrate to GVHD target organs decreasing disease severity [+Foxp3+ Treg cells and reduction of CD4+CD25− conventional T cells [+ memory T population with high expression of CD11c and α4β7 in gut that plays a pivotal role in initiating gastrointestinal GVHD via promoting Th1 response and cytokine production [In addition to Th1, Th2 and Th17 cells that produce associated cytokines and affect GVHD, there are several important T cell populations that also impact GVHD via cytokines and other mechanisms. Many studies have documented the roles of Treg cells in GVHD. Both natural and induced Treg cells are able to restrain conventional T cell proliferation and attenuate GVHD with multiple mechanisms including IL-2, IL-10 and TGF-β . CD4+CD1severity . Notablyseverity . IL-2 tr T cells . In addi T cells ,71. Furtoduction .Taken together, these studies indicate that a wide range of cytokines and T cell populations are involved in the pathogenesis of GVHD. A thorough understanding of the complex molecular and cellular networks will provide better insights for more effective GVHD management.in vitro study using dendritic cell (DC) culture indicated that bortezomib can inhibit the proliferation of DCs and also blocked expression of costimulatory molecules CD80 and CD86. This drug was also found to diminish IL-12 and TNF-α release in DCs following treatment of LPS [in vitro and in vivo [Recent years witnessed a significant growth in findings related to the role of various cytokines in the pathogenesis and management of GVHD. Several new drugs have been used to treat GVHD based on cytokine regulation. For example, zinc supplementation was reported to be beneficial in the induction of tolerance that amelioratesTh1-dominated allogeneic immune response . A recent of LPS . Another in vivo . The use in vivo . Erlotin in vivo . Moreove in vivo . AlthougIn vivo deletion of miR-142 does not affect T cell development. But in vitro and multiple models of GVHD targeting miR-142 leads to attenuated T cell proliferation [Micro RNAs (miRs) play important roles in regulation of various immune responses such as infection, tumor, and autoimmunity . Recentlferation . Recentlferation . Moreoveferation . These sin vivo. Apart from the expansion of Treg cells, αDR3 also down-regulated proinflammatory cytokines such as IFN-γ, IL-1β, and TNF-α. Notably this GVHD alleviating effect was achieved by a single dose of αDR3 [Uses agonistic antibodies have shown proved efficacy in GVHD management. Recently, αDR3, an antibody to death receptor 3 (DR3), has been investigated for its role in the management of GVHD. DR3 is mainly present on Treg cells, lymphoid tissue inducer cells and NKT cells . Studies of αDR3 . Another of αDR3 . In GVHD of αDR3 . These sCytokines also find uses as biomarkers for GVHD. For example, tear cytokine profile was found associated with systemic chronic GVHD. This finding is evident by the presence of enhanced levels of IL-2, IL-10, IL-17α, IFN-γ, IL-6, and TNF-α tear cytokines in chronic GVHD patients . RecentlIn summary, the role of cytokines in GVHD is indispensable. Several therapeutic interventions based on targeting cytokines have already shown promising results in GVHD amelioration. As Th1, Th2 and Th17 cytokines are associated with the pathogenesis and management, exploitation of these immune mediators will produce practical approaches to combat GVHD. Further studies are required to understand the underlying mechanisms associated with the roles of cytokines in GVHD in order to achieve optimum diagnostic and therapeutic benefits.


