NF1, CDKN2A, and SUZ12/EED tumor suppressor gene (TSG) inactivation, and presented no gain-of-function mutations. In contrast, sporadic cell lines collectively displayed different TSG inactivation patterns and presented kinase-activating mutations, fusion genes, altered mutational frequencies and COSMIC signatures, and different methylome-based classifications. Cell lines re-classified as melanomas and other sarcomas exhibited a different drug-treatment response. Deep genomic analysis, methylome-based classification, and cell-identity marker expression, challenged the identity of common MPNST cell lines, opening an opportunity to revise MPNST differential diagnosis.Malignant peripheral nerve sheath tumors (MPNSTs) are soft-tissue sarcomas of the peripheral nervous system that develop either sporadically or in the context of neurofibromatosis type 1 (NF1). MPNST diagnosis can be challenging and treatment outcomes are poor. We present here a resource consisting of the genomic characterization of 9 widely used human MPNST cell lines for their use in translational research. NF1-related cell lines recapitulated primary MPNST copy number profiles, exhibited   •A comprehensive genomic resource of widely used MPNST cell lines is presented•Genomic, epigenomic, and marker information improve MPNST differential diagnosis•Genomic analysis detected misidentified and misdiagnosed MPNST cell lines•Misdiagnosed cell lines exhibited a different drug-treatment response Biological sciences; Neuroscience; Cancer; Omics TP53 is also frequently lost or mutated. MPNSTs also show recurrently altered chromosomal regions, particularly constituting somatic copy number gains , around forty different MPNST cell lines may have been established by different laboratories, derived from both sporadic and NF1-related MPNSTs. Some of these MPNST cell lines are well distributed among labs,,,Established cell lines are an important tool for gaining insight into cancer biology and treatment. However, there are also different caveats in their use as faithful and useful models, with issues including misidentification and cross-contamination and poor characterization of similarity to their tumor source.,In this work, we performed a deep genomic characterization of 8 commonly used MPNST cell lines,NF1 germline (c.1649dupT) mutation and somatic copy number alteration landscape, and dismissed the use of the T265 cell line, which we assume was misidentified at some point after its establishment and expansion. Note that ST88-14 NF1 germline mutation is not correctly described in certain repositories and publications.We started with 9 NF1-associated and sporadic MPNST cell lines for a short tandem repeat (STR) authentication analysis and a comprehensive genomic characterization. We first performed an interspecies PCR of all cell lines to identify any possible interspecies cross-contamination . Then weNF1 mutational status. It also provides a reference to the original description of each cell line. With them, we performed flow cytometry, SNP-array analysis, whole-exome sequencing, and whole-genome sequencing techniques. We compiled information about their ploidy, global copy-number profile and loss of heterozygosity (LOH) status, structural rearrangements, single nucleotide variants (SNVs), and mutational signatures, and summarized the mutational status of a set of selected MPNST-related genes. With all these data we elaborated a practical summary sheet for each cell line, containing the most relevant information  and G-banding staining. Both techniques produced results difficult to summarize consistently (data not shown), possibly due to the high degree of variability when analyzing multiple metaphases from the same cell line and to the highly rearranged nature of MPNST genomes. Therefore, we decided to analyze the ploidy by propidium iodide staining analysis using flow cytometry A, which Next, we performed a global copy number analysis using SNP-array data from the 8 MPNST cell lines. To verify whether these cell lines were capturing the copy number profiles present in primary MPNST tumors, we also analyzed SNP-array data from an independent set of primary MPNSTs previously analyzed,We also performed a copy number analysis from all cell lines based on WGS data. In addition, we obtained a B-allele frequency (BAF)-like profile from variant-allele frequencies (VAF) and a log-R ratio (LRR) from coverage. We plotted them together with the resulting LOH determination and copy number calling from SNP-array comparing both independent sets of data . Part ofCDKN2A, PRC2 genes, and TP53  and small indels, uncovered a relatively moderate impact of these alterations in MPNSTs. We used WES and WGS data from all cell lines to call small variants. Since we did not have available non-tumoral tissue counterparts for these cell lines, we filtered the small variants to partially remove germline variants and obtain a call set enriched in somatic variants. The number of variants with a potential impact on protein function in this dataset was modest , particuNF1 mutations, somatic mutations also affected NF1, as well as other genes including TP53, PRC2 genes, and PTEN. Remarkably, we did not identify gain-of-function mutations in oncogenes, except a BRAF V600E mutation in the STS-26T cell line. In contrast, we identified gains in genomic regions containing receptors, especially a highly gained region containing PDGFRA and KIT in two NF1-related cell lines (S462 and NF90-8)  B. The moThis complete description of the genomic status of MPNST cell lines uncovered a fair degree of diversity among them, prompting us to question the MPNST identity of the sporadic cell lines and to perform a further characterization. Since sarcomas comprise a morphologically heterogeneous class of tumors and their diagnosis has been hampered by a high misclassification rate, we decided to perform methylome