BNLF2a and BCRF1 genes which may participate in the survival of infected cells. These results support the involvement of EBV in AITLs.Angioimmunoblastic T cell lymphoma (AITL) is probably the most common peripheral T-cell lymphoma. This pathology, although rare, is more common in Europe than in other regions. This lymphoma has a poor prognosis. AITL is very commonly associated with the Epstein-Barr virus (EBV) although the virus is not often found in neoplastic T cells but rather in adjacent B cells. Our objective was to study the transcriptome of EBV in AITLs comparatively to other EBV-associated lymphomas and to compare the results with those obtained for cell lines. We showed in AITLs a strong expression of Bam-HI A rightward transcripts (BARTs) more expressed than in the other lymphomas and especially than in cell lines. BARTs can participate in tumor development. We also showed a latency IIc in AITLs with the expression of BNLF2a gene expression. For most AITLs, BCRF1, which encodes a homologous protein of human interleukin 10, vIL-10, was in addition expressed. This co-expression can contribute to immune escape and survival of infected cells. Considering these results, it can be assumed that EBV plays a pathogenic role in AITLs.The Epstein–Barr virus (EBV) is associated with angioimmunoblastic T cell lymphoma (AITL) in more than 80% of cases. Few studies have focused on this association and it is not clear now what role the virus plays in this pathology. We used next-generation sequencing (NGS) to study EBV transcriptome in 14 AITLs compared to 21 other lymphoma samples and 11 cell lines including 4 lymphoblastoid cell lines (LCLs). Viral transcripts were recovered using capture probes and sequencing was performed on Illumina. Bam-HI A rightward transcripts (BARTs) were the most latency transcripts expressed in AITLs, suggesting they may play a role in this pathology. Thus, BARTs, already described as highly expressed in carcinoma cells, are also very present in AITLs and other lymphomas. They were poorly expressed in cell lines other than LCLs. AITLs showed a latency IIc, with  Epstein–Barr nuclear antigen-2 (EBNA-2), -3A, and -3C genes) [The Epstein–Barr virus (EBV) is a widespread human gamma-herpesvirus of which two types can be distinguished, EBV type 1 (EBV-1) and EBV type 2 (EBV-2), according to geographic distribution, virulence, and differences in the latent genes  and non-coding RNAs, Epstein–Barr virus-encoded small RNAs (EBERs), are also transcribed, plus a set of miRNAs. This latency pattern, defined as latency III (Lat III), drives B cell growth transformation resulting in the establishment of permanent in vitro lymphoblastoid cell lines (LCLs). Lat III is found in immunocompromised lymphomas.First resting B-cell EBV infection leads to expression of the Epstein–Barr nuclear antigen-2 (EBNA-2) and EBNA-Leader Protein (EBNA-LP) proteins as well as latent BHRF1, a bcl2 homolog protein, driven by the activated viral promoter Wp . The upsOther latency patterns have also been described in which the latency gene expression is more restricted ,7,8. LatIn contrast to the latent state, entering the lytic cycle corresponds to the activation of more than 80 genes . Gene exEBV is involved in various B cell malignancies, such as endemic BL where it is found in about 80% of cases , HL withBNLF2a. A group of six late genes was also expressed .For these reasons, in this study, we examined the EBV transcriptome in 14 AITL biopsies compared to other EBV positive lymphomas and cell lines by RNA-seq, We demonstrated for AITLs a significant expression of BARTs superior to that observed for other lymphomas. In addition, we reported that AITLs exhibited latency IIc with a strong expression of in situ hybridization (ISH). Unfortunately, we did not have enough material to study which cells carried the virus. NGS results are shown in We performed massive parallel sequencing by NGS after mRNA enrichment then EBV transcripts capture, to evaluate the EBV transcriptome in 11 EBV positive human lines, which included 4 LCLs, and 35 EBV positive lymphomas from patients, including 14 AITLs, selected based on EBER positivity by EBNA-1, LMPs, EBNA-2, and BARTs), and 17 other genes were expressed for the majority of patients while the other genes were only slightly or not expressed at all.Analysis of results for all patients showed that samples from the 14 AITL patients did not form a cluster . The samWe analyzed the viral transcripts present in samples and, because we performed a poly(A)+ capture before sequencing, we did not detect any non-polyadenylated transcripts, for example, the EBERs.For AITLs, the results obtained show that the most expressed latency transcripts are BARTs B, which BNLF2a. Among the lytic genes, it seems that six of them, the late genes BCRF1, BSRF1, BVRF1, BNRF1, BFRF3, and BOLF1, present a clustered expression in the majority of AITLs. Interestingly, BCRF1 encodes for a protein that shows great homology with human interleukin-10 (IL-10) and is referred to as v-IL-10.AITL biopsies and more largely all patient samples tested carried latency II EBV as evidenced by the lack of EBNA-3 expression A. NevertWe studied the EBV transcriptome in B95-8 line, initially obtained from a primary infection, in four BL lines , in two NK/TL lines (SNK6 and MEC04), characteristics of which are mentioned in BHRF1 which codes for a bcl2 homolog and miRNAs. EBNA-LP was also highly expressed in all cell lines except MEC04. It can be noticed that MEC04 has a very different profile from other lines.It may be noted that the cell lines expressed little or no EBNA-3 and that only LCL and Raji expressed EBNA-2 . BARTs wLF1, LF2, and LF3 for B95-8 or EBNA-2 for P3HR1 , resulting in down-regulation of the HLA class I proteins, thus blocking antigen presentation to cytotoxic T lymphocytes ,66. BNLFytokines , and proytokines . Jochum ytokines  demonstrBCRF1, five other late genes were expressed in almost all AITL samples. This strongly expressed gene cluster is made up of BSRF1, BVRF1, BNRF1, BFRF3, and BOLF1. Apart from BCRF1 and BNRF1, which would play a role in cell immortalization, the others, as far as is known today, encode integument or capsid proteins. Clusters of lytic genes activated during latency have been described by others in NPC, GC, or BL [In addition to C, or BL , but theEBNA-LP , or latency III for Raji, with a weak EBNA-3 expression. This latency change for Raji has already been reported and is due to growing conditions [To better understand the behavior of EBV in lymphoma tissues, we decided to compare the results we obtained for our patients with those obtained for cell lines. We, therefore, studied the EBV transcriptome in the following lines: B95-8, four BL lines, two NK/TL lines, and four LCLs. Surprisingly, we did not find viral behavior specific to a pathology, just as LCLs do not all behave in the same way. Latency gene expression was particularly interesting, and EBNA-LP was highly expressed in all lines except MEC04. BARTs were somewhat more expressed in LCLs than in other lines, although the expression was low. As expected, our LCLs were found to be in latency III although, depending on the lines, the latency genes were expressed to varying degrees. The BL lines, that are derived from tumors exhibiting latency I, were mostly in latency I, while expressing nditions ,71.Interestingly, our lines showed much lower BART expression than patient tumors apart from the MEC04 line which had a very unique behavior. The MECO4 cell line was initially established from a patient with a fatal nasal NK-cell lymphoma at a leukemic stage . MEC04 iBHRF1 which otherwise was very weakly expressed in patient samples. It encodes a protein whose role is poorly defined but which is a bcl-2 homolog and therefore exhibits anti-apoptotic properties essential for cell transformation [LMP2A. In this study, the MEC04 line is the only one that expressed, albeit very weakly, BHRF1 and LMP2A were, in contrast, highly expressed.Among the most expressed genes in all lines was ormation ,75. EBV ormation . They coormation . InteresSpontaneously growing EBV-positive B-cell lines (LCLs), CoAN, DPL, KREB2, and MLEB2 cell lines, were established from the peripheral blood of four subjects whose characteristics are reported in 2 atmosphere. MEC04 and SNK6 cell lines were cultured under the same conditions and supplemented with 100 U/mL of human IL-2 .In addition to the LCLs established in our laboratory, seven other lines were used, the characteristics of which are mentioned in The population included in this study was composed of 14 patients with AITL and 21 other patients suffering from HL or B or T non-HL lymphoma . All pat®  on the Benchmark XT automatonTM (Roche Diagnostics). This was followed by staining with Ventana ISH iVIEW blue plus detection kit®  on the same apparatus.Detection of EBER1 by ISH was used to determine the presence of EBV allowing sample selection. Briefly, the formalin-fixed paraffin-embedded (FFPE) tissue sections were deparaffinized, rehydrated in a graded solution of xylene and alcohol, then deproteinized with proteinase K before incubation with the Ventana EBER 1 DNP ProbeFor EBV positive samples, further work was performed on frozen material collected at the same time as the FFPE tissue.To determine the type of EBV present in each patient sample, we aligned sequences obtained against the unique EBNA-2/EBNA-3 regions of the prototype genomes of EBV type 1 (NC_007605) and type 2 (NC_009334) EBV. The prototype strain for which a greater number of reads matched for a given sample corresponded to the type of that sample.