Microtubules are cellular targets for a variety of anticancer therapies because of their critical function in mitosis. Taxol belongs to a class of microtubule targeting agents that suppresses microtubule dynamics and interferes with the functioning of the mitotic spindle, thereby effectively blocking cell cycle progression of rapidly proliferating tumor cells. Despite its antitumor activity, drug resistance remains a common obstacle in improving its overall clinical efficacy. Previous studies have shown that the expression of a specific β-tubulin isotype, βIII-tubulin/TUBB3, is dysregulated in drug-refractory tumors. However, whether enhanced TUBB3 expression is directly involved in promoting taxol resistance remains a subject of debate. Here, we have used several approaches to assess the functional relation of TUBB3 overexpression and taxol resistance. First, we generated a number of taxol-resistant cell lines, to find that TUBB3 expression was elevated in a resistant cell line (RPE-20) derived from untransformed retinal pigment epithelial (RPE) cells, but the abundance of TUBB3 remained unchanged in four other cell lines after taxol treatment. However, although RPE-20 cells displayed enhanced TUBB3 levels, we find that simultaneous up-regulation of the P-glycoprotein (P-gP) drug-efflux pump is responsible for the resistance to taxol. Indeed, we could show that TUBB3 levels were dynamically regulated upon taxol exposure and withdrawal, unrelated to the resistance phenotype. Next, we generated cell lines in which we could induce robust overexpression of TUBB3 from its endogenous locus employing the CRISPRa system. We demonstrate that solely enhancing TUBB3 expression results in a very minor decrease in the sensitivity to taxol. This was further substantiated by selective depletion of TUBB3 in a series of breast cancer cell lines expressing high levels of TUBB3. We find that TUBB3 depletion had a minimal effect on the sensitivity to taxol in one of these cell lines, but had no effect in all of the others. Based on these findings we propose that TUBB3 overexpression can only marginally affect the sensitivity to taxol in cultured cell lines. Microtubules, polymers of α/β heterodimers, are dynamic cytoskeletal structures that are essential for many cellular functions, including cell movement, intracellular transport and cell division. Particularly during cell division, cells depend on the formation of a highly dynamic microtubule network, the mitotic spindle, which facilitates faithful segregation of chromosomes to the two new daughter cells . Since uPaclitaxel (hereafter referred to as taxol) is an MTA that suppresses microtubule dynamics and thereby disrupts mitotic progression. This mode of action is thought to be responsible for the potent ability of taxol to prevent cell proliferation in tumors , 5. TaxoTaxol specifically targets the β-subunit of tubulin , of whicin vitro studies demonstrated that TUBB3 enhances the rate of tubulin depolymerization in the presence of taxol  > 1 | [x/y] < −1.Raw data were analyzed by MaxQuant (version 1.5.8.3)  using st


It also exhibits strong suppression of tumor growth. In our previous study, we assessed the anti-neoplastic effects of 7-O-succinyl macrolactin A tromethamine salt  on a glioma cell line. Moreover, according to our data, SMA salt might be contributed to the inhibitory effects on migration and invasion, as well as a cytotoxic effect on the glioblastoma cell lines. In the present study, we investigated the anti-tumor effects of combination therapy with SMA salt and temozolomide (TMZ) in glioblastoma cell lines. The combination therapy affected cell viability significantly, decreasing in glioblastoma cell lines. In cell migration assays, combination therapy showed more inhibitory effects than TMZ in these cell lines. The tumor volume was significantly decreased in the combination group compared with both TMZ and control groups by using the orthotopic mouse model. The effects of combination therapy with SMA salt and TMZ attributed to the inhibition of migration, invasion activities and anti-tumor effects. SMA salt could be one of the promising candidates for combination therapy in clinical settings. Glioblastoma (GBM) is the most frequent primary malignant brain