analysis of all cell lines in comparison to established reference cohorts of different peripheral nerve sheath and soft tissue tumorsAltogether, the fine landscape of genomic alterations, methylome-based classification, marker expression, and particular informative gain-of-function mutations, captured a fair degree of variability among MPNST cell lines C uncoverNF1 gene (Selumetinib) and a BET inhibitor targeting the inactivation of PRC2 (JQ1), since both TSGs had different inactivation patterns in the two groups of cell lines . The use of WGS also allowed the identification of fusion genes, such as EML4-ALK, generated by an inversion affecting both genes in the HS-PSS sporadic cell line. The presence of fusion genes is common in other types of soft tissue sarcomas that otherwise contain genomes with few genomic alterations. However, fusion genes are not common in the karyotypically complex MPNSTs.Despite the diverse ploidies exhibited by the MPNST cell lines, NF1-related cell lines faithfully reproduced the copy number profiles present in primary MPNST tumors, something that was not true for sporadic cell lines. These results reinforce the idea that despite the high degree of genomic alterations, MPNSTs contain quite stable genomes, as shown by comparing primary tumors with derived orthotopic PDX.TP53. Notably, the frequency and the type of mutation signatures exhibited provided an important differential indicator. While all cell lines exhibited a similar number of mutations and similar mutational signatures, the STS-26T cell line contained a much higher number of mutations and an important contribution from mutational signature 7, predominantly found in skin cancers.In contrast to the importance of somatic copy number alterations and structural rearrangements, MPNST cell lines exhibited a modest frequency of mutations, with moderate functional impact, mainly involving the inactivation of PDGFRA and KIT receptors, consistent with previous reports,BRAF V600E mutation in the STS-26T cell line and the already mentioned EML4-ALK fusion gene in the HS-PSS cell line.Genomic alterations or mutations constituting a gain-of-function were not common in NF1-related MPNST cell lines. In fact, we identified only two cell lines (S462 and NF90-8) with a highly gained region in chromosome 4 containing the BRAF V600E mutation. It also exhibited a much higher mutation frequency than the other cell lines and an important COSMIC signature 7, predominantly present in skin cancers as previously described by Hayward et al. (2017).BRAF V600E mutations described in the literature,,NF1 and CDKN2A. It was classified as melanoma by the methylome classifier and expressed the markers SOX10 and S100B. The expression of these two markers is lost in transitions from atypical neurofibromas to MPNSTs and is commonly significantly reduced or absent in MPNSTs.NF1 and CDKN2A,,,,Our deep genomic characterization  questioned the MPNST identity of the analyzed sporadic cell lines. Methylome-based classificationPreliminary data on drug treatment using these cell lines showed a different impact on cell viability between genuine MPNST cell lines and the ones potentially misidentified, highlighting the importance of correct MPNST diagnostics for better clinical management.Despite the potential misdiagnosis of the three sporadic cell lines studied here, it is important to remark that these results do not imply that all sporadic MPNSTs (and derived cell lines) are not genuine MPNSTs. In fact, deep genomic analysis of primary MPNSTs , confirmed the existence of sporadic MPNSTs exhibiting the same genomic characteristics as the NF1-related MPNST cell lines reported here. Conversely, our results neither discard the misidentification of MPNSTs in the NF1 setting.In summary, the new genomic and epigenomic characterization of MPNST cell lines provided in this work uncovered the misidentification of the commonly used NF1-related T265 MPNST cell line and, in addition, compiled multiple pieces of evidence to question the identity of the three sporadic MPNST cell lines analyzed here, proposing alternative identities for all of them: a melanoma for STS-26T; a desmoplastic melanoma for the HS-Sch-2 cell line; and an epithelioid inflammatory myofibroblastic sarcoma for the HS-PSS. These results may imply the need of determining the impact of their use in previous and probably current works being performed, considering the new information provided. It also alerts us, as a scientific community, that we need to improve the characterization and control of the cell lines and tissues we use in our research. But above all, it provides an opportunity to look ahead and improve our understanding of what is an MPNST and which types might exist. In this regard, a systematic combination by different laboratories of histological characterization together with these new ways of analyzing genomes and epigenomes opens the door to revising the manner we perform differential diagnostics of MPNSTs and related tumors.Our results, in addition to generating a valuable resource for the study of new therapeutic strategies for MPNSTs, uncover the need to systematically analyze MPNSTs, combining pathology with genomic and molecular techniques. Genomic analysis such as copy number profiles, structural variants, mutation frequencies and signatures, presence of gain-of-function mutations, and the inactivation of specific TSGs, together with methylome-based sarcoma classification and cell identity marker analysis, emerge as valuable tools for a better differential diagnosis and classification of MPNSTs.Different limitations of this study exist. The cell lines used here may not completely represent the repertoire of recurrent genomic alterations present in MPNSTs due to their type and limited number. In addition, the use of established MPNST cell lines makes difficult the availability of the original MPNST for comparison purposes or of a normal pair tissue for the identification of a reliable set of somatic variants. Finally, all sporadic cell lines used here may be misclassified, which urges for the deep genomic characterization of additional MPNST cell lines.This work has been approved by the Germans Trias i Pujol Hospital (HUGTiP) Ethics Committee.eserra@igtp.cat) and Bernat Gel (bgel@igtp.cat).Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Eduard Serra . We studied six NF1-associated cell lines: S462 (RRID:CVCL_1Y70),Total DNA was extracted from cell lines using the Gentra Puregene Kit (Qiagen). DNA was quantified with Nanodrop 1000 spectrophotometer (Thermo Scientific). For SNP array, whole exome and genome sequencing and methylome experiments, a fluorescence-based quantification of DNA was performed either by using the Quant-iT PicoGreen dsDNA Assay (Thermo Fisher Scientific) or a Qubit fluorometer (Life Technologies).DNA fingerprinting of short tandem repeats (STRs) was conducted for all MPNST cell lines using the AmpFlSTR Identifiler Plus Amplification kit (Applied Biosystems) following the manufacturer’s instructions. This kit is based on the analysis of 16 microsatellites, including the nine STRs used by the ATCC.6 cells from each cell line were trypsinized, washed with PBS, and fixed in ice-cold 70% ethanol for 2h at −20°C. Then, cells were washed with PBS and resuspended in a citrate-phosphate buffer for at least 30 min, up to 2h. Cells were then washed with PBS- 1% FBS and propidium iodide (PI) was added. Cells in PI solution were treated with DNAse-free RNAse A for 30–45 min at 37°C and were ready for flow cytometry analysis. All samples were analyzed on a FACSCanto II flow cytometer  and a total of 10,000 single cells were analyzed for each sample. Aggregated cells were excluded by gating out on a biparametric plot with DNA content pulse area versus width. Data was analyzed using FlowJo software . HFF-1 were used as 2n control cells.About 1–2x10NF1 constitutional pathogenic variant of the cell lines S462,NF1 pathogenic variant of NMS-2 cell line, and the NF1 pathogenic variants present in the HS-Sch-2 cell line by whole-exome sequencing (see below) which were also confirmed by Sanger sequencing. We used specific primers targeting the mutation region in each case and the BigDye Terminator v.3.1 Sequencing Kit (Applied Biosystems). Sequences were generated with the ABI Prism 3100 Genetic Analyzer (Applied Biosystems) and analyzed with CLC Main Workbench 6 software.Sanger sequencing was used to confirm previously described SNP-array data from the different cell lines and tumors was obtained from Magallon-Lorenz et al. (2021).WES from the 8 MPNST cell lines was also previously analyzed in Magallón-Lorenz et al. (2021).The whole genome of two cell lines (ST88–14 and S462) had already been sequenced for Magallón Lorenz et al. (2021).MUC3A, MUC5AC, OR52E5, OR52L1, SMPD1, PRAMEF and LILR). Finally, we filtered out the variants present in dbSNP except for those included in COSMIC somatic mutations (https://ftp.ncbi.nlm.nih.gov/snp/others/rs_COSMIC.vcf.gz) or the International Cancer Genome Consortium (ICGC) (https://ftp.ncbi.nlm.nih.gov/snp/others/snp_icgc.vcf.gz) variant lists. WES data was processed using the same approach and used to validate the variants identified in WGS data.Small nucleotide variants were called with strelka2MUC3A, MUC5AC, OR52E5, OR52L1, SMPD1, PRAMEF and LILR). We also filtered out the variants in dbSNP except for those present in COSMIC and ICGC. We used this call set enriched in somatic variants with the mutSignaturesRaw variants called by Strelka2 in WGS data were also the basis for the mutational signature analysis. Since normal pairs were not available, we applied a series of filters to approximate a somatic callset: we filtered out the variants with a population frequency (AF_popmax) higher than 1%, called in more than one cell line, with a variant allele frequency (VAF) lower than 0.1 and, variants in highly variable genes (https://github.com/Boyle-Lab/Blacklist/blob/master/lists/hg38-blacklist.v2.bed.gz), -no-edge option and 1000 bp bins). To obtain the exact copy number profile of each sample we used the threshold method with sample-specific thresholds defined considering the ploidy of each cell line obtained by flow cytometry. Summarized and per-cell line copy number profiles were plotted using the CopyNumberPlots (10.18129/B9.bioc.CopyNumberPlots) and karyoploteRWe called copy-number alterations from WGS using CNVkithttps://github.com/brentp/smoove) as a structural variant (SV) caller with parameters for small cohorts and excluding the problematic regions defined in https://github.com/hall-lab/speedseq/blob/master/annotations/exclude.cnvnator_100bp.GRCh38.20170403.bed.https://github.com/TranslationalBioinformaticsIGTP/CliffHunteR), an in-house developed sensitivity-oriented R package for breakpoint detection, and a thorough visual inspection using Integrative Genomic Viewer (IGV)NF1, CDKN2A, SUZ12, EED, TP53, PTEN, RB1). To discard germline structural variants, we filtered out SVs present in the Database of Genomic Variants (DGV)https://cancer.sanger.ac.uk/census).We used LUMPYDNA methylation profiles were generated using the Infinium MethylationEPIC (850k) BeadChip array  according to the manufacturer’s instructions. The data was processed as previously described.Inter-chromosomal rearrangements detected by LUMPY or CliffHunteR affecting genes commonly altered in MPNST were validated by PCR and Sanger sequencing. PCR primers, annealing temperatures and amplicon lengths are summarized in EML4-ALK v5a fusion gene breakpoints were detected by LUMPY in HS-PSS cell line. EML4-ALK fusion gene was validated by RT-PCR and Sanger sequencing. Total RNA from HS-PSS cell line was extracted using the 16 LEV simplyRNA Purification Kit (Promega) following the manufacturer’s instructions