®  according to the manufacturer’s instructions and treated with DNase I by using RNase-free DNase set® . The frozen biopsies were previously disrupted and homogenized by adding lysis buffer and using the Precellys 24 tissue homogenizerTM  while the lines were directly lysed by lysis buffer. Extracted RNA was re-suspended in RNAse-free water. RNA was then quantified and quality was determined using the Agilent RNA 6000 Nano Kit®  on the Agilent 2100 BioanalyzerTM to obtain the RNA integrity number (RIN).Total RNA was extracted from cell lines and frozen biopsies by using the RNeasy mini kit®  according to the manufacturer’s protocol.In brief, 700 ng high-quality total RNA, with a RIN ≥ 9, was used as input for each sample. Poly(A) mRNAs were selected by using oligo(dT) beads in a KAPA mRNA capture kitThe prototype genomes of EBV type 1 (NC_007605) and type 2 (NC_009334) EBV were used as references for designing EBV probes . Probes (100 to 120 bp) were designated to be overlapping and cover the entire viral genomes a minimum of 5 times, without matching the human hg19 genome (GRch38.p13), as determined by the SSAHA algorithm. Coverage for EBV-1 and EBV-2 genomes was estimated at 99.7% and 99.9%, respectively . The vast majority of designated probes were unique, although some probes had a higher degree of multi-locus homology to increase coverage of all regions.TM system  to analyze the EBV transcriptome in the cell lines and selected patient samples (Bronner). The technique consists of an EBV mRNA capture from the poly(A) mRNAs by using NimbleGen SeqCap RNA Enrichment SystemTM (Roche Diagnostics) according to the manufacturer’s protocol. Briefly, sample libraries were prepared using the KAPA Stranded RNA Library Preparation kit® . Selected poly(A) mRNAs were first fragmented by enzyme digestion. Then first and second cDNA strands were synthetized and the obtained fragments were blunt-ended. A tailed and ligated to specific adapters from SeqCap Adapter kits® A and B . Subsequently, sample libraries were amplified for 11 cycles by a ligated mediation PCR (LM-PCR). At this step, a qualitative control using the High Sensitivity DNA kit®  on the Agilent 2100 Bioanalyzer and a quantitative one using Qubit dsDNA HS Assay Kit®  were performed. The amplified sample libraries were then pooled in equal molar quantities and hybridized to EBV biotinylated probes at 47 °C for 3 days consecutively using SeqCap Hybridization and Wash Kit® . At that time, the hybridized fragments were recovered by magnetic streptavidin-beads  and enriched by 14 cycles of LM-PCR using SeqCap EZ Accessory kit v2® . Size selection was performed during library preparation using a single Agencourt AMPure XP treatment from SeqCap Pure Capture Bead kit®, to remove DNA fragments below 200 bp. The final concentration was measured by the Agilent High Sensitivity DNA kit and libraries were finally 2 × 300-base paired-end sequenced on an Illumina MiSeq instrumentTM .We used the high-throughput Illumina MiSeqThe sequencing of all cell lines was carried out in its entirety in triplicate.Data obtained were checked for quality using FastQC v 0.11.5  and no trimming was done. Raw sequencing reads were aligned against hg19 (GRch38.p13), EBV1 (NC_007605.1), and EBV2 (NC_009334.1) reference genomes using bwa mem 0.7.17-r1188. Data were then quantified using featureCounts 1.6.0 . Samples were then averaged and put in log. Statistically, differentially-expressed genes were visualized with heatmaps  using pheatmap , tidyverse , hrbrthemes , and viridis  R packages (built under R version 4.0.2).In summary, our EBV transcriptome study on 14 AITL biopsies was compared to the results obtained for the other lymphomas tested and showed that BARTs were much more strongly and more frequently expressed for AITLs suggesting that they might play a role in this lymphoma. We have shown that AITLs exhibited a latency corresponding to the latency IIc described by Strong et al.  and that


DUSP4 and PPP2R2A reduces the proliferation of melanoma cells. DUSP4 encodes an inhibitor of ERK, suggesting that further activation of MAPK signaling activity through its loss is selectively deleterious to melanoma cells. Collectively, these data present a resource of genetic dependencies in melanoma that may be explored as potential therapeutic targets.Targeting the MAPK signaling pathway has transformed the treatment of metastatic melanoma. CRISPR‐Cas9 genetic screens provide a genome‐wide approach to uncover novel genetic dependencies that might serve as therapeutic targets. Here, we analyzed recently reported CRISPR‐Cas9 screens comparing data from 28 melanoma cell lines and 313 cell lines of other tumor types in order to identify fitness genes related to melanoma. We found an average of 1,494 fitness genes in each melanoma cell line. We identified 33 genes, inactivation of which specifically reduced the fitness of melanoma. This set of tumor type‐specific genes includes established melanoma fitness genes as well as many genes that have not previously been associated with melanoma growth. Several genes encode proteins that can be targeted using available inhibitors. We verified that genetic inactivation of  DUSP4. The melanoma‐specific genetic dependencies identified might be explored as novel specific therapeutic targets.Genome‐wide CRISPR‐Cas9 genetic screens in tumor cell lines allow systematic identification of genetic dependencies that may be cancer type‐specific. Here, we analyzed available data from such screens aimed at identifying genes, inactivation of which impairs proliferation of melanoma cells. A subset of the melanoma fitness genes encode proteins that can be inhibited with available small molecule inhibitors. Our data show that for growth melanoma cells not only depend on activators but also on inhibitors of MAPK signaling activity such as 1BRAF, NRAS, KIT, or NF1 genes. These oncogenic mutations commonly occur in the early stages of melanoma development . Gene expression data for the 28 melanoma cell lines were available for analysis from the CCLE data portal. The data sets are available at https://data.broadinstitute.org/ccle/CCLE_RNAseq_081117.rpkm.gct , penicillin (100 I.U./ml)/streptomycin (100 μg/ml), and GlutaMAX (Thermo Fisher Scientific). A375, IGR1, and WM983B melanoma cell lines were obtained from ATCC/Rockland, and HEK293T cells were available from lab stocks (LUMC). Cell lines were STR profiled, tested negative for mycoplasma, and cultured in a humidified incubator at 37°C and 5% CO5 cells were seeded in 6‐well plates or 2.5 × 105 cells in 6‐cm dishes. Cells were transduced next day in 2 ml or 5 ml culture medium supplemented with 8 μg/ml polybrene to reach a MOI of 3. Cells were incubated overnight at 37°C and cultured for 4 days in fresh medium supplemented with blasticidin‐S (5 μg/ml) and puromycin (2 μg/ml) for selection and further analysis.Lentivirus for stable Cas9‐expressing cell lines was produced by transfecting pKLV2‐EF1a‐Cas9Bsd‐W (Addgene #68343) into HEK293T cells together with packaging vectors (psPax2 and pMD2.G). For gene inactivation experiments, two sgRNA sequences per gene of interest were cloned into a plasmid DNA vector (U6‐sgRNA‐PGKpuro‐2A‐BFP) from the Sanger CRISPR‐Cas9 genome‐wide arrayed sgRNA library Table  containi2.4A375‐Cas9, IGR1‐Cas9, and WM983B‐Cas9 cells were transduced with lentiviral sgRNA expression vectors and cell lysates were prepared for immunoblot analysis as described previously , 2,000 cells/ml (IGR1), and 3,000 cells/ml (WM983B) for colony formation assays. Cells were fixed for 5–10 min in 4% paraformaldehyde (PFA) and stained for 30 min with crystal violet (0.05%) when control wells transduced with negative control sgRNAs targeting 2.6p‐value<.01).IGR1‐Cas9 and A375‐Cas9 cells were seeded in 96‐well plates to monitor cell confluency using the IncuCyte live‐cell analysis system (Essen BioScience). For this assay cells were seeded at a density of 50 cells/well (A375) and 200 cells/well (IGR1) in 6 replicates and scanned images of each well were taken every 12 hr. Data were analyzed using IncuCyte software and confluency was calculated over 6 days (A375) and 8 days (IGR1) and normalized to the confluency on day 1 to correct for seeding variation. Colony formation and cell confluency assays were performed in biological duplicates, and combined data were analyzed using GraphPad Prism version 8. A 2‐way ANOVA and Bonferroni's multiple comparisons test were performed to detect statistically significant differences in cell confluency  were extensively processed and analyzed. In these genome‐wide CRISPR‐Cas9 dropout screens, the depletion of sgRNAs from a genome‐wide library was measured in populations of Cas9‐expressing tumor cells following 21 days of culture after transduction , and the remainingline was triple‐wild‐type. There were no KIT mutations in any of the tested melanoma cell lines , effect size , and gene function, we proceeded with functional in vitro studies of two genes encoding for proteins with a reported inhibitory effect on MAPK signaling activity, DUSP4 and PPP2R2A. DUSP4 was a significant fitness gene in 16 melanoma cell lines including one NRAS‐mutant, and PPP2R2A significantly affected fitness of 16 melanoma cell lines, including one NRAS‐mutant and one NF1‐mutant   as well as an independent melanoma cell line (A375). This suggests that DUSP4 could be an effective target across a larger panel of melanoma cell lines.Consistent with findings from the CRISPR‐Cas9 screens, inactivation of DUSP4 did not induce a growth disadvantage in the IGR1 cell line, showing heterogeneity between different melanoma cell lines with respect to fitness effects. In addition, not only BRAF‐mutant but some NRAS‐mutant melanoma cell lines were sensitive to inhibition of DUSP4 and PPP2R2A. PPP2R2A was confirmed to be a fitness gene in the two cell lines included in the initial CRISPR‐Cas9 screens, IGR1 and WM983B as well as in the independent A375 cell line. PPP2R2A is a PP2A regulatory subunit that has been reported to inhibit MAPK signaling by dephosphorylating ERK (Koutsioumpa et al., Genetic inactivation of DUSP4 and PPP2R2A results in decreased cell proliferation. Collectively, these data present a resource of genetic dependencies in melanoma that may be explored as potential therapeutic targets.The generation of effective treatment strategies in melanoma remains a challenge. Here, we present an analysis of CRISPR‐Cas9 screen data aimed at melanoma cell lines, identifying 33 genes that specifically affect the fitness in this tumor type. In vitro experiments in human melanoma cell lines confirmed that inactivation of The authors declare that there is no conflict of interest to disclose.Fig S1Click here for additional data file.Fig S2Click here for additional data file.Fig S3Click here for additional data file.Table S1Click here for additional data file.Table S2Click here for additional data file.Table S3Click here for additional data file.Table S4Click here for additional data file.Table S5Click here for additional data file.Supplementary MaterialClick here for additional data file.