tumor in adults , 2. AlthTMZ is a well-known alkylating agent and is applied widely with GBM as a first-line treatment –11. TMZ In recent years, the anti-angiogenic agent has gained popularity from researchers and physicians. Some groups worked on anti-angiogenic agents, such as cilengitide or bevacizumab –17. HoweBacillus polyfermenticus KJS-2 [7-O-succinyl macrolactin A (SMA) and macrolactin A (MA) are macrolactins generated from us KJS-2 . Macrolaus KJS-2 . Macrolaus KJS-2 . Then, Sus KJS-2 , and Romus KJS-2 . Accordius KJS-2 , 23. In us KJS-2 .In the present study, we investigated the anti-tumor effects of combination therapy with TMZ and SMA salt in GBM cell lines.We assessed the effects of combination therapy on GBM cell lines for 48 hours. In the U87 cell line, the viability of the combination group (39.9%) was significantly lower than other two groups  at the concentration of SMA salt at 25 μM (P < 0.001 and P <0.0001). In U251 and LN229 cell lines, the combination groups (46% and 40.3%) also showed significantly lower cell viability than the TMZ group  and SMA salt group  at a 25 μM concentration of SMA salt Figure . MoreoveWe performed the cell migration assay with the 24-well Transwell apparatus and followed the manufacturer's protocol. Eventually, the stained migrating cells were counted to assess the effects of SMA salt and TMZ. The cell migration abilities in U87 cell lines were significantly decreased in the combination group (12.2%) than in the SMA salt (25.03% and P < 0.001) and TMZ groups (44.01% and P < 0.001) Figure . SimilarWe also evaluated the inhibitory effect of invasion in three cell lines, U87, U251, and LN229. The combination group showed greater inhibitory effects than the SMA salt and TMZ groups on cell invasion in the three cell lines Figure . In the 3) was significantly smaller than in the TMZ group  and the control group  . However, there was no significant difference between the combination group and TMZ group. We assumed that the main reason there was no difference between the two groups was due to the blood-brain barrier (BBB) and time. SMA salt may or may not cross the BBB. To discover this, we would like to do more research on the BBB and SMA salt. Furthermore, we should prolong the period of survival analysis to observe the difference between the temozolomide group and the combination group. Overall, the Another strong effect of SMA was anti-angiogenesis. Anti-angiogenesis was explored in publications by Judah Folkman in the 1970s, which is an interesting issue for those researching tumors. According to some reports, SMA exhibited stronger anti-angiogenic activity than MA and showed suppression of tumor growth and tumor-induced angiogenesis in an MDA-MB-231 human breast cancer cell-inoculated CAM assay . The maiin vitro and in vivo experiments to confirm anti-tumor effects and combination therapy with cilengitide and belotecan to prolong the survival of mice. These results showed a favorable expectation for the treatment of glioblastoma. In a multicenter randomized open-label phase 3 trial for cilengitide combined with standard treatment, however, they demonstrated dismal results [Recently, many papers on chemotherapy treatment, especially anti-angiogenesis, were published –16, 28.  results . Patientin vitro and in vivo. Although the effect of combination therapy in survival analysis is not as satisfactory as our previous study, we might suggest that the effects of combination therapy with SMA salt and TMZ attributed to the inhibition of migration and invasion activities and anti-tumor effects on GBM cell lines. SMA salt could be one of the promising candidates for combination therapy in clinical settings. The exact mechanisms of combination therapy of TMZ and SMA salt in GBM cell lines will need more examination in future studies.In conclusion, this study has shown the feasibility of combination therapy on GBM cell lines 2.The U87MG, U251MG, and LN229 cell lines were human glioma cell lines purchased from American Type Culture Collection . Cells were cultured in complete Dulbecco's Modified Eagle Medium (DMEM), which was supplemented with 10% fetal bovine serum (FBS) and incubated at 37°C and 5% CO3 cells per well to allow for overnight adhesion. The next day, the