in the Maxwell 16 Instrument (Promega). RNA was quantified with a Nanodrop 1000 spectrophotometer (Thermo Scientific). RNA (0.5 μg) was reverse transcribed using the Superscript III reverse transcriptase enzyme (Life technologies) according to the manufacturer’s instructions. PCR primers, annealing temperatures, and amplicon lengths were previously described by Takeuchi et al. (2008).Cells were fixed in 4% paraformaldehyde (PFA)  in PBS for 15 min at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 10 min, blocked in 10% FBS in PBS for 15 min, and incubated with the primary antibodies, SOX10, SOX9 and S100B overnight at 4°C. Secondary antibodies were Alexa Fluor 488- and Alexa Fluor 568- (Thermo Fisher Scientific). Nuclei were stained with DAPI . Slides were mounted with Vectashield (Vector laboratories), and coverslips were secured with polish nail.Compounds (Selumetinib and JQ1) were prepared at 10 mmol/L in DMSO, and were diluted 5-fold from 40 μmol/L to 0.064 μmol/L, with DMSO. Cells were seeded in 96-well plates (Corning) at a density between 2.000 and 10.000 cells/well. 24 h later, drugs were added in three replicates. After 48 h of incubation with the drugs, cell viability was analyzed using MTT assay (Sigma-Aldrich), following manufacturer’s instructions. The percentage of cell viability was calculated by normalizing the values to DMSO control cells.Bioinformatic analysis is thoroughly described in the


Inula viscosa is widely distributed throughout Europe, Africa, and Asia. Used as a medicinal plant in different countries, I. viscosa has been characterized for its complex chemical composition in order to identify the bioactive compounds responsible for its biological activities, including anticancer effects. Sesquiterpene lactones (SLs) are natural, biologically active products that have attracted considerable attention due to their biological activities. SLs are alkylating agents that form covalent adducts with free cysteine residues within enzymes and key proteins favoring cancer cell cytotoxicity. They are effective inducers of apoptosis in several cancer cell types through different molecular mechanisms. This review focuses on recent advances in the cytotoxic effects of I. viscosa and SLs in the treatment of neoplastic diseases, with a special emphasis on their proapoptotic molecular mechanisms.Cancer is a complex disease including approximately 200 different entities that can potentially affect all body tissues. Among the conventional treatments, radiotherapy and chemotherapy are most often applied to different types of cancers. Despite substantial advances in the development of innovative antineoplastic drugs, cancer remains one of the most significant causes of death, worldwide. The principal pitfall of successful cancer treatment is the intrinsic or acquired resistance to therapeutic agents. The development of more effective or synergistic therapeutic approaches to improve patient outcomes and minimize toxicity has become an urgent issue.  Inula viscosa (L.) Aiton. (Dittrichia viscosa L.) is a medicinal perennial plant belonging to the genus Inula and family Asteraceae and is considered one of the most crucial pharmaceutical plants in the Mediterranean Basin [I. viscosa has been characterized in different countries for its complex chemical composition to identify the bioactive compounds responsible for its biological activities [I. viscosa has long been used in folk medicine owing to its anti-inflammatory [I. viscosa, which are based on the inhibition of COX1, COX2, and iNOS enzymatic activity [I. viscosa leaves growing in Algeria. They identified different phytochemical components, including phenolic acids, flavonoids, lignans, and terpenoids; some of which were recognized for the first time and belong to different subfamilies of compounds [Traditional herbal medicines are excellent sources of natural biologically active products with powerful therapeutic effects that can be utilized in the treatment of various diseases, including cancer. an Basin . As a metivities . I. viscammatory , anthelmammatory ,5, and iammatory  and diabammatory . Hernandammatory . Recentlactivity ,10. Kheyompounds .I. viscosa have been demonstrated in various cancer cell lines [I. viscosa were tested alone or in association with cancer cell lines [I. viscosa extracts from three different regions of Morocco, revealing the presence of sesquiterpene lactones (SLs), tomentosin, and inuviscolide as major representative compounds, with tomentosin concentrations ranging from 22% to 64% and inuviscolide concentrations ranging from 0% to 58% in different Moroccan regions [Asteraceae family, SLs have been identified in several families of flowering plants, including Solanaceae, Araceae, and Cactaceae [Inula (Asteraceae) genus [Recently, cytotoxic and anticancer activities of ll lines ,13,14. Tll lines ,16,17,18ll lines ,20. Mess regions . Most seactaceae . As repoI. viscosa extracts, characterize previous molecular mechanisms. SLs induce cell cycle arrest in the G2/M phase and appearence of a sub-G0 fraction evocative of apoptotic cell death in melanoma cell lines [I. japonica, performs antineoplastic activity against Burkitt lymphoma (BL) cells [I. viscosa on cervical cancer cells [Recently, several studies have reported the potential anticancer effects of SLs and have shown that tomentosin and inuviscolide exert antiproliferative effects on human cancer cell lines ,24. In pll lines . JaponicL) cells . Additioer cells .I. viscosa and its prevalent SLs in the treatment of neoplastic diseases, with special emphasis on their proapoptotic molecular mechanisms.This review summarizes the emerging roles of I. viscosa and SLs in solid and hematologic cancers. A literature search for original and review articles was performed using electronic databases of Medline  by using the terms ‘Inula