Real-time quantitative PCR (RT-qPCR) has been widely applied in uncovering disease mechanisms and screening potential biomarkers. Internal reference gene selection determines the accuracy and reproducibility of data analyses. The aim of this study was to identify the optimal reference genes for the relative quantitative analysis of RT-qPCR in fourteen NF1 related cell lines, including non-tumor, benign and malignant Schwann cell lines. The expression characteristics of eleven candidate reference genes  were screened and analyzed by four software programs: geNorm, NormFinder, BestKeeper and RefFinder. Results showed that GAPDH, the most frequently used internal reference gene, was significantly unstable between various cell lines. The combinational use of two reference genes (PPIA and TBP) was optimal in malignant Schwann cell lines and the use of single reference genes (PPIA or PRLP0) alone or in combination was optimal in benign Schwann cell lines. These recommended internal reference gene selections may improve the accuracy and reproducibility of RT-qPCR in gene expression analyses of NF1 related tumors. Neurofibromatosis type 1 (NF1) is an autosomal dominant tumor predisposition syndrome that affects multiple organ systems and has a wide range of variable clinical manifestations, such as pigmentary lesions, skeletal abnormalities . The majCurrently, Omics studies play an increasingly important role in uncovering disease mechanisms and screening potential therapeutic biomarkers. Among different omics studies, transcriptomics is the bridge between genomics and proteomics, which could provide deep and precise measurements of transcriptional levels. Several studies have been conducted to figure out the molecular mechanisms of NF1 and to advance the development of targeted drugs –7. TheseIn the present study, eleven candidate reference genes were selected, including 18S Ribosomal RNA (RPS18), β-actin (ACTB), β-2-microglobulin (B2M), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Peptidylprolyl isomerase A (PPIA), Hypoxanthine phosphoribosyltransferase 1 (HPRT1), TATA binding protein (TBP), Ubiquitin C (UBC), Ribosomal protein large P0 (RPLP0), Transferrin receptor  (TFRC), and Ribosomal protein 32 (RPL32). These genes were the most frequently used for relative quantification analysis in other neoplastic diseases –17.NF1+/- Schwann cell lines, five benign pNF cell lines and seven malignant MPNST cell lines. We analyzed the data obtained as two separate groups: (1) benign NF1 tumor study including Schwann cell (SC) + pNF cell lines and (2) malignant NF1 tumor study including SC + pNF + MPNST cell lines, aiming to identify the most qualified reference genes for gene expression analysis in benign and malignant NF1 tumor study respectively.We investigated the stability of these eleven reference genes in fourteen different NF1 related cell lines, including two non-tumor ® CRL3388™), hTERT NF1 ipNF95.6 (ATCC® CRL3389™), hTERT NF1 ipNF95.11b (ATCC® CRL3390™), hTERT NF1 ipnNF95.11c (ATCC® CRL3391™), hTERT NF1 ipn02.3 2λ (ATCC® CRL3392 ™), sNF96.2 (ATCC® CRL2884 ™) and sNF02.2 (ATCC® CRL2885 ™) were obtained from the American Type Culture Collection (ATCC). All the cell lines used in this study were cultivated in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37°C in a humidified incubator at 5% CO2 and were confirmed negative for mycoplasma prior to use. Verification of cell lines was performed by Short Tandem Repeat (STR) DNA profiling .Five MPNST cell lines  were generous gifts from Dr.Vincent W Keng  and Jilong Yang . pNF cell line A68 was derived from a surgically excised pNF specimen of a 51-year-old female with NF1. pNF cell line WZJ was derived from a surgically excised pNF specimen of a 12-year-old boy with NF1. These two pNF cell lines were immortalized as described previously . Ethics ™ RT Master Mix kit (TaKaRa RR036A) according to the manufacturer’s instructions. After the reaction, the cDNA was diluted to 20 ng/ml. All RT-qPCR experiments were performed with the same batch of cDNA.Total RNA was extracted from all NF1 related cell lines using AxyPrep Multisource RNA Miniprep Kit  according to the manufacturer’s instructions. The quality and quantity of RNA were measured using a NanoDrop 2000 Spectrophotometer  through OD260/280 and OD260/230 ratios. 500 ng of total RNA was used for the cDNA synthesis reaction using PrimeScript® Premix Ex Taq™ Kit (TaKaRa RR420A) on an Applied Biosystems 7500 Real-Time PCR System, as described previously   of 0.48  and TBP (1.26) were the most stably expressed reference genes in SC + pNF + MPNST cell lines  . And PRLRT-qPCR was extensively applied in identifying key pathogenic genes in various diseases, for RT-qPCR is considered as the golden standard for gene expression analysis due to its accuracy and sensitivity . It is aThe ease to generate RT-qPCR data is in sharp contrast to the challenges to guarantee that the results obtained are reliable. In the absence of proper reference genes, data obtained are possibly inaccurate and irreproducible, as it has been shown that the use of a single reference gene without validation results in a significant bias (ranging from 3-fold in 25% of the results up to 6-fold in 10% of the results) .Prior to this study, there is no comprehensive and systematic validation of suitable reference genes for the relative quantification analysis of target gene expression in NF1 related peripheral nerve sheath tumor-derived cell lines. In previous studies, GAPDH and ACTB have been most frequently used in gene expression analysis of NF1 without solid validation –31. HoweNF1+/- Schwann cell lines, five pNF cell lines and seven MPNST cell lines. The expression stability of eleven frequently used reference genes, including RPS18, ACTB, B2M, GAPDH, PPIA, HPRT1, TBP, UBC, RPLP0, TFRC, and RPL32 were investigated and analyzed separately in these cell lines.In our study, in order to find the internal reference genes stably expressed in different NF1 samples, we investigated fourteen different NF1 related cell lines, which include two non-tumor t. Different algorithms were applied to estimate both the intra- and the intergroup expression variations and rank candidate genes based on the instability score. GeNorm and NormFinder calculate the stability  value, ΔCt uses the average of S.D. and BestKeeper uses the S.D. of the crossing points [Four different mathematical and statistical models were utilized to analyze the data obtained, including geNorm, NormFinder, BestKeeper and ΔCg points –22. In ag points .t, indicating the consistency of these statistical models and the reliability of the results. According to the results of RefFinder, the same conclusion can be drawn that PPIA and PRLP0 are the best reference genes for normalization in benign NF1 tumor study, while PPIA and TBP are as well in malignant NF1 tumor study.Due to the fact that the software applied are based on different mathematical models, the ranking orders of reference gene stabilities varied slightly among these tools. However, the top two positions of reference genes in SC + pNF group and SC + pNF + MPNST group determined by geNorm were identical to those determined by NormFinder, BestKeeper and ΔCM values > 0.5). As a multifunctional gene, the use of GAPDH as a reference gene has been also questioned and challenged in other cancers including lung cancer [PPIA is a gene encoding for a cyclosporin binding-protein, TBP is a gene encoding for a TATA-binding protein, and PRLP0 encodes a ribosomal protein that is a component of the 60S subunit. In NF1 related Schwann cell lines, they were stably expressed irrespective of tumor pathology and severity. In a previous study, TBP was also proven to be a stable reference gene in Schwann cells derived from neurofibromas . Howeverg cancer , breast g cancer  and bladg cancer . Accumulg cancer –39.M value of 0.225, is acceptable for data normalization. In contrast, Maertens et al suggested that the combination of three reference genes  was the optimal choice for normalization by using the geNorm software [In addition, considering the difference of opinion on the minimal number of reference genes required for RT-qPCR, we explored the necessity of selecting multiple reference genes for data normalization in our samples. In previous studies, some investigators showed that the combination of more than one reference gene improved the accuracy of results ,20,40–42software . The disLimitations existed in this study that only SC, pNF and MPNST cells were used to identify the most stable reference genes. whether these reference genes are also applicative to normalize RT-qPCR experiments using tumors containing a more complex cellular composition, such as cutaneous, plexiform and atypical neurofibromas, or MPNST tumors still needs experimental verification.NF1+/- Schwann cell lines, pNF cell lines and MPNST cell lines. According to the results, we recommend using two reference genes (PPIA and TBP) in combination for gene expression analyses in MPNST cell line-based researches and using single reference genes (PPIA or PRLP0) alone or in combination in pNF cell line-based studies.In this study, we systematically explored the suitability of fourteen candidate reference genes for normalization of gene expression in different NF1 related cell lines, including non-tumor S1 Checklist(XLS)Click here for additional data file.S1 FigAgarose gel electrophoresis of RT-qPCR amplification products for each of the internal reference genes provided by Sangon (M: Sangon DNA Marker {B600335}).(TIFF)Click here for additional data file.S1 TableThe expression levels of eleven candidate reference genes  in fourteen NF1 related cell lines.(XLSX)Click here for additional data file.S2 Table(XLS)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S1 File(XLSX)Click here for additional data file.


Furthermore, inhibition of histone modifying enzyme activity confirms the view that the MIEP is predominantly regulated by the activity of cellular chromatin. Finally, we observe that ip2-derived IE transcription is cycloheximide-sensitive in reactivating DCs, behaviour consistent with an early gene designation. Taken together, these data argue that MIEP activity is still important for HCMV reactivation but ip2 activity could play cell-type-specific roles in reactivation.Human cytomegalovirus latency and reactivation is a major source of morbidity in immune-suppressed patient populations. Lifelong latent infections are established in CD34+progenitor cells in the bone marrow, which are hallmarked by a lack of major lytic gene expression, genome replication and virus production. A number of studies have shown that inhibition of the major immediate early promoter (MIEP) – the promoter that regulates immediate early (IE) gene expression – is important for the establishment of latency and that, by extension, reactivation requires reversal of this repression of the MIEP. The identification of novel promoters (termed ip1 and ip2) downstream of the MIEP that can drive IE gene expression has led to speculation over the precise role of the MIEP in reactivation. In this study we show that IE transcripts arise from both the MIEP and ip2 promoter in the THP1 cell macrophage cell line and also CD14+monocytes stimulated with phorbol ester. In contrast, we show that in  A hallmark of human cytomegalovirus infection is the establishment of a lifelong latent infection of the host . In healper se, as well as a number of inflammatory stimuli, which are known to promote virus reactivation [The establishment of HCMV latency is dependent on the eventual silencing of immediate early (IE) gene expression from the major IE promoter (MIEP). Given the central role IE gene expression plays in productive infection it has long been considered that an induction of previously silenced IE gene expression represents the first stage in the reactivation from latency. Consequently, the differential regulation of the MIEP is likely to be a key molecular mechanism governing this switch. Consistent with this, the MIEP is responsive to myeloid differentiation signals, tivation . Furthertivation . Studiestivation . Finallytivation . Taken tin vitro and thus it was suggested they could have reactivation-specific function during reactivation [A recent report using an experimental model of HCMV latency in CD34+cells and the CD14+monocytic THP1 cell line has challenged this view and suggested that induction of IE gene expression during reactivation is triggered by alternative promoters to the canonical MIEP that are encoded within an intron of the MIE reading frame (designated ip1 and ip2) . The acttivation . Indeed,tivation .in vitro from primary myeloid progenitors using well-established protocols is MIEP-derived. Importantly, both macrophages and DCs have been demonstrated as important sites of HCMV reactivation in vivo [In our ongoing studies of HCMV reactivation, which focus on the biology of dendritic cells (DCs) in this process, it became evident that THP1-derived macrophages did not respond in an equivalent manner to DCs when challenged with inhibitors of HCMV IE gene expression . Thus, i in vivo  – cell tThe collection of venous blood samples from anonymous donors was approved and performed in accordance with established guidelines for the handling and processing of said tissue by the UCL and Cambridge Local Research Ethics committees. All studies with human material abide by Declaration of Helsinki principles.The HCMV isolates Merlin and TB40/e were purified from infected human retinal pigment epithelial cells using sorbitol gradients as previously described . VirusesTo test for inhibition of HCMV reactivation, histone acetyltransferase inhibitors (HATi) C646 and MG149 were added 1 h prior to IL-6 stimulation. p300: C646 , MOZ: MG149  or cells were treated with DMSO solvent control.