cells were treated with SMA salt at a concentration of 25 and 50 μM and TMZ at a concentration of 500 μM. After 2 days, we added 10 μL of the CCK-8 solution per well and then incubated for 2 hrs at 37°C and 5% CO2. The optical density (OD) of the sample plate was measured at 450 nm by an ELISA reader . The viability of the tumor cells was assessed by calculating the OD ratio of the specific OD in each sample to the OD of the control sample.The cytotoxicity of SMA salt , TMZ and a combination of SMA salt and TMZ was measured using the Cell Counting Kit-8 . Each cell line was seeded into 96-well plates at a density of 5 × 105 cells/mL) were treated with SMA salt, TMZ, and the combination of SMA salt and TMZ and prepared in serum-free medium. FBS-containing medium (750 μL) was added to the lower chamber and 200 μL of prepared cell suspension was added to the insert. After 24 hrs, cells that were remaining in the insert  were gently retrieved using a cotton-tipped swab and allowed to air dry for 20 min. A solution of 0.4% crystal violet (500 μL) was added to each well of the apparatus. After 10 min, the migrated cells that traversed the membrane separating the insert from the lower chamber were stained by dipping the lower surface of the membrane into the stain. The stained membranes were washed several times with water and allowed to air dry. The results of cell migration assays are described as a percentage .The insert of a 24-well Transwell apparatus  was incubated at 37°C for 1-2 hrs. The U87MG, U251MG and LN229 cells (2 × 105 cells/mL) were prepared in serum-free medium, and 200 μL of prepared cell suspension was added to each insert. Medium containing FBS (750 μL) was added to the lower chamber of the Transwell apparatus. After 24 hrs, non-invading cells were removed, and the invading cells were quantified as described above [Matrigel  was thawed overnight at 4 ng and diluted (1-5 mg/mL) in serum-free cold DMEM. A 100 μL volume of the diluted Matrigel was added to the upper chamber of a Transwell apparatus and incubated at 37°C for 4-5 hrs to allow the gel to swell. The treated U87MG, U251MG and LN229 cells . Finally, the hole was sealed and the skin was sutured. All of these steps were followed in our lab protocol. This study was approved by the Institutional Animal Care and Use Committee of the Medical Science Research Institute, Seoul National University Bundang Hospital (authorization number: BA1411-164/058-01).Six-week-old Female Balb/c-nu mice were ordered from Orient Bio . Thirty mice were used for tumor volume and survival analysis. Each experiment utilized 15 mice. Fifteen mice were divided into 3 groups in each experiment. All of them were anesthetized and the head was fixed in a stereotactic frame, followed by the creation of a midline scalp incision (1 cm). A small hole was made 0.5 mm anterior and 2 mm lateral from the bregma. A sterile 10 μL Hamilton syringe with a #26S needle was inserted at a depth of 3.5 mm from the surface of the skull and withdrawn by 0.5 mm to inject 2 × 10The orthotopic mice were randomly divided into three groups: a control group, TMZ group and combination group . The mice in the control group were injected with saline only, and both SMA salt and TMZ were administered at a dose of 50 mg/kg daily intraperitoneally in the TMZ and combination groups. The drug treatments began 7 days after the implantation of tumor cells and for 5 days per week. Half of the mice were sacrificed 5 weeks after the implantation of the tumor cells for tumor volume analysis; the remaining mice were observed for another 3 months to analyze survival. Death was defined as a weight reduction to 75% of their initial weight or an unexpected sudden death before that weight was reached.L), width (W) and height (H) of each tumor sample was measured and used the following formula to calculate the tumor volume [When we obtained mice brains, the mice were perfused with phosphate-buffered saline (PBS) and then fixed in 4% paraformaldehyde; after that, the brain was removed for paraffin embedding. The fixed brains were coronally sectioned into slices of 5 μm thickness. The slices were mounted and stained with hematoxylin and eosin. The maximal length .We used Student's