viscosa’, ‘tomentosin’, ‘inuviscolide’, ‘cancer’, ‘carcinoma’, ‘apoptosis’. Combinations of these terms were used to screen the mentioned databases for relevant content: title, abstract, and full content, respectively. Only studies written in English were considered for evaluation. Pre-screening and screening selection removed duplicate studies, foreign language studies, irrelevant studies, and studies for which updated research was unavailable. We considered both clinical and experimental studies (in vivo and in vitro).The purpose of this review is to provide an overview of the current knowledge regarding the antineoplastic role of Cytotoxic screening models represent crucial preliminary data for analyzing the antineoplastic properties of selected plant extracts. Early tests were cell-based assays performed on established cell lines in which the toxic effects of plant extracts or isolated compounds could be measured. Conventional antitumor agents display significant cytotoxic activities in cell culture systems .I. viscosa hexane extract showed pronounced cytotoxic effects against both cervical cancer cell lines, inducing dose-dependent cell growth inhibition by stimulating apoptosis, which is related to the decrease in mitochondrial membrane potential (ΔΨm) and increase in intracellular reactive oxygen species (ROS) production. Thus, I. viscosa extracts showed significant cytotoxic effects against cervical cancer cell lines through the inhibition of proliferation and induction of apoptosis involving a mitochondria-mediated signaling pathway by pro-caspase activation, BCL-2 expression, and PARP cleavage [I. viscosa extracts target the telomerase machinery and induce apoptosis in human cervical carcinoma cell lines (SiHa and HeLa) and that the molecular mechanism underlying I. viscosa extract-induced apoptosis includes a caspase-3 mediated signaling pathway [Benbacer et al., with the purpose of developing new anticancer drugs against cervical cancer, applied the human cervical carcinoma SiHa and HeLa cell lines as a model system to screen the anticancer effects of plants from traditional Moroccan medicine. In particular, they demonstrated that cleavage . The sam pathway .I. viscosa extracts harvested from three different regions of Morocco  in two breast cancer cell lines, MCF7, an estrogen receptor-positive cell line, and MDAMB-231, an estrogen receptor-negative cell line. These two I. viscosa extracts showed different toxicity on breast cancer cells, suggesting that the different cytotoxic activity can be an integral effect of the combination of three major compounds, tomentosin, inuviscolide, and isocostic acid which are present in variable concentrations in plants from different regions. Furthermore, the reduced toxicity exerted by the two extracts on MCF-7 cells when compared with MDA-MB-231 cells suggests that heterogeneous susceptibility could be dependent on the activation of different signaling pathways [I. viscosa from Taounate, Imouzzer, and Sefrou had different rates of polyphenols associated with different antioxidant activities [I. viscosa on MCF-7 cells [An interesting study by Messaoudi et al. showed the cytotoxic activity of ethyl acetate and ethanolic pathways . In additivities . Further-7 cells ,31.I. viscosa aqueous extract on colon cancer cell growth in vitro and tumor growth in vivo, using a xenograft mouse model. In vitro experiments revealed that exposure of colorectal cancer cells to I. viscosa extract significantly reduced cell viability in a dose- and time-dependent manner. Interestingly, the analysis of the molecular mechanisms underlying the I. viscosa effect showed the activation of caspase-9 in HCT116 well-differentiated cells and of caspases-8 and -9 in colo320 poorly-differentiated cells. These findings suggest that I. viscosa extract induces apoptosis through the intrinsic mitochondrial pathway in well-differentiated cells, and through both the intrinsic and extrinsic pathways in poorly-differentiated cells. In vivo studies revealed that treatment with I. viscosa extract inhibited tumor growth in mice transplanted with MC38 cells, showing a strong reduction in the weight and volume of neoplastic lesions. Interestingly, no side effects such as weight loss, behavioral changes, ruffled fur, or changes in kidney and liver function were observed, suggesting the absence of toxicity from I. viscosa [Bar-Shalom et al. examined the possible therapeutic effects of I. viscosa collected from an uncontaminated area of the National Park on Asinara Island, Sardinia, revealed powerful anti-lymphoma activity. Specifically, Raji cells treated with increasing concentrations of I. viscosa ethanolic extract demonstrated a dose- and time-dependent decrease in cell viability, displaying a reduction in cell proliferation obtained by the induction of cell cycle arrest in the G2/M phase, and a dose-dependent increase in cell apoptosis. A gene expression analysis of signal transduction and apoptotic pathway players involved in B-lymphocyte functions showed that the molecular mechanisms involved in I. viscosa anticancer activity were characterized by the downregulation of genes involved in cell cycle and proliferation , as well as in the inhibition of cell apoptosis  [ BCL11A) .I. viscosa methanol and aqueous extracts on the malignant melanoma cell lines A2058 and MeWo, and normal fibroblasts. Cytotoxicity, apoptosis induction, and migration suppression were strongly induced in malignant melanoma cell lines by I. viscosa methanol extracts compared to the aqueous extracts [I. viscosa has been demonstrated. Specifically, I. viscosa methanol extract promotes the downregulation of miRNAs related to epithelial-mesenchymal transition and poor prognosis in malignant melanoma, such as miR-191 and miR-193, while favoring the overexpression of miR-579 and miR-524, which mainly repress the MAPK signaling pathway in malignant melanoma [An interesting study