−1; SIGMA), which was added 6 h prior to stimulation of reactivation.Inhibition of histone deacetylase activity was achieved using histone deacetylase inhibitor (HDACi) Romidepsin (3 nM), which was dissolved in DMSO. Inhibition of protein translation was achieved using cycloheximide , an equivalent dilution of DMSO, or PMA (20 nM); 4d with M-CSF/IL-1β ; or 6d with GM-CSF/IL-4 (both 1000 U ml−1) and a further 6–24 h with LPS (500 ng ml−1) or IL-6 (500 ng ml−1) to promote reactivation.CD14+monocytes were isolated from apheresis cones  by MACS CD14+positive cell separation (Miltenyi Biotec) before seeding on plastic and subsequent feeding with X-VIVO-15 supplemented with 2 mM THP1 cells were infected at an m.o.i.=5 and cultured in 2 % RPMI-10 for 5 days incubated with PMA (20 nM) for 48 h to promote differentiation to a macrophage-like phenotype.To isolate RNA, cells were washed once with PBS before direct harvest of samples with RLT buffer (Qiagen). RNA extraction was then performed as per the manufacturer’s instructions using the RNeasy Mini Kit (Qiagen). RNA (or RNA from historic studies) was converted to cDNA, then amplified by Sybr green quantitative real time-PCR using previously published gene-specific primers : UL123 5n>2. All scatter plots and bar charts depict the mean and one standard deviation from the mean. Significance was assumed if P<0.05.The Mann–Whitney U test was applied to test for significance between the means as a non-parametric distribution was assumed. Statistical analyses were only applied if To investigate the origin of IE gene expression in reactivating cells we analysed historic samples  availablInterestingly, the detection of UTR70 was variable and often at the threshold of detection in both CD34+ and CD14+derived DCs . FurtherNext, we performed the same analysis in THP1 cells stimulated with PMA to confirm we could re-capitulate previously published observations  and alsoIt was possible that our experimental system of monocytes and their differentiation to DCs promoted the use of the MIEP in reactivation studies from experimental latency and thus we investigated the impact of stimulating monocytes down alternative differentiation pathways . For theThese observations supported an inference of the previous study  that sugConsequently, we asked whether differences in the origin of IE transcription may explain the cell-type-specific effects of the HATi . First wIn the same study that defined the cell-type-specific effect of the C646 HATi we reported that a second HATi, MG149, inhibited IE expression in both cell types . MG149 tOne major aim of using chromatin reversing agents, such as HDACi to induce HCMV IE gene expression from latent virus, is to promote immune clearance of otherwise latently infected cells by so-called shock-and-kill approaches . Thus, wOur inability to consistently detect ip2-driven UTR70 transcription in reactivating DC samples led us to further investigate the regulation of the ip2-derived IE transcripts in these cells . SamplesThe expression of viral IE gene products is crucial for lytic infection and their induction is considered pivotal for reactivation from latency. This concept holds true for all members of the herpesvirus family and thus the mechanisms that control the initiation of lytic infection or the molecular switch from latency to reactivation have been widely studied.in vitro models and, importantly, during natural latency, the MIEP is associated with methylated histones during latent infection and acetylated histones during the reactivation phase [The prevailing hypothesis for HCMV has long been that the differential regulation of the viral MIEP, in a cell-type-specific manner, is the basis of MIEP regulation that dictates latency and reactivation. For the establishment of latency, the MIEP is required to be eventually silenced and thus, for reactivation to occur, the silencing of the MIEP must be reversed. This model is built on the view that host cell factors, involved in chromatin-mediated regulation of transcription, underpin this virus transcriptional control (reviewed in ). As sucon phase . Indeed,on phase , stronglon phase . The datet al. that the ip2 promoter is active in cells in which MIEP activity is limited – i.e. latently infected cells [Importantly, qPCR analyses suggest that the IE transcripts detectable in DCs differentiated from primary myeloid cell precursors are derived from the canonical MIEP. The data also suggest that the activity of the ip2 promoter is less sensitive to the activity of inhibitors of histone-modifying enzymes. Indeed, in the original study of ip2 activity, UTR70 transcription is evident in both latent and lytic infection of THP1 cells with PMA promoting an elevation of basal UTR70 RNA expression . Indeed,ed cells . The deted cells .The original identification of novel promoters that drive HCMV IE gene expression, which were non-essential for lytic infection, led to the logical consideration that they played a key role in HCMV reactivation. Intriguingly, a deletion of the ip2 promoter (and thus a deletion in the MIE locus) was observed to generate a reactivation defect in latently infected THP1 cells and a CD34+model system of latency . Indeed,Our data in no way dismisses a role for the non-canonical IE promoters. Indeed, we clearly observe ip2 driven UTR70 transcripts, particularly in THP1 cells and also in primary monocytes stimulated with PMA. Furthermore, it is well established that the MIEP is subject to complex patterns of regulation underpinned by the cell type used. For example, deletion of the CREB response elements from the proximal canonical MIEP resulted in a defect in HCMV reactivation in DCs whereas a HCMV virus with NF-kB site MIEP deletions was less affected . Howevercis repression sequence present in the MIEP [We accept that in this study that we have not directly assessed the impact of the ip2 deletion virus on reactivation of HCMV in DCs. However, our data clearly show that in CD34+ or monocyte derived DCs the origin of the IE transcription is derived from the canonical MIEP derived and is not inhibited by cycloheximide. In contrast, we note that, when detectable in our hands, ip2-derived IE transcription in DCs was cycloheximide dependent, which would be consistent with it being considered to be an early/late lytic promoter as suggested in the original studies in fibroblasts . That sathe MIEP ) is not What is not clear is whether the ip2 promoter is required for lytic infection of differentiated THP1 cells; if this was the case, this would partially explain the importance of ip2 activity for viral replication upon reactivation in these cells as major IE gene expression represents the only first step towards viral reactivation. Additionally, it will be important to investigate the very plausible possibility that this is not an either/or scenario. We noted that in the studies of the ip2 deletion virus, a defect in MIEP-derived transcription was also evident upon reactivation in the CD34+cells . It is aIn summary, we report that we observe that alternative ip2-driven IE transcription (generating UTR70 transcripts) clearly occurs in the THP1 cell model of HCMV latency and reactivation and also is evident in CD14+cells treated with phorbol ester (PMA) and this is entirely consistent with the previous identification of a novel reactivation associated IE promoter . However


Here we report that the knockout of GPx1 in HAP-1 cells has no significant effect on cell size, viability, growth and metabolic rates. Significant increases in the cytotoxic potency of hydrogen peroxide and tert-butylhydroperoxide, the anticancer drugs cisplatin and carboplatin as well as the alkylating agents lomustine and temozolomide were found. While a concentration dependent increases in intracellular reactive oxygen species (ROS) levels were observed for both HAP-1 cell lines treated with either cisplatin, lomustine or temozolamide, no significant enhancement in ROS levels was observed in the GPx1 knockout compared to the native cell line except at the highest concentration of temozolamide. On the other hand, a ca. 50% decrease in glutathione levels was noted in the GPx1 knockout relative to the native line, suggesting that factors other than ROS levels alone play a role in the increased cytotoxic activity of these drugs in the GPx1 knockout cells.The role of glutathione peroxidases (GPx) in cancer and their influence on tumor prognosis and the development of anticancer drug resistance has been extensively and controversially discussed. The aim of this study was to evaluate the influence of GPx1 expression on anticancer drug cytotoxicity. For this purpose, a GPx1 knockout of the near-haploid human cancer cell line HAP-1 was generated and compared to the native cell line with regards to morphology, growth and metabolic rates, and oxidative stress defenses. Furthermore, the IC In addition to catalase and superoxide dismutase, glutathione peroxidases (GPx) represent the first line defenses to protecting cells from oxidative stress and regulating cellular redox balance . In the Glutathione peroxidases have been found associated with tumor progression, malfunction of anticancer therapy and development of drug resistance ,11,12,1350 values of sixteen widely used anticancer drugs were determined in the native and GPx1 knockout variant, and the knockout index of every drug was calculated. The effects on reactive oxygen species (ROS) and GSH levels in both native and knockout cell lines were also evaluated.The goal of the current study was to evaluate the effect of GPx1 expression on cell viability and anticancer drug cytotoxicity. For these studies, the near-haploid HAP-1 cell line with a GPx1 knockout variant were obtained and characterized regarding various oxidative defense systems. The ICtert-butyl-hydroperoxide, thiotepa and vinblastine. IMDM medium stable glutamine and penicillin/streptomycin were from PAN Biotech , 3--2,5-diphenyltetrazolium bromide (MTT) and paclitaxel were from Alfa Aesar , lomustine and temozolomide from Biomol , NADPH and p-nitrophenyl phosphate were from Carl Roth , cisplatin and carboplatin from Chempur , doxorubicin from Pharmacia & Upjohn , oxaliplatin from Sanofi Aventis  and bortezomib from Selleckchem . All chemicals, kits and equipment for western blotting were obtained from Bio-Rad . The Annexin-V-FITC/propidium iodide kit was from Miltenyi Biotec  and the ATP CellTiter-Glo kit was from Promega . For microtiter plate absorption measurements a Spectramax 384 Plus plate reader from Molecular Devices  was used. The antibody anti-GPx1 (#3206) was purchased from Cell Signaling , anti-GPx4 (AB125066) from Abcam , anti-Prdx-1 (LF-MA0214) from A-frontiers , anti-rabbit-HRP (A-6154) from Sigma Aldrich, whereas the antibodies for anti-Prdx-2, anti-Trx-1 and Trx-2 were produced and validated in the working group of Dr. Christopher H. Lillig  within and according to the following publication [The following materials were purchased from Sigma Aldrich : camptothecin, chlorambucil, colchicine, 5,5′-dithiobis(2-nitrobenzoic acid (DTNB), 2′,7′-dichlorofluorescine diacetate (DCF-DA), fetal bovine serum (FCS), glutathione (GSH), glutathione reductase, glucose 6-phosphate, glucose 6-phosphate dehydrogenase, melphalan, methotrexate, podophyllotixin, -GPx4 AB1066 from 2 atmosphere. Cells were transferred weekly into new culture flasks when confluence reached 75% (7500 cells in 5 mL medium into a T25 flask) to ensure growth in exponential phase. Once a week, the medium in culture was replaced by fresh one. Cell cultures were routinely tested for mycoplasma. Microscopy was performed with a DMi8 inverse microscope  fitted with an HC PL Fluotar L 20x/0.40 dry objective in a transmitted light phase contrast.The GPx1 knockout cell line  and its parental HAP-1 strain (cat # C631) were obtained under license from Horizon Discovery . Cells were grown in IMDM medium supplemented with 10% FCS, 1% penicillin  + streptomycin (10 mg/mL) and 1% stable glutamine in a humidified incubator with 5% COTM counter. First, the cells were detached from the growth surface with 1.5 mL trypsin-EDTA solution and the reaction stopped with 3.5 mL complete growth medium. For measurements with the Coulter Counter Z2 , 100 µL of the cell suspension were added to 10 mL of Coulter Counter Isoton in triplicate. Each sample was measured and subsequently analyzed with help of the corresponding software Z2 AccuComp (ver. 3.01a). Three biological replicates with three technical replicates were analyzed and mean