CYCD7;1  using the imprinted FWA promoter overcomes cycle arrest of the central cell in the Arabidopsis female gametophyte in the unfertilized ovule, leading to multinucleate central cells at high frequency. Unlike FERTILIZATION‐INDEPENDENT SEED (fis) mutants, but similar to lethal RETINOBLASTOMA‐RELATED (rbr) mutants, no seed coat development is triggered. Unlike the case with loss of rbr, post‐fertilization endCYCD7;1 in the endosperm enhances the number of nuclei during syncytial endosperm development and induces the partial abortion of developing seeds, associated with the enhanced size of the surviving seeds. The frequency of lethality was less than the frequency of multinucleate central cells, indicating that these aspects are not causally linked. These larger seeds contain larger embryos composed of more cells of wild‐type size, surrounded by a seed coat composed of more cells. Seedlings arising from these larger seeds displayed faster seedling establishment and early growth. Similarly, two different embryo‐lethal mutants also conferred enlarged seed size in surviving siblings, consistent with seed size increase being a general response to sibling lethality, although the cellular mechanisms were found to be distinct. Our data suggest that tight control of CYCD activity in the central cell and in the developing endosperm is required for optimal seed formation.In angiosperms, double fertilization of the egg and central cell of the megagametophyte leads to the development of the embryo and endosperm, respectively. Control of cell cycle progression in the megagametophyte is essential for successful fertilization and development. Central cell‐targeted expression of the D‐type cyclin  CYCD activity is needed for seed development.Controlling cell cycle progression in the female gametophyte is essential for fertilization and development. Here we use targeted activation of a cyclin in the central cell to show that division of the central cell nucleus can be uncoupled from egg cell proliferation. Further phenotypes of these lines show that accurate control of  Seeds consist of three compartments – the mature embryo, the endosperm and the encapsulating seed coat – carrying different balances of maternal and paternal genome  mutants also triggers seed coat differentiation . In complex with their cyclin (CYC) regulatory subunit , allowing the activation of imprinted genes in the maternal germ line . Previous work showed that a protein fusion of GFP to the FWA promoter fragment and the N‐terminal homeodomain and nuclear localization signal of the FWA protein (dFWA) is sufficient to target the GFP to the nuclei of the central cell in the female gametophyte, prior to fertilization, and the nuclei of developing endosperm, until cellularization  ovules, the gametophyte central cell contains a single nucleus, located towards the egg apparatus on the micropyle pole of the ovule Figure a. In endt Figure b,c. In ln Figure e, the nun Figure d, displafie mutants is a consequence of the fertilization‐independent proliferation of the central cell that occurs together with the acquisition of endosperm fate and the fertilization‐independent differentiation of the seed coat. We therefore examined ovules for evidence of premature seed coat differentiation using vanillin staining , a high proportion of endCYCD7;1 ovules contain 16–20 nuclei in the endosperm, compared with between seven and nine in the WT ovules , only 10% were at the 2/4‐cell stage and no embryos were still at the embryo‐proper stage. At 4 DAP, the majority of WT embryos (54%) were in the globular stage, only 27% had progressed beyond this point and 19% were still in the 16‐cell stage. endCYCD7;1 embryos were mainly at the triangular and globular stages. At 5 DAP, line A had 72% of embryos at the heart stage and 17% of embryos at the triangular stage. Line D had 28% at the heart stage and 41% at the triangular stage. A high proportion of endCYCD7;1 embryos reached the heart phase faster than in the WT, but the cellular patterning of the heart stage embryo was unaffected .The rate of embryo development progression was also influenced by 1 Figure . At 3 daendCYCD7;1 seed development slowed, with a marked effect on the transition from the heart phase to the torpedo phase. This could be consistent with a