evaluated the cytotoxic and anticancer effects of extracts , confirmextracts ,30. For melanoma .I. viscosa extract in a mouse model of DMBA/croton oil-induced skin carcinoma. Animals received treatment with the extract before and after the induction of skin carcinogenesis, showing that I. viscosa extract inhibited the development of papilloma in mice. Furthermore, ingestion of I. viscosa extract delayed the formation of cutaneous papillomas in animals and simultaneously decreased the size and number of papillomas. A structure–activity study showed that I. viscosa extract contains bioactive molecules with much greater inhibition of the subunits of the proteasome, as well as a decrease in the concentration of proteasome and its catalytic activity in serum and intracellularly when compared to chemically synthesized inhibitors, thus emerging as a new candidate for targeted therapy against skin carcinoma. Specifically, molecular docking analysis revealed that tomentosin, inuviscolide and isocostic acid compounds obtained from I. viscosa extract were stabilized in the pocket of the 20S proteasome β5 receptor subunits by various interactions, mirroring the same mechanisms exerted by carfilzomib, a potent second-generation proteasome inhibitor with significant anti-myeloma activity [I. viscosa extracts also increased cell cycle arrest and cell death in the glioblastoma LN229 cell line, characterized by a TP53 mutation, compared to U87MG cells with wild-type TP53. SW620 cells were more sensitive to I. viscosa extracts, suggesting that they may contain molecules with high therapeutic potential against MDR cell lines. PC-3 are androgen-insensitive and apoptosis-resistant prostate cancer cells, on which I. viscosa extracts induce growth inhibition, cell cycle arrest and apoptosis, supporting the idea that active compounds effectively targeting extrinsic and intrinsic apoptosis pathways are present in the plant [I. viscosa treatment with in vitro and in vivo models.The ubiquitin–proteasome system plays a key role in intracellular proteolysis, particularly in the degradation of abnormal proteins. In fact, it is directly involved in the regulation of most biological processes, such as cell cycle, apoptosis, muscle differentiation, and immune response . Many stactivity . I. vische plant . Table 1I. viscosa were tested alone or in association in cancer cell lines [Different biologically active compounds, belonging to several families, isolated from ll lines ,16,17,18ll lines ,20, showll lines ,22.In SLs, it is assumed that the electrophilic ab-unsaturated carbonyl structures, such as α-methylene-γ-lactone, represent bioactive functional groups interacting in a Michael-type addition with the nucleophilic sites of biological molecules. SLs are thought to inhibit tumor cell proliferation by selective alkylation of cysteine sulfhydryl groups in growth-regulatory biological macromolecules, such as key enzymes that control cell division by restoring the ability of tumor cells to undergo apoptosis . In addiI. viscosa leaves and analyzed their anticancer effectiveness against human melanoma cell lines with the aim of developing new agents for melanoma treatment, taking into consideration the aggressiveness and chemotherapeutic resistance of this tumor. Tomentosin and inuviscolide reduced cell viability in three different human melanoma cell lines by favoring cell cycle arrest at the G2/M phase, associated with an increase in the sub-G0 fraction indicative of apoptotic cell death, as demonstrated by changes in membrane phospholipids, mitochondrial membrane potential, and activation of caspase-3. The molecular mechanism of SL-mediated G2/M arrest and apoptosis in melanoma cell lines suggests that SLs are possible alkylating agents that might cause DNA damage, which activates the kinase ATM/ATR. This activation is followed by early phosphorylation of TP53 (Ser15) and CDC2 (Thr14 and Tyr15) providing early G2/M arrest. The activation of TP53 transactivates p21waf1, which also reduces the protein concentration of CDC2/Cyclin B1, favoring the elongation of G2/M arrest, and ultimately resulting in apoptosis [Rozenblat et al. purified the SLs, tomentosin and inuviscolide, from poptosis . To bettpoptosis . In humapoptosis .I. viscosa causes a reduction in the mitochondrial membrane potential and an increase in ROS levels in human cervical cell lines. It leads to the downregulation of pro-caspase-3 protein, cleavage of PARP, and enhanced caspase-3 activity associated with BCL2 downregulation in tomentosin-treated SiHa and HeLa cells [To explain possible mechanisms underlying tomentosin-induced apoptosis, Merghoub et al. studied the effects of tomentosin on telomere lengthening by hybridization with a telomeric C-rich probe (21C) under non-denaturing conditions, which drastically induced telomere G-overhang shortening in human cervical cell lines. This mechanism was validated on JW10 cells in which tomentosin induced a significant anti-proliferative effect acting on hTERT expression, while it exhibited a low cytotoxic effect on Wi38 fibroblast cells, a primary cell culture without telomerase expression and activity . FurtherLa cells .FOXO3 and p27 expression, thus suggesting that FOXO3 upregulation controls G2/M phase cell cycle arrest through p27 overexpression after tomentosin treatment in an osteosarcoma cell line [p27 overexpression regulated by the upregulation of FOXO3. In addition, cell cycle arrest and apoptosis induction are boosted by the overexpression and phosphorylation of TP53 and activation of the ERK signaling pathway [ROS are commonly produced by mitochondrial oxidative metabolism in response to cellular stress . They arell line . A simil pathway .BAX overexpression, BCL-2 downregulation, and inhibition of inflammation, as revealed by the downregulation of IL-6, TNF-α, IL-1β, and IL-8 which modulate cell growth proteins in gastric cancer