values calculated.Two different methods were used to estimate the size of the HAP-1 and KO.HAP-1.GPx1 cells: changes in impedance with the Coulter Counter Z2 and image analysis with the EVETM counter , 10 µL of the cell suspension were diluted with 10 µL of phosphate buffered saline (PBS) and subsequently added to 10 µL of a Trypan blue solution. After mixing, 10 µL were transferred to measurement slides and inserted into the counter. Data analysis was performed with the corresponding software EvePC (ver 1.01). Mean values of both cell size and live/dead ratios were calculated from three biological replicates with three technical replicates each.For measurements with the EVETo determine growth rates and, subsequently, doubling times of both HAP-1 and KO.HAP-1.GPx1 cells the crystal violet cell proliferation assay was performed . Cells w(N(t) = OD at time t; N (0) = OD at time 0; t = time in h).To compare the metabolism of HAP-1 and the corresponding GPx1-knockout cell line, two different microplate-based assays were performed. Both include the conversion of a substrate into a photometrically detectable product by intracellular enzymes which may be used to represent the cellular metabolism. The rate of conversion was monitored by detecting the corresponding ODs of the products over a course of three and four hours, respectively.The acidic phosphatase (APH) assay is based on the principle that intracellular APH hydrolyze p-nitrophenyl phosphate (PNPP) to p-nitrophenol (PNP). An increase of PNP can subsequently be detected at a wavelength of λ = 405 nm. Additionally, the MTT assay was used to assess whether there are any differences between the two cell lines’ metabolisms. The tetrazolium bromide compound (MTT), water soluble and of yellow color, will be reduced to the corresponding insoluble MTT-formazan product by mitochondrial dehydrogenases of viable cells. The presence of this purple product can be detected at λ = 570 nm.2, humidified atmosphere).For both the MTT and the APH assay, 20,000 cells were seeded in 0.1 mL medium per well of a 96-well plate and allowed to adhere for 24 h at standard culturing conditions  and related to the used protein concentration. GSH/GSSG content is expressed in ratios of µg to mg of total protein.For the determination of the total glutathione (GSH + GSSG) content of cell lysates, an enzymatic recycling assay was used, which is based on the oxidation of GSH to GSSH by Ellman’s reagent and the restauration of GSH by a NADPH/GR and G-6-P/G-6-P-DH system . This me3VO4, 0.1% Triton-X and freshly added 1% protease inhibitor cocktail (PIC), on ice for 30 min followed by a sonication for 10 min. After a centrifugation step at 18,000× g for 10 min at 4 °C the protein concentration was quantified via Bradford method against BSA as standard. Protein samples were stored at −80 °C till usage. For the electrophoretic separation 30 µg of total protein amount were diluted in 22.5 µL deionized water and 7.5 µL 4x Laemmli Buffer containing 0.65 mM 1,4-dithiothreitol, 18.66 µM bromophenol blue, 0.25 mM Tris (pH 6.8), sodium lauryl sulfate 7.5%, and 37.5% glycerol in water. Each slot of the precast gels was loaded with protein samples and after electrophoretic separation blotted onto PVDF-membranes by using the Trans-Blot Turbo System from Bio-Rad. Blots were blocked with 10% non-fat milk powder in Tris buffered saline/tween buffer (TBST) containing 0.02 mM Tris, 0.145 mM NaCl and 0.5% Tween 20 in water for 2 h and incubated with primary antibody dilution  in TBST plus 1% BSA over night at 4 °C. After a washing procedure with TBST, blots were incubated with horse radish peroxidase conjugated secondary antibody dilution (1:5000) in TBST plus 1% BSA for 2 h at room temperature. Selected protein bands were detected with Clarity Western ECL Substrate and recorded with an Advanced Fluorescence Imager . As a loading control, total protein normalization was done by using the TGX Stain-Free gel system [For the determination of the protein expression, western blotting was done following the instructions for the precast Criterion TGX Stain-Free Gels and the corresponding Trans_Blot Turbo Pack Midi PVDF membranes from Bio-Rad . The cell line samples were collected and lysed with a buffer containing 50 mM Tris (pH 7.4), 100 mM NaCl, 100 mM NaF, 5 mM EDTA, 0.2 mM Nal system ,29.50 values were determined with MTT cell viability assay as described previously [N,N-dimethylformamide  or dimethyl sulfoxide , then serial diluted at 1000-fold the intended concentration before being diluted 1:500 into culture medium. To each well was added 100 µL test solution, giving a total volume of 200 µL/well with final concentrations of DMF or DMSO of 0.1% (v/v). Final concentrations are shown in the figure. After an incubation period of 48 h, 40 µL of a 2.5 mg/mL solution of MTT in Dulbecco’s buffer was added to each well and incubated for 4 h at 37 °C. Then the medium was removed and replaced by 50 µL DMSO, then placed on a plate shaker 5 min to dissolve the formazan crystals. The optical density was measured with a plate reader set at λ = 570 nm; ODs were expressed as ratios to the ODs of the test wells to the OD of the untreated control (T/C%). The results of T/C% were plotted against the concentration of the tested drug on a logarithmic scale. The inhibition of viability to 50% (IC50) was calculated via interpolation at 50% with GraphPad Prism Software 6.0.For the determination of the anticancer potency of various anticancer drugs on HAP-1 cells, the ICeviously . Briefly50 value and incubated for 24 h. After removal of the medium, cells were harvested with trypsin/EDTA and washed with PBS by a centrifugation and resuspension step, then treated with 500 µL of a 20 µM DCF-DA solution in PBS for 30 min in the dark. The staining solution was replaced with 500 µL PBS, followed by flow cytometry determination at λexcitation/λemission = 488/527 nm with a MACSQuant Analyser 10 . Debris was excluded in the dot plot of the forward vs. sideward scatter. The mean fluorescence signal of DCF in treated cells was related to the untreated control population.For the determination of the ROS-content of cells after incubation with anticancer drugs, a cell cytometer based technique with the intracellular ROS sensor reagent DCF-DA was used as previously described . Briefly50 values of the anticancer drugs and the peroxides in the knockout cell line (KO.HAP-1.GPx1), compared to the parental HAP-1 cell line as well as for comparison of the GSH content in both cell lines. For the determination of varying ROS accumulation, a two-way ANOVA test was performed with the Sidak’s adaption of a multiple comparison.The GraphPad Prism 6.0 program  was used for all statistical evaluations. A two-tailed paired t-test was used for the comparison of ICThe cell line used in this work was the HAP-1 line, which is a near-haploid cell line derived from the KBM-7 cell line, a line obtained from a chronic myeloid leukaemia (CML) patient . HaploidTM automated cell counter. While the sizes of the cells varied depending on the method used, both methods revealed no differences in the diameters of the two cell lines  is the most abundant non-protein thiol in cells and an important non-enzymatic defense against oxidative stress. It is also a substrate for GPx1, with two GSH equivalents being consumed for each peroxide reduced. The total GSH + GSSG content of both HAP-1 cell lines was measured by an enzymatic assay, based on the oxidation of GSH to the disulfide GSSG by reaction with DTNB followed by its recycling back to GSH via a NADPH/glutathione reductase (GR) and glucose-6-phosphate (G6P)/glucose-6-phosphate dehydrogenase (G6P-DH) system . Surpris2O2 and tert-butyl hydroperoxide were also performed as positive controls. Representative dose-response curves over more than 100-fold concentration ranges are shown in 50 values of each compound was determined in the parental and in the GPx1 knockout HAP-1 variant, KO.HAP-1.GPx1. The knockout-index (KOI) is the quotient of the IC50 value of the knockout cell line divided by the IC50 of the parental cell line. A KOI value of one indicates that the knockout of GPx1 had no effect on the cytotoxic potency of the compound. Is the KOI less than one, then the toxicity of drug is enhanced, whereas a KOI greater than one indicates a diminished potency of the anticancer drug in knockout cells. 50 values of tested anticancer drugs and peroxides for the viability inhibition in both cell lines along with their corresponding KOI values.The MTT viability assay was then used to evaluate the effect of a GPx1 knockout in cancer cells towards the cytotoxicity of anticancer drugs. Treatments with two peroxides, Htert-butyl hydroperoxide were toxic to the HAP-1 cell line with IC50 values of 92.4 and 22.1 µM, respectively  and the spindle poisons vinblastine (IC50 ~ 1.65 nM), paclitaxel (IC50 ~ 2.75 nM), camptothecin (IC50 ~ 3.85 nM), colchicine (IC50 ~ 13.5 nM) and podophyllotoxin (IC50 ~ 20 nM) all showed not differences in IC50 values between the two cell lines. The proteasome inhibitor bortezomib and the anthracycline doxorubicin, a drug known to be redox active, gave a trend to increasing toxicity in the knockout cell line with KOIs around 0.84, but these were not significant. For the DNA platinating agent cisplatin and its prodrug form carboplatin, significant enhancements in cytotoxicity in the knockout cell line were also found; cisplatin toxicity increased from an IC50 value of 1.06 µM in parental cells to 0.52 µM in knockout cell line with a KOI of 0.49 while for the cisplatin prodrug carboplatin a KOI of 0.83 was determined. For oxaliplatin a non-significant trend to increased potency in knockout cell line was detectable, with a KOI of 0.71.The DNA alkylating agents chlorambucil, melphalan and thiotepa had similar IC2O2 but other cellular oxidants can also bring about fluorescence [50 values between knockout and native lines. Both cell lines were incubated with either cisplatin, lomustine or temozolomide for 24 h at concentrations above and below their IC50 values . DCF-DA is widely used for measuring oxidative stress in cells, being particularly sensitive to Hrescence . Only th0 values . A repreThe aim of this work was to assess the influence of the key antioxidative enzyme GPx1 on anticancer drug cytotoxicity. In this proof-of-concept study, we used a GPx1 knockout cell line, obtained by a CRISPR-Cas9 deletion of the only GPX1 gene in HAP-1 haploid human cancer cells, to analyze for differences in potency of widely used anticancer drugs to the parental line. Characterization of both cell lines showed slight differences in the morphology but no differences in size, doubling-time, viability or rates of metabolism. Our next focus was on the expression of various redox associated enzymes and intracellular GSH + GSSG content. It was anticipated that the knockout cell line would adapt to the loss of GPx1 function by increasing the expression of other antioxidant enzymes and/or GSH + GSSG. However, with the exception of a complete loss of GPx1 expression, we found no other significant alterations in the knockout cell line with respect to catalase, GPx4, peroxiredoxin-1 and -2 and thioredoxin-1 and -2. This suggests that under normal growth conditions, GPx1 is not essential for maintenance of a healthy redox balance. However, against our intuition we found a significant decrease in GSH + GSSG content in the knockout cells relative to the native cell line, suggesting a more complicated relationship between GPx1 and glutathione metabolism.Pursuant to our question of the influence of GPx1 expression on anticancer drug sensitivity, the cytotoxic potency of two peroxides and sixteen anticancer were examined in HAP-1 and KO.HAP-1.GPx1 cells. Not surprisingly, we detected knockout indices below one for both peroxides. The association between loss of GPx1 function with an increased sensitivity to peroxides has long been recognized . ImportaChen et al. recently showed that an increased GPx1 expression leads to resistance to cisplatin in non-small cell lung cancer cells whereas a downregulation via silencer RNA sensitizes tumor cells towards the drug . These fOn the basis of the varying potency of the anticancer drugs in the GPx1 positive and negative cell lines, we expected differences in ROS accumulation after incubation with cisplatin, lomustine and temozolomide. It is known that an incubation of these drugs can bring about an enhancement in ROS levels, which is connected to the anticancer drug potency ,39,40. WIn conclusion, it could be shown that GPx1 is not required for cell viability under normal cell culture conditions; this finding is consistent with earlier studies with knockout mice. Thus, therapeutic inhibition of GPx1 might not be expected to be toxic to cells under normal conditions. For the first time an association has been shown between GPx1 expression and anticancer potency of lomustine. Finally, inhibition of GPx1 would appear to be a worthwhile therapeutic goal to potentiate the cytotoxic activity of a number of anticancer drugs. Recently identified pentathiepins as potent inhibitors of GPx1 are being investigated with this strategy in mind .