reduction of CYCD7;1 expression in the endosperm at this stage; however, detailed analysis of the different lines showed that from 7 DAP onwards, the line with the strongest level of expression (line A) tends to develop more slowly than the WT, whereas seeds from the line with the lowest expression level are still somewhat further ahead in seed development than WT seeds , it therefore appears that the accelerated endosperm development in endCYCD7;1 lines stimulates the early phases of embryo development, but may subsequently lead to a slower progression to later stages beyond the heart phase.In conclusion, although reaching heart phase faster, et al., et al., et al., et al., et al., et al., et al., endCYCD7;1 seeds was compared with seeds from untransformed Col‐0 WT plants and from WT segregants derived from the primary transformants, all obtained from plants grown alongside each other under the same conditions  × 103 μm2. WT segregant lines produce seeds with areas ranging from 110 × 103 to 128 × 103 μm2, and were no significantly different from the untransformed WT . In contrast, homozygous endCYCD7;1 plants produced significantly larger seeds , 143 × 103 μm2 , 125 × 103 μm2 , 116 × 103 μm2 . The seed size increases were similar between manual and natural occurring endCYCD7;1 self‐crosses (Table S1). When endCYCD7;1 pistils were pollinated with WT pollen, seed sizes were similar to the seeds produced by endCYCD7;1 self‐fertilization , and for the four endCYCD7;1 lines, seeds were larger than WT × WT crosses . In contrast, when WT pistils were pollinated with endCYCD7;1 pollen, the average seed area did not significantly differ from the average for seeds from WT × WT (P = 0.35), and was significantly smaller than the for seeds from endCYCD7;1 self‐fertilized plants (P = 3.9 × 10−4).The endCYCD7;1 genotype of the female gametophyte.Taken together, these results show that the production of enlarged seed is conferred by the endCYCD7;1 lines, however : WT mature siliques measured on average 1.61 ± 0.17 cm in length, whereas endCYCD7;1 siliques were significantly shorter . As the siliques were shorter, the total number of healthy and aborted seed in the pods was recorded . In mature WT siliques, 5% of the total number of seeds produced were aborted, whereas in endCYCD7;1 siliques, 80% of seeds were aborted in line A, 65% in line B, and 50% in lines C and D .A negative effect on silique length was observed in the D Figure d,e. HencendCYCD7;1 embryos had aborted  that had progressed beyond globular phase, suggesting that the early stages of embryo development are particularly susceptible to the effects of endCYCD7;1.To understand better the stage at which this lethality arises, we examined siliques 29 h after pollination and found that a significant proportion of developing d Figure d, althouendCYCD7;1 plants, and the proportion aborted was recorded in both mature siliques and during early stages , similar to the number of seeds developing and the proportion aborting in the WT pistil pollinated by WT pollen . Similar results were found for the three other endCYCD7;1 lines . CDKA1;1 encodes the catalytic partner of CYCD and is required for embryo development . In the nclpp2+/− mutant, 20% of ovules showed an abnormal ovule development. Of the properly developed ovules in nclpp2+/−, 96% of ovules showed a single central cell nucleus and 4% displayed two or three nuclei in the central cell of the gametophyte. The presence of extra nuclei in the central cell of unfertilized ovules suggests a lack of fusion, uncoordinated cellularization or division in the formation of the female gametophyte and ovules in the nclpp2 background. These results were in contrast to the endCYCD7;1 ovules with the penetrant supernumerary central cell nucleus phenotype . Although each of these mutant alleles conferred a significant lethality in the mature silique of 45 and 75%, respectively, the timing of the abortion appeared to differ. At 48 h after pollination, 75% of ovules in the nclpp2+/− progeny were already degenerating and not progressing in embryogenesis, whereas 25% progressed, albeit with a delay in embryogenesis. In contrast, in the cdka1;1–1+/− pistils no ovule degeneration was observed at this point in time, although embryogenesis