cells [A recent study validated the hypothesis that tomentosin-induced oxidative stress is involved in apoptosis via the mitochondria-mediated signaling pathway in gastric carcinoma cell lines. The molecular mechanisms that explain the antiproliferative effect of tomentosin are based on the reduction in PCNA and cyclin D1, which are able to control cell growth. The induction of apoptosis is related to er cells .BCL2A1 and CDKN1A, and upregulation of the proapoptotic PMAIP1 gene [ATF4 and DDIT3 genes, suggesting that tomentosin treatment the activation of the protective unfolded protein response signaling might induce cell apoptosis. Functional connection analysis executed by the connectivity map tool indicated that tomentosin acts as the heat shock protein inhibitors on MM cells [Recently, the antineoplastic activity of SLs has also been evaluated in hematologic tumors such as LB, multiple myeloma (MM), and leukemia cancer cell lines. Virdis et al. demonstrated that tomentosin exerts strong antitumor activity on human BL  and MM IP1 gene . In totaMM cells . Furthermore, Yang et al. showed that tomentosin stimulates intracellular ROS production, causing mitochondria-centered death in leukemia MOLT-4 cells, revealing significant cytotoxicity. Tomentosin induces apoptosis in MOLT-4 cells by suppression of NF-κB and proinflammatory cytokines, as revealed by the complete blocking of anti-apoptotic proteins (cyclin D1 and BcL-2) and activation of proapoptotic proteins (caspase-3 and BAX) via the mTOR/PI3K/AKT pathway . These fCASP8, PPARG, FAS, FADD, TNF, and TNFR1 genes for extrinsic pathway and BAX, BCL2, CASP3, CASP7, CASP9, and CYCS genes for intrinsic pathway and increases caspase-3 and caspase-9 protein levels [Recently, Güçlü et al. analyzed the anticancer effects of tomentosin on PANC-1 and MIA PaCa-2 human pancreatic cancer cells on which the treatment induces suppression of proliferation, migration, invasion, and colony formation capacity. Tomentosin increases apoptosis rate and ROS production and decreases mitochondrial membrane potential apoptosis in pancreatic cancer cells. At a molecular level, tomentosin induces overexpression of apoptosis-related genes, such as n levels . Despite the advances in the development of innovative antineoplastic drugs, cancer remains one of the most important causes of death worldwide. The principal pitfall of successful cancer treatment is the intrinsic or acquired resistance to therapeutic agents. Among the conventional treatment options, chemotherapy is most often applied to different types of cancer. Several chemotherapeutic molecules induce cell cycle arrest but not apoptosis, permitting neoplastic cells to repair their damaged DNA and therefore potentially limit treatment effectiveness . These aI. viscosa and its natural biologically active compounds, such as tomentosin and inuviscolide. Moreover, it provides a detailed description of the molecular mechanisms of action in the context of in vitro experiments. Interestingly, cytotoxic effects against human cancer cell lines induced by I. viscosa and SLs are characterized by cell growth inhibition and apoptosis induction, related to the decrease in mitochondrial membrane potential and increase in intracellular ROS production. Various molecular mechanisms are responsible for the activation of apoptosis. In particular, cell treatment induces activation of pathways known to lead to cellular death, downregulation of anti-apoptotic genes such as BCL2A1 and CDKN1A, and upregulation of the proapoptotic PMAIP1 gene. In addition, SLs can target multiple signaling pathways, such as NF-κB, PIK/AKT/mTOR, and MAPK, which control apoptosis, growth, proliferation, migration, and invasion. In vivo tests within preclinical animal studies and subsequently in the context of controlled clinical trials should also be encouraged to shed further light on the exact molecular mechanisms by which tomentosin induces pharmacological effects in human cancer and to assess possible side effects. These preliminary steps could allow the development of potential natural products with limited toxicity and relevant antitumor activity, making them available among the therapeutic armamentarium offered to patients affected by solid and hematologic tumors.This review describes recent advances in our understanding of the potential anticancer activity of


Patients with intrahepatic cholangiocarcinoma (ICC) require chemotherapy due to late detection, rapid disease progression, and low surgical resection rate. Tumor cell lines are extremely important in cancer research for drug discovery and development. Here, we established and characterized a new intrahepatic cholangiocarcinoma cell line, ICC-X1. STR testing confirmed the absence of cross-contamination and high similarity to the original tissue. ICC-X1 exhibited typical epithelial morphology and formed tumor spheres in the suspension culture. The population doubling time was approximately 48 h. The cell line had a complex hypotriploid karyotype. The cell line exhibited a strong migration ability in vitro and cell inoculation into BALB/c nude mice led to the formation of xenografts. Additionally, ICC-X1 cells were sensitive to gemcitabine and paclitaxel but resistant to 5-fluorouracil and oxaliplatin. RNA sequencing revealed that the upregulated cancer-related genes were mainly enriched in several signaling pathways, including the TNF signaling pathway, NOD-like receptor signaling pathway, and NF-κB signaling pathway. The downregulated cancer-related genes were mainly enriched in the Rap1 signaling pathway and Hippo signaling pathway among other pathways. In conclusion, we have created a new ICC cell line derived from Chinese patients. This cell line can be used as a preclinical model to study ICC, specifically tumor metastasis and drug resistance mechanisms.The online version contains supplementary material available