Here we show that the frequency of cells with detectable NR2F2 and NR2C2 nuclear foci varies considerably between ALT+ cell lines and does not correlate with the level of protein expression. In addition, four of five ALT+ cell lines lack (TCAGGG)n repeats in some telomeres, indicating that direct NR binding does not play a role in ALT at these telomeres. NR2F2-depletion altered the abundance of C-circles and APBs but the direction of the response was inconsistent between three ALT+ cell lines. Moreover, transcriptome analysis following NR2F2-depletion in the ALT+ cell lines revealed different very responses. For example, NR2F2-depletion down-regulated many genes in U2OS cells, consistent with the cell cycle arrest and changes to ALT markers, but these features were not shared by the other two ALT+ cell lines. Among 86 ALT-associated genes, only MND1 showed consistent down-regulation across three NR2F2-depleted ALT+ cell lines. Altogether our data suggest that NR2F2 does not play a direct role in ALT and we speculate about an alternative role for this NR in a DNA damage response at telomeres.The alternative lengthening of telomeres (ALT) facilitates telomere lengthening by a DNA strand invasion and copying mechanism. The nuclear receptors (NRs), NR2F2 and NR2C2, can bind to (TCAGGG) Telomeric DNA is terminated by a G-rich single strand overhang, which is required for t-loop formation and capping5. Degenerate or variant repeats that maintain the 6-base pair periodicity and contain three consecutive G-residues tend to be more abundant in telomeric DNA than others6. Repeats such as (TGAGGG), (TCAGGG) and (TTGGGG) often have complex interspersion patterns with (TTAGGG) repeats and these patterns vary between alleles at each chromosome end7. Furthermore short arrays of (CTAGGG)n, cause localised, replication-dependent instability in telomeres8. Variant repeats are unlikely to bind to TRF1 or TRF2 in vivo, although some may bind to POT18, rendering these proximal regions of telomeres only partially functional in normal cells. In summary, the proximal telomere that includes variant repeats, varies in length and composition between alleles, chromosomes and individuals.Telomeres are the protective DNA–protein complexes that cap the ends of linear chromosomes. In human cells the double-stranded DNA component can be subdivided into a proximal region (closest to the centromere) comprising variant or degenerate repeats interspersed with the canonical telomeric repeat (TTAGGG)9. Bypass of the M1 checkpoint, through disruption of TP53 and RB1 expression, extends the replicative lifespan until the cell reaches crisis . Telomere: telomere fusions that form at crisis11, contribute to a high level of genome instability and cell death12. To survive crisis a TMM must be activated, either telomerase or the Alternate Lengthening of Telomeres (ALT).In the absence of an active telomere maintenance mechanism (TMM), telomeres shorten during each cell division until the Hayflick limit  is reached when short telomeres signal DNA damage and the cell enters senescence14. There are a variety of biomarkers that can be used to determine if a cell line or tumour shows ALT activity. These include: highly heterogenous telomere length; the presence of ALT-associated PML bodies (APBs)15; high frequency of recombination at telomeres17 often described as telomere sister-chromatid exchanges19; extensive presence of Telomere Induced DNA damage Foci (TIFs) that persist through metaphase and cell division20; presence of both G-and C-overhangs21; altered chromatin modifications in subtelomeric regions associated with increased expression of telomeric repeat containing RNA (TERRA)25; and a high level of extrachromosomal linear and circular telomeric DNA, in particular C-circles. C-circles are variable sized, partially double-stranded molecules that arise from DNA damage-induced replication fork collapse, which results in sudden cleavage and circularisation during lagging strand synthesis of telomeres in ALT+ cells27.The ALT mechanism is utilised by 10–15% of cancers, in particular some sarcomas and tumours of the central nervous system29. It involves strand invasion of one telomere that will be lengthened (recipient) into another telomere or sister-telomere (donor), which is used as a template for copying and extension of the invading telomere. As a result of this process, variant repeats, normally located in proximal regions, can become distributed throughout the full length of telomeres and between telomeres32. A variety of genes are required for telomere length maintenance in ALT including but not limited to the MRN complex (MRE11/RAD50/NBS1)33; POLD334; BTR (BLM/TOP3A/RMI1/2) and its interaction with FANCM37. ALT also requires regulation and localisation TRF1 and TRF2 proteins to APBs via sumoylation by the SMC5/6 complex38. Frequent inactivation or loss of ATRX, or its partner DAXX40, along with histone H3.3 mutations, demonstrate that telomeric chromatin organisation and correct histone deposition play significant roles in suppressing ALT42.ALT is a homology directed repair (HDR) mechanism that shows strongest resemblance to Break-Induced Replication (BIR)43; (2) a slower long-range homology searching process that requires RAD51 single-strand filament formation and the HOP1-MND1 heterodimer44 and (3) orphan nuclear receptors (NRs) binding to variant (TCAGGG)n repeats within telomeres, facilitating recruitment of ZNF827, a zinc-finger DNA binding protein and the NuRD, nucleosome remodelling and deacetylase protein complex45.Recent studies have identified a variety of routes that are thought to bring telomeres together to facilitate strand invasion during ALT. These include (1) recruitment of RCF-PCNA-POLD3, as expected for BIR47. Here we show that multiple telomeres appear to lack binding sites for NR2F2 and NR2C2 in four of five ALT+ cell lines. This is consistent with a lower frequency of NR2F2 and NR2C2 foci at telomeres in the nuclei of four out of five ALT+ cell lines. We propose that telomeres lacking NR2F2 and NR2C2 binding sites are unlikely to engage in direct recruitment of ZNF827, the NuRD complex, or other NR2F2 interacting proteins, such as FANCD248. siRNA mediated NR2F2 down-regulation in three ALT+ cell lines had a modest to severe effect on the cell cycle but the effect on various ALT markers was highly inconsistent between the cell lines. Transcriptome analysis showed that NR2F2 depletion had a dramatic effect on gene expression in U2OS cells that was consistent with a reduction in cells in S-phase and near cell cycle arrest. Notably only one ALT-related gene, MND1, was down-regulated upon NR2F2 depletion in all three cells lines, possibly suggesting an indirect role for NR2F2 in the regulation of MND1. In summary, NR2F2 has a known role in cell cycle regulation that our data corroborate, but its role in the ALT mechanism appears to be more indirect than has been proposed previously. Nevertheless NR2F2 and NR2C2 do bind to some telomeres where they recruit other proteins48 and we speculate that this may serve a different function.NRs are a large family of diverse transcriptional activator /repressors that bind to DNA in a sequence specific manner and play many roles in cell regulation, differentiation and organogenesis. The class of NRs that can bind to (TCAGGG) repeats include NR2F2 (COUP-TFII) and NR2C2 (TR4). NR2F2 in particular is known to play significant roles in tumorigenesis through its impact on angiogenesis and the tumour microenvironment30. The other ALT+ cell lines showed much lower percentages of cells with NR2F2 or NR2C2 foci, some approaching the low level observed in the HT1080 telomerase + cell line. Distribution analysis was conducted on the nuclei with NR2F2 or NR2C2 foci to investigate colocalisation with telomeres. This demonstrated that, when present, NR2F2 foci colocalised with telomeres in all the cell lines, although ALT+ cell lines (in particular VA13/2RA30) showed significantly higher proportion of NR2F2 foci at telomeres compared to HT1080. In addition, all cell lines also had foci that did not colocalise with telomeres. Similar trends were observed in nuclei with NR2C2 foci again with VA13/2RA showing the highest proportion of cells with foci at telomeres , U2OS, SUSM-1 and SAOS2 ALT+ cell lines and in HT1080 (telomerase +). This showed a fairly consistent NR2C2 expression but considerable variation in NR2F2 expression between cell lines, from barely detectable in SAOS-2 to highly expressed in W-V n repeats in approximately 50% of amplified molecules from some telomeres, for example XpYp in W-V, suggested this arose from allelic differences in the progenitor cell line2. This was confirmed using allele specific XpYp STELA primers in W-V cell line  and hybridisation to DNA probes that detect the canonical (TTAGGG) or the variant repeats (TCAGGG) and (TGAGGG). Only the VA13/2RA cell line showed the presence of (TCAGGG)6. In contrast, C-circles were significantly increased in the W-V and U2OS NR2F2 depleted cells  and in contrast, both measures were significantly increased in the treated U2OS cells  in the siNR2F2 treated W-V cells and a highly significant decrease in cells in S-phase in siNR2F2 treated U2OS cells  that was accompanied by increases in G1 and G2-M, suggesting a cell cycle arrest  versus control (CNT) separated the U2OS from the W-V and VA13/2RA cells, recapitulating the PCA analysis. There was also good separation between NR2F2-KD vs CNT replicates for each cell line  analysis was conducted between control and siNR2F2 treated cells and comparisons made between cell lines. Following checks on the RNA-seq data , rRNA processing , and sister chromatid segregation   showed that the highest number of DE genes , with fewer DE genes identified between the VA13/2RA KD vs CNT cells (n = 3317) and even fewer between the W-V KD vs CNT cells  Fig. . In contP = 2.80e−10; Fig. P = 1.47e−07; Fig. HSPA1A)  was upregulated in WV and downregulated U2OS; while the Bone Morphogenetic Protein 4 (BMP4)  a secreted ligand of the TGF-beta, was down regulated in WV but upregulated in U2OS  was used to determine how NR2F2 depletion influences cell cycle regulation. This revealed down-regulation of 42 cell cycle related genes in the W-V KD vs CNT (FDR = 0.0177), 57 in the VA13/2RA KD vs CNT (FDR = 0.0096), and 122 in U2OS KD vs CNT (FDR = 0.0024) , with the highest level in VA13/2RA, making it an outlier. There was no obvious relationship between the level of NR2F2 protein expression and the percentage of cells showing nuclear foci however, when present, the foci where significantly more likely to colocalise with telomeres in ALT+ cell lines compared to the telomerase + HT1080 cell line, although the VA13/2RA cell line was again an outlier. Consistent with the variability of those features, we also demonstrated that four of the five ALT+ cell lines have some telomeres that lack (TCAGGG) variant repeats making then unlikely sites for NR2F2 or NR2C2 binding. Following siRNA mediated depletion of NR2F2 there were some changes to the level ALT markers but the direction of change was inconsistent between cell lines. In brief, APB frequency increased in U2OS, decreased in VA13/2RA and was unchanged in W-V siNR2F2 depleted cells, whereas C-circle abundance increased in W-V and U2OS but was unchanged in VA13/2RA following NR2F2 downregulation. Western blot and differential gene expression analyses between siRNA control and siNR2F2 treated cells verified significant down-regulation of NR2F2 but no significant change in NR2C2 expression, indicating no compensatory effect in the cell lines examined.The two orphan nuclear receptors NR2F2 and NR2C2 have the potential to bind to telomeric DNA that contain (TCAGGG) variant repeats. The binding of NR2F2 and/or NR2C2 to telomeres in cells that utilise the