was delayed . Nevertheless, despite these substantial differences in the phenotypes leading to lethality, in both cases a substantial increase in average seed size was observed compared with either the progeny of a Col‐0 WT or the progeny of a WT sibling of the heterozygote mutant , similar to endCYCD7;1 lines. These observations suggest that, at least under growth‐room conditions, sibling lethality has a strong effect on the size of the remaining seeds, consistent with either the removal of silique space constraints or a strong sink effect of the ovules normally limiting the growth of Arabidopsis Col‐0 seeds in the silique.The proliferation of the endosperm post‐fertilization was, on average, delayed in endCYCD7;1 expression, we examined the different seed compartments. A WT embryo had a volume of 25 (±4.7) × 106 μm3, whereas endCYCD7;1 embryos had volumes ranging from 61 (±10.1) × 106 μm3 (line A) to 38 (±11.3) × 106 μm3 (line D), correlated with levels of CYCD7;1 activation , indicating that the enlarged overall seed size is associated with larger embryos.In order to understand the larger seed phenotype and the effect of endCYCD7;1 radicles have no extra layers, and have a radial pattern similar to that of WT. The length of the first 16 cortical cells, upwards from the cortical–endodermis initial (CEI), was measured. The average length of first cortical cell in a WT root was 6.5 μm. In endCYCD7;1, cortical cell lengths ranged from 5.5 to 6 μm depending on the line observed . Overall, the cell length increased up the root, and for both genotypes the cortical cell length is statistically different, depending on the position with respect to the CEI .No defects in embryonic anatomy were observed Figure a. Confoc3 μm2 , 116 × 103 μm2 (line B), 106 × 103 μm2 (line C), and 97 × 103 μm2 (line D). As the embryo volume increased, the cotyledon area in endCYCD7;1 was significantly enlarged, with a 29–60% increase . The average surface areas of epidermal pavement cells were 194 μm2 for the WT and 210, 196, 200 and 199 μm2 for endCYCD7;1 lines A, B, C and D, respectively . A Bonferroni multi‐comparison test revealed that only from endCYCD7;1 line A were the pavement cells significantly larger, by 8% . The ratio of cotyledon surface:epidermal pavement cell surface showed that the WT area consisted of 388 cells, and that the endCYCD7;1 area consisted area of 572, 589, 529 and 448 cells for lines A, B, C and D, respectively . Given the small effect on cell size, but the large increase in cell number, we conclude that in endCYCD7;1 lines the major driver towards additional embryo growth is an increase in cell number in the mature embryo and hence cell production during embryo growth.The cotyledon areas and the average pavement cell areas were also measured in the mature embryo Figure a, making Figures a and S5aendCYCD7;1 seed coat. The size of cells in the outer layer of the outer integument was measured on mature dry seeds. Cells of the outer integuments had on average an area of 778 ± 220 μm2 for the WT . The average cell areas for the endCYCD7;1 lines were 854 ± 256 μm2 (line A), 857 ± 261 μm2 (line B), 821 ± 285 μm2 (line C) and 807 ± 237 μm2 (line D), which do not significantly differ from the WT cell area . The WT seed area was 12.3 × 104 μm2, whereas endCYCD7;1 seed areas were significantly larger, with averages of 17.4 × 104 μm2 (line A), 17.3 × 104 μm2 (line B), 16.2 × 104 μm2 (line C) and 14.3 × 104 μm2 (line D) . Therefore, the estimated number of cells in the outer integument inferred by the ratio of seed and cell area was 159 for WT. The endCYCD7;1 lines contained significantly more cells, with averages of 204, 203, 197 and 181 for lines A, B, C and D, respectively . Therefore, the larger endCYCD7;1 seed is linked to more cell proliferation in the various seed compartments rather than increased cell expansion.A similar effect was seen in the cdka1;1–1+/− and nclpp2+/− plants are larger, but on average contain fewer cells in the outer integuments, suggesting that cell expansion is underpinning the increase of seed size in these mutants .Our analysis of surviving siblings in the other embryo‐lethal mutants examined, however, showed that an increase in cell number is not a prerequisite for larger seeds. The surviving