at 10.1186/s12935-022-02840-3. Intrahepatic cholangiocarcinoma (ICC) is a malignant tumor, which originates from the intrahepatic bile duct epithelial cells. This cancer ranks second in occurrence to hepatocellular carcinoma and accounts for 10–15% of all cases of primary liver cancer . The onsThe majority of patients are hard to resect or develop distant metastases because of local tumor infiltration; therefore, treatment options other than surgery are required. Currently, gemcitabine-based chemotherapy combination regimens are used as first-line treatments for patients with unresectable or recurrent and metastatic cholangiocarcinoma. However, these regimens have limited effect on improving the long-term survival of patients .The choice of an appropriate course of treatment for patients and improving their survival requires a thorough understanding of the pathogenesis of ICC, the complex interactions between the pathogenic genes, and the tumor microenvironment.Tumor-derived cell lines provide important cell experimental models for basic and clinical research on human tumors. However, with an increase in the number of passages in vitro, some unique biological characteristics of the cell line gradually change or disappear and even characteristics that the original cell does not have may be produced. Cell cross-contamination has also been detected in many cell lines. HeLa, T-24, and M14 are the most common cross-contaminating cell lines; therefore, studies using these cell lines may produce erroneous results –12.In the United States, the lack of reproducibility of preclinical studies costs about $56 billion annually. The cost of biological reagents and reference materials accounts for about one-third of the total cost. Several studies are not reproducible because of the misidentification, contamination, genetic drift, and clonal evolution of cell lines, thereby hindering cancer research .ICC is a highly heterogeneous disease with multimolecular profiles and diverse clinical outcomes. Tumor heterogeneity hinders patient prognosis and treatment. Notably, the lack of availability of ethnically diverse cell lines has impeded the development of drugs beneficial to patients of different ethnicities. Therefore, ethnic differences should also be factored into tumor research –18.At present, there are many ICC cell lines available for research, but the number of ICC cell lines of Chinese origin is very small. This is incompatible with China’s huge population. To further study the characteristics of ICC in the Chinese population, more Chinese-derived ICC cell lines need to be established. The new ICC cell lines can enrich the existing cell line bank and can provide a preclinical model for the development of new drugs to treat patients with ICC .In this study, we established a new ICC cell line of Chinese origin and named it ICC-X1. Further, we determined its biological and molecular characteristics. Our model can be used as an effective tool to study the mechanisms underlying the occurrence, development, metastasis, and drug resistance of ICC.ICC tissues were obtained from surgical specimens resected in the Fourth Department of General Surgery, the First Hospital of Lanzhou University, China. The patients did not receive any preoperative treatment. Fresh ICC tissue specimens were cut under sterile conditions and transported to the laboratory for primary cell isolation and culture in the shortest possible time. The postoperative pathology report revealed a poorly differentiated ICC in the specimens. This study was approved by the Ethics Committee of the First Hospital of Lanzhou University . Informed consent was obtained from the patient.Six-week-old sterile BALB/C female nude mice were purchased from Changzhou Cavens Laboratory Animal Co., Ltd  and raised in the Animal Experiment Center of Lanzhou University, China. Mice were provided ad libitum access to sterile water and food. Animal experiments were carried out per the requirements of the Laboratory Animal Protection Committee of Lanzhou University, China. When an animal became seriously ill during the experiment, it was euthanized by carbon dioxide exposure followed by cervical dislocation.2. The digested solution was filtered using a 200-mesh sieve and the filtrate was centrifuged at 300×g for 3 min. The supernatant was discarded, the pellet was resuspended in 5 mL PBS, and centrifuged again at 300×g for 3 min. The final pellet was resuspended in the RPMI-1640 medium containing 10% fetal bovine serum , 1% penicillin–streptomycin. The suspension was inoculated into culture plates and incubated at 37 °C in a 95% air and 5% CO2 atmosphere. The medium was changed twice a week. When the cells reached 70–80% confluence, cell passage and cryopreservation were performed under optimal conditions.The freshly obtained tissue samples were placed on an ultraclean bench and rinsed three times with phosphate-buffered saline (PBS) at 4 °C; the blood vessels, necrotic tissues, and interstitial tissues on the surface were removed using sterile forceps. The tissue sample was finely chopped and incubated with collagenase type II and neutral protease solution at 37 °C for approximately 2 h in a humidified atmosphere containing 5% COThe human cholangiocarcinoma RBE cell line was obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China in 2016. The cell line was cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum , 100 U/mL penicillin, and 100 µg/mL streptavidin-mycin .2 for 2 weeks. The cells were harvested every 24 h and analyzed using an inverted microscope to observe their general morphology.ICC-X1 cells were seeded in the 6-well plates and incubated at 37 °C in a humidified atmosphere containing 5% COFifteen passages of logarithmic growth phase ICC-X1 cells were used for these experiments. STR analysis was performed according to the protocols published by the American National