ALT mechanismTP53 and RB1 signaling pathways54, giving rise to the W-V and VA13/2RA cell lines that have evolved independently to manage the disruption caused to the cell cycle. In contrast NR2F2 depletion had a significant effect on the cell cycle in U2OS cells, such that the U2OS cells were unable to enter S-phase, they accumulated in G1 or G2 consistent with the increase in APBs and C-circles. This conclusion is supported by the GO term analysis as the DE genes in the U2OS siNR2F2, which showed enrichment for biological processes linked to DNA replication, aspects of RNA processing and sister-chromatid segregation.The consequence of siNR2F2 treatment on gene expression varied considerably between the three ALT+ cell lines with the greatest perturbation in U2OS cells. PCA analysis of differential expression data following NR2F2 depletion showed a greater similarity between the VA13/2RA and W-V cell lines than to the U2OS. This is consistent with the GO term analysis showing enrichment for genes involved in defense against viruses, as both W-V and VA13/2RA cell lines are derived from SV40-LT transformed fibroblast cells. Depletion of NR2F2 in W-V and VA13/2RA affected the cell cycle to a limited extent compared to the effect in U2OS cells. This may be explained by SV40-LT expression, which disrupts the MND1, which showed significant down-regulation in all three cell lines. The MND1-HOP1 heterodimer is required for long-range homology searching by RAD51-bound telomeres in ALT+ cells so facilitating strand invasion and break-induced replication (BIR) that lengthens telomeres44. Interestingly, RAD51 was significantly down-regulated in two of the NR2F2-depleted cell lines (U2OS and VA13/2RA), Fig. 43 . Expression of the PCNA and POLD genes were significantly down-regulated in U2OS siNR2F2 treated cells but not in VA13/2RA and W-V. In the third proposed ALT mechanism, NR2F2 bound to telomeres recruits ZNF827 and the NuRD complex, preventing a DNA damage response and facilitating chromatin remodelling and telomere-telomere interactions31. Two components of the NuRD complex showed DE but in opposite directions (CHD3 up-regulated and RBBP7 down-regulated) and only in U2OS NR2F2 depleted cells. The ZNF827 gene was also significantly up-regulated in U2OS treated cells, which is surprising as the zinc finger protein has been reported to interact with NR2F2 at telomeres31. Recently it has been shown that FANCD2 can form a complex with NR2F2 or NR2C2, independently from the Fanconi anaemia (FA) core complex or monoubiquitination of FANCD2. When the FANCD2-NR2F2/C2 complex localises to telomeres in ALT+ cell lines, the complex recruits the MUS81 endonuclease and stimulates DNA synthesis at telomeres suggesting a role in telomere maintenance in this setting48.There was almost no overlap between genes that showed DE following NR2F2 depletion and ALT-related genes across the three cell lines. The one exception was 32. Recently it has been shown that ZBTB10, a zinc finger containing protein, preferentially binds to (TTGGGG) another variant repeat commonly found at the proximal ends of telomeres14. ZBTB10 binds to telomeres, particularly in the ALT+ U2OS cell line, where it facilitates interaction with the TRF2/RAP2 complex but inactivation of ZBTB10 had a limited effect on gene expression, and no clear effect on telomere length or ALT markers55. It was proposed that the (TTGGGG)–ZBTB10–TRF2/RAP1 interaction may play a role in DNA damage response at telomeres in normal cells55. We speculate that NR2F2 and NR2C2 could play a similar roles at very short telomeres that contain (TCAGGG) but lack (TTGGGG) repeats. Localisation of NR2F2 or NR2C2 at such telomeres might recruit FANCD2 or ZNF827 in a DNA damage response rather than a performing a specific role in the ALT mechanism.If the effect of NR2F2 depletion is to block or reduce entry into S-phase when that checkpoint is intact, this would be expected to have an indirect effect on ALT markers (APBs and C-circles), as seen in U2OS cells. Moreover, the lack of consistency in response to NR2F2 depletion among the ALT+ cell lines (with the exception of MND1 down-regulation) suggests that NR2F2 only plays an indirect role in ALT. Nevertheless, it is reasonable to ask why NR2F2 and NR2C2 bind to telomeres and recruit ZNF827 and FANCD2, especially as this may lead to insertion of telomeric DNA elsewhere in the genome and cause instabilityn repeats plays an important role at short dysfunctional telomeres in normal cells remains to be determined.In summary we have shown that NR2F2 depletion has very variable effects on differential gene expression, the cell cycle and ALT markers in U2OS versus WV and VA13/2RA cell lines. This points towards the known role of NR2F2 in regulation of the cell cycle but suggests only an indirect role on the ALT mechanism. Whether or not NR2F2/NR2C2 binding to (TCAGGG)W-V is an SV40 transformed, ALT+ cell line derived from normal fibroblasts from a 45-year-old male with Werner’s syndrome (WRN−/−). W-V was a gift from Roger R. Reddel. The WI38VA13/2RA (VA13/2RA) ALT+ cell line, derived from normal female lung fibroblasts transformed by SV40 large T antigen, was obtained from the European Collection of Authenticated Cell Cultures (ECACC). SUSM-1 ALT+ cell line, derived from male liver fibroblasts immortalized by chemical (4NQO) transformation was a gift from Olivia M. Pereira-Smith. SAOS-2 and U2OS are both female osteosarcoma derived ALT+ cell lines obtained from Paolo Salomoni. HT1080, a telomerase-positive cell line derived from a male fibrosarcoma was obtained from ECACC.2 and ambient O2 and subcultured by standard methods. Dead cells were assessed using Trypan Blue stain .W-V, U2OS and HT1080 were grown in DMEM medium  with 10% Fetal Bovine Serum ; SAOS2 in RPMI 1640  with 10% FBS and VA13/2RA in MEM medium  with 10% FBS and 1X Non-Essential Amino Acids . The cells were culture at 37 °C in 5% CO6 cells) were made in RIPA buffer, quantified using the Bradford assay and diluted to 1–3 mg/ml in Laemmli loading buffer. SDS-PAGE electrophoresis and western blots were prepared using standard procedures56. The X-ray films were converted to TIFF images and analysed using ImageJ v1.5e software, with normalisation57.The primary antibodies used were: NR2C2/TR4 (PPH0107B-00) and NR2F2/COUP-TF2 , GAPDH  and Anti IgG-Horseradish peroxidase (HRP)-conjugated secondary antibodies . Cell lysates (~ 1 × 103 PNA probe (PANAGENE) in PNA hybridization solution , 10 mM Tris–HCl, pH7.2)6. Coverslips were mounted with ProLong Gold containing 5 μg/ml of DAPI. APBs were detected by colocalisation of PML  with telomeres58.Cells on coverslips (70–90% confluency) were processed for antibody detection of NR2F2 or NR2C2 with visualization using fluorescence conjugated secondary antibodies . Telomeres were detected by in situ hybridization to Cy3–OO-(CCCTAA)https://imagej.nih.gov/ij/plugins/colocalization.html).For colocalization, cells were visualised using an Olympus FV1000 confocal microscope and intensity profile plot analysis. Nuclei at interphase were captured in 15–20 Z planes in 0.25 μm increments for the green (AlexaFluor488), red (Cy3-Tel-PNA) and blue (DAPI) channels. For quantitative analysis, slides were screened using the Olympus cell scanning system to capture images of 100–1000 s of nuclei in blue, green and red channels. Co-localisation analysis was conducted using ImageJ v1.5e software with the Java plugin tool by Pierre Bourdoncle  or 66.5 °C  for 30 s, 68 °C for 10 min. Each reaction was split into three identical 0.8% LE agarose gels that were processed into Southern blots with variant-repeat detection by hybridization to 32P-dCTP labeled DNA probes that detect (TTAGGG)n, (TCAGGG)n, or (TGAGGG)n repeats. Phosphor-image analysis was conducted using a Typhoon 9400  and ImageQuant software. N.B. the 17p6 primer annealing site is polymorphic (presence/absence) in populations. Consequently, productive amplification with 17p6 in STELA reactions behaves as a Mendelian trait (NJR unpublished).STELA-PCR was conducted as reported previously26 was conducted and C-circle abundance quantified by dot-blot hybridisation of native and denatured products to 32P-dCTP labelled telomere and Alu probes respectively. The signal from the telomeric probe hybridization was normalised to the Alu probe using the ImageQuant software .The C-circle assay6 cells were harvested in 1 ml of 70% ice-cold ethanol. The cells in PBS were treated with RNase, stained with PI, and processed using a FACSCantoII cell analyser (BD Biosciences). The analysis was carried out using the FACSDiva software (BD Biosciences).Cell cycle analysis was carried out by Propidium Iodide (PI) staining and FACS at 72 h after transfection with the siNR2F2 or control siRNAs. Approximately 1 × 10Data from western blots, c-circle assays, co-localisation of NRs with telomeres, APBs, and cell cycle analyses were assessed using ANOVA, pairwise multiple comparison T tests (with Benjamini–Hochberg correction), or Pearson correlations using programs in R v3.5.1.60. It is predicted to target five known splice variants of NR2F2 that are protein coding  with other genes and includes 5 mismatches with NR2F1 transcripts6), C-circle (1 × 106), RNA-Seq (1.5 × 106 ) and FACS (1 × 106) analyses.For each biological replicate nine 10 cm petri-dishes (each containing three coverslips) were seeded with cells and cultured for 24 h to reach 40–50% confluence. Prior to transfection, the medium was replaced. Three plates were used as untreated controls, or transfected with the NR2F2 targeting or control Stealth siRNAs. Transfection of the double-stranded siRNAs (25 to 50 pmol) was carried out in low toxicity Lipofectamine RNAiMAX Transfection Reagent with Opti-MEM Medium (Thermo Fisher Scientific) following the manufacturer’s instructions. The siRNA/lipofectamine mix was added to growth medium in a 1:5 ratio. After 72 h, the coverslips were removed for APBs analysis. The rest of the cells were detached from the petri-dish by trypsinisation and harvested for western blot  were sent to Novogene Corporation (Hong Kong) for library preparation using an Illumina TruSeq protocol, including mRNA poly-A selection, mRNA random fragmented, and first strand cDNA synthesis with random hexamers and reverse transcriptase. The cDNA libraries were sequenced using an Illumina-HiSeq 4000, generating > 12 GB data.61 and the fastq files assessed before and after removing the adaptors using FASTQC v0.11.5 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Remaining reads were aligned to the human genome with TopHat262 using the human genome assembly (Homo_sapiens.GRCh38.82) as reference. Only uniquely mapped reads were used in the downstream analysis  reads. Fastq files were trimmed using skewer v. 0.2.264 by countToFPKM v.1.0 was used to obtain the normalised gene expression. Differential expression (DE) between the groups were determined with DESeq2 v1.16.165. GO analysis was performed on gene subsets identified by overlap analysis of DE genes  using R/Bioconductor cluster Profiler v3.10.166 and the top 10 terms for each gene set was plotted. Pathway enrichment analysis, using Gene Set Enrichment Analysis (GSEA) with the Reactome database of biological pathways, was conducted using DOSE v.3.10.1: an R/Bioconductor package for Disease Ontology Semantic and Enrichment analysis67.The resulting count matrix from Rsubread.v.1.26 was imported into R and, to filter out low expressed genes, only genes with at least 1 count per million (CPM) reads in a minimum of three samples were used for further analysis. The Fragments Per Kilobase of transcript per Million mapped reads (FPKM) methodhttps://www.ebi.ac.uk/arrayexpress/.The RNA-seq data has been deposited at EMBL-EBI ArrayExpress: accession number E-MTAB-9599 available at: Supplementary Information.