seeds derived from heterozygote endCYCD7;1 plants would influence the early development of the seedlings, we compared germination rate and seedling development up to 7 days in WT and endCYCD7;1 lines   and msi1 , and this is proposed to integrate genetic and epigenetic regulation of endosperm growth  in 70% ethanol, washed three times with 70% ethanol. Sterilized seeds were dispersed on GM medium . The seeds were stratified (4°C for 48 h) and germinated on GM, then transferred onto soil. Plants were grown at 21°C under a 16‐h light/8‐h dark photoperiod.Seeds were surface sterilized with 5 g LArabidopsis thaliana ecotype Columbia (Col‐0) was used. pFWA:NLS‐3xVENUS and pFWA:CYCD7;1 constructs were introduced using floral‐dipping methods  or d,l‐phosphinothricin (15 μg ml−1). The transgenic line carrying pBCH2‐pFWA:dFWA‐GFP was used  translational start was amplified by PCR from genomic Arabidopsis Col‐0 DNA and subcloned HindIII‐BamHI in the pBluescript SK‐II (pBSC_pFWA) . The CYCD7;1 coding sequence was amplified by PCR from cDNA derived from Col‐0 seedlings. The CYCD7;1 coding sequence was then inserted in BamHI‐SacI in the pBSC_pFWA. The pFWA:CYCD7;1 cassette was excised from HindIII‐SacI and inserted in the plant binary vector pGPTV‐bar  and pCUC2:3xVENUS (BamHIII‐NotI), respectively . Total RNA (0.5 μg) was used to perform a OneStep RT‐PCR (Qiagen). ACTIN2 transcripts were used as a reference. Primer sequences are available on request, and the intensity of the amplicons was compared on agarose gels.Total RNA was extracted from siliques emerging from flowers using the RNeasy Plant mini Kit  from the WT were pollinated and left for 1.5 days before sampling.WT and endCYCD7;1 were dissected from pistils, and siliques were incubated in an acidic solution (6 N HCl) of 1% (w/v) vanillin (Sigma‐Aldrich) at room temperature (20°C) for 20 min and rinsed in H2O prior to differential interference contrast (DIC) microscopy. Vanillin reacts with the proanthocyanidins synthesized by the seed coat of the fertilized ovules, producing a red reaction product . Cleared samples were observed using a Zeiss Axio Imager M1 microscope equipped with DIC optics (http://www.zeiss.com).For examination with DIC optics, seeds and ovules were cleared and mounted in chloral hydrate/30% glycerol , approximately 230 seeds were scored for each line. The percentage of seeds at each embryo stage was recorded .Mature dry seeds were photographed under a Leica microscope (MZ16F) using a LeicaFire Cam digital camera , the following light‐path settings were used: YFP‐VENUS was excited with 514 nm and emission was recorded by a 519–621 nm bandpass filter; and for propidium iodide, excitation was set at 543 nm, and emission recorded through a 493–572 nm bandpass filter).et al., m sodium metabisulphite, 0.15 N HCl, propidium iodide 100 μg ml−1) for 1 h at room temperature. Embryos and seeds were rinsed with water and cleared with a chloral hydrate solution. Embryos and seeds were examined with a confocal laser microscope (Zeiss LSM 710).Dry seeds were imbibed for 2 days in the dark at 4°C. Mature embryos were extracted from the seed coat and fixed with 50% ethanol/10% acetic acid for 24 h, whereas dried mature seeds were fixed directly with 50% ethanol/10% acetic acid for 24 h . Differences between means for different populations were assumed to be significant when P > 0.05.Statistical analysis using one‐ and two‐way Figure S1. Heart‐stage embryos in WT and endCYCD7;1 lines.Click here for additional data file.Figure S2. CYCD7;1 expression under the activity of FWA induces an increase of seed abortion.Click here for additional data file.Figure S3. Developmental characterization of cdka1;1–1+/− and nclpp2+/− mutant‐derived ovules and seeds.Click here for additional data file.Figure S4. Characteristics of mutant and endCYCD7;1 seeds.Click here for additional data file.Figure S5. Features of enlarged endCYCD7;1 mature seeds.Click here for additional data file.Figure S6. Germination of seeds derived from endCYCD7;1, WT and nclpp2+/−.Click here for additional data file.Table S1. Reciprocal crosses between Col‐0 WT and endCYCD7;1 lines reveal a maternal origin of seed size increase .Click here for additional data file. Click here for additional data file.