Cyanobacteria  are ubiquitous, Gram-negative photoautotrophic prokaryotes. They are considered as one of the most efficient sources of bioactive secondary metabolites. More than 50% of cyanobacteria are cultivated on commercial platforms to extract bioactive compounds, which have bene shown to possess anticancer activity. The chemically diverse natural compounds or their analogues induce cytotoxicity and potentially kill a variety of cancer cells via the induction of apoptosis, or altering the activation of cell signaling, involving especially the protein kinase-C family members, cell cycle arrest, mitochondrial dysfunctions and oxidative damage. These therapeutic properties enable their use in the pharma and healthcare sectors for the betterment of future generations. This review provides a baseline overview of the anti-cancerous cyanobacterial bioactive compounds, along with recently introduced nanomaterials that could be used for the development of new anticancer drugs to build a healthy future for mankind. Nostoc sp. was first used to treat gout, fistula and different types of cancers . It. ItDolabll lines  showed iLyngbya sp., contains a unique N-methyl-3-bromotyrosine, a modified 4-methylproline, a 2-(1-oxopropyl) pyrrolidine and an N-pivalamide unit. Based on cell morphological profiling analysis, bisebromoamide was identified as an actin filament stabilizer [50 value of 0.04 µg/mL, and exhibited 50% growth inhibition against a panel of 39 human cancer cell lines with a GI50 value of 40 nM  and human cervical adenocarcinoma (KB) cell lines [Borophycin (boron-containing metabolite) is an acetate-derived polyketide  isolatedll lines   and colon (HCT-116) cancer cell lines, as well as exquisite antiproliferative effects in the NCI-60 cell lines [Carmaphycins A and B , isolatell lines   < 100 nM in human SF-295 and U87-MG glioblastoma cells and mouse embryonic fibroblasts (MEFs) (50 < 23 nM). Previously, it was evaluated against an in vitro panel of 60 selective human cancer cell lines, including colon, breast and ovarian malignant tumors cells. The results indicated that coibamide A was found to be more effective against the MDA-MB-231 breast cancer cell line than against other cell lines [Coibamide A  is a novs (MEFs) . It indull lines . Recentlll lines .Nostoc sp. var. ATCC 53789 and GSV 224, is a potent anticancer agent. It inhibits microtubule assembly, and exhibited anti-tumorigenic activity against several solid tumors implanted in mice, including multidrug-resistant cancer cells  showed antiproliferative effects against M21 and M21-L human melanoma cell lines at nanomolar concentrations [Cryptophycin, a depsipeptide isolated from er cells . The IC5ll lines ,40. Sevell lines . A clinill lines , but faill lines . Cryptopll lines . The anttrations .Lyngbya majuscula, was screened against the NCI-60 panel of human tumor cell lines. It inhibits the binding of tubulin polymerization into the colchicine binding pocket, and arrests the cells in the G2/M phase of the cell cycle  as the antibody, Dol10-containing ADCs have been synthesized and investigated for in vitro cytotoxicity assays against HER2-positive (SK-BR-3) human tumor cells. Additionally, ADCs derived from Herceptin® and PEG8-Dol10 effectively delayed tumor growth at a dose of 10 mg kg−1, and prolonged the survival time in mice bearing human ovarian SKOV-3 xenografts [Dolastatins are peptides initially isolated from the sea hare t cancer . Dolastat cancer a,b are lp. VP642 . They shectively . Additioectively . Furtherll lines . Recentlnografts .Lyngbya confervoides, which reveal cytotoxicity against several cancer cells. The IC50 values for grassypeptolides A were evaluated against HT29 and HeLa cancer cell lines, and were found to be 1.22 and 1.01 µM, respectively , human lung cells (SW1271 and A529) and murine melanoma cells (B16-F10), with IC50 values ranging from 0.13 to 0.72 µg/mL [Hormothamnin A , a cycli72 µg/mL  . Itralamide A showed lower cytotoxicity than Itralamide B against HEK293 cells with an IC50 of 6 µM [Itralamides A and B , cyclode of 6 µM ,63 , P388 (murine leukemia), PC3 (human prostate cancer), A549 (human lung cancer) and SK-OV3 (human ovarian cancer), with IC50 values ranging from 1.6 nM to 6.4 nM  inhibitor, and is currently being used for the development of anticancer drugs. It has been screened to evaluate its cytotoxicity against many epithelial and fibroblastic cancer cell lines [2+ and inhibits class I histone deacetylase. Beside its HDAC inhibitory property, largazole exhibits a wider range of in vitro and in vivo biological activities, for example antitumor, antiosteogenic and antifibrotic activities [+) lymphoma cells to ganciclovir  at nanomolar concentrations [Largazole , a cyclill lines   and colon (LoVo) cancer cell lines with IC50 values of 0.03 µg/mL and 0.5 µg/mL, respectively , while lyngbyabellins K, L, M and 7-epi-lyngbyabellin L , exhibited antiproliferative activity against the above cell lines with IC50 values of 3.3 and 1.7 µM, respectively [Moorea producens and, in particular, 6,8-di-O-acetylmalyngamide-2 showed strong anticancer activity by activating adenosine monophosphate-activated protein kinase (AMPK) [Malyngamides , small atrations . Isomalytrations b, showedectively . Additioectively . Recentle (AMPK) .Nostoc, Nodularia and Anabaena genera, induces cell death and exhibits anti-proliferative activity against various human cancer cells, specifically against p53-mutated cell lines with IC50 values ranging from 0.7 to 4.5 µM [Nocuolin A a, a natuo 4.5 µM   and neuro-2a neuroblastoma cells (IC50 = 29 nM) by inhibiting serine proteases activity (IC50 = 0.38 µM) [Symplocamide A d, a cycl0.38 µM)   [Veraguamides A–C and H–L cyclodepsipeptides  were isoc effect . Veragua 141 nM) . Moreove 141 nM) .Lyngbya sp.), Bouillomides A and B (depsipeptides from Lyngbya bouillonii), Molassamide (depsipeptide from Dichothrixutahensis), Largamides (cyclic peptides from Lyngbya confervoides), Pompanopeptin A, (cyclic peptide from L. confervoides), and all dolastatin 13 analogs, strongly inhibit non-caspase proteases’ activity with IC50 values ranging from 0.32 to 25.0 μM [Symploca sp., exhibits cytotoxicity against HCT-116 colon cancer with an IC50 of 0.74 µM by disturbing microtubule formation [Kempopeptins A and B , liver cancer (HepG-2), colon cancer (HCT116), and lung cancer (A549) cell lines. However, its crude extract BS1-EG exhibited various effects on all tested cell lines [Moreover, dragonamide b, a lipo 1 μg/mL   cell lines with IC50 values ranging from 0.3 to 0.7 nM [L. majuscula, exhibits cytotoxicity on Jurkat (IC50 3nM) and CEM leukemia (IC50 14 nM), A549 lung carcinoma (IC50 46 nM), Molt4 T leukemia (IC50 60 nM), M21 melanoma (IC50 1.3 nM) and U266 myeloma (IC50 5.8 µM) cell lines by inducing apoptosis via caspase 8 [Lyngbya sp., induce cytotoxicity in cancer cell lines, such as KB cells, with IC50 values ranging from 2.14 to 7.28 µM, 0.63 µM and 0.58 µg/mL, respectively, and later inhibit the serine proteases pathway [L. semiplena, showed anticancerous potential against NCI-H460 lung cancer with an LD50 of 0.4 µM [Malevamide D and E  are pepto 0.7 nM  , design flexibility and large surface-to-volume ratio make these materials of significant use. With the appreciation of the enhanced properties of metals at nanosizes, extensive research on different nanometals has been carried out in recent years for exploring their applications in cancer research. Emphasizing the biological synthesis, cyanobacteria are considered as one of the best biological systems for nanoparticle (NP) synthesis, both intra-cellularly as well as extra-cellularly . Howeverya, etc. . Moreoveya, etc. . Furtherd Au NPs . Oscillatoria limnetica have exhibited cytotoxic effects against human breast (MCF-7) and colon (HCT-116) cancer cell lines with IC50 values of 6.15 μg/mL and 5.37 μg/mL, respectively [L. majuscula were screened against three leukemic cell lines, i.e., K562, MOLT-3, and REH, and showed dose- and time-dependent anticancer activity. The REH cells showed the maximum sensitivity to AgNPs, with an IC50 value of 620 ± 3.73 μg/mL [Nostoc sp. strain HKAR-2 showed a dose-dependent cytotoxic activity against MCF-7 cancer cell lines, with an IC50 of 27.5 μg/mL [Nostoc linckia synthesizes silver nanoparticles (9.39 to 25.89 nm). These AgNPs exhibit in vitro antitumor activity against the human breast MCF-7 cancer cell line (IC50 = 27.79 ± 2.3 μg/mL), and were shown in vivo to inhibit tumor growth in Ehrlich ascites carcinoma-bearing mice [Nostoc sp. EA03 exhibit less cytotoxicity when used in lower concentrations against the human adenocarcinoma alveolar basal epithelial cells (A549 cells) and human lung fibroblast (MRC-5 cells) cancer cell lines [Very few studies on the above have suggested that these NPs could be used for the treatment of cancer . Recentlolon HCT- cancer c73 μg/mL . Moreove.5 μg/mL . The phying mice . ZnO NPsll lines .Arthrospira platensis with anti-fungal biofilm activity [Aphanizomenon flosaquae , the delivery of which to the deep skin layers was improved when using propylene glycol-containing nanovesicles [Cells and inhibited tumor growth. Consequently, they showed notable effects as anticancer agents and could be used as nanomedicines in cancer research. However, some cyanobacterial nanoformulations could also be used to deliver therapeutic drugs to cancer sites and monitor the tumor tissues, but limited data are available. As mentioned above, numerous studies show that, even at pico to nano molar concentrations, several cynobacterial secondary metabolites, such as apratoxin, calothrixin, curacin A, dolastatins, etc., are effective, and have shown potent inhibition of several cancer cell lines in vitro, however in vivo studies are limited. As with camptothecin, whose clinical application is limited due to its low solubility, although it has shown potent anticancer activity in vitro ,121, mosactivity  and the vesicles . HoweverSpirulina showed protective effects against the cardiotoxicity induced by doxorubicin, and thus improve the therapeutic index of doxorubicin [Spirulina platensis against oxidative stress caused by doxorubicin were also evaluated [Previously, green carbon nanotags (G-tags) developed from cyanobacteria were shown to be an advanced and efficient imaging platform for anticancer therapy due to their high solubility, excellent photostability and low cytotoxicity. These nanotags, when conjugated with doxorubicin, not only induced cell death in cancer cells (HepG2 and MCF-7), but also, the fluorescence of G-tags enables the monitoring of doxorubicin uptake by cancer cells and their intracellular location . Similarorubicin . Similarvaluated . These cvaluated .In the last few decades, the use of cyanobacteria as a novel source of therapeutics has been realized. Several chemically diverse compounds have been screened against different cancer cell lines, but still, very few cyanobacterial compounds have entered clinical trials, as much is not known at the molecular level. Though their economic cultivation expanded their utilization in drug discovery, still there is a need to explore more deeply into cyanobacterial compounds in order to clarify the specific targets and the mechanisms involved in cancer. Thus, more scientific attention and interdisciplinary research would have to be devoted to finding novel compounds by exploring cyanobacterial strains from extreme unexplored habitats. Furthermore, we do not explore in this review paper the structure–function relationship that might be established and would link a structural motif to a particular target. To this end, the reader may refer to the comprehensive review published previously by Salvador-Reyes and Luesch , which dOne of the main subjects of interest in the field of natural biomedicines and their nanoformulations is their toxicity and/or that of contaminating toxic substances. Indeed, cyanobacteria could also be the sources of toxin production . AlthougIn addition, the biosynthesis of NP by cyanobacteria is a relatively new idea to which promising results are attributed and, in the future, it will be possible to build datasets for marine pharmaceuticals by developing nanoformulated drugs based on cyanobacteria. However, to bring it to the platform of drug development there is a need to optimize biosynthesis pathways involving the futuristic approach of green synthesis by controlling the size and shape of particles and achieving monodispersity in the solution phase. As cyanobacteria act as natural therapeutic and have pharmaceutical potential, it could be expected that they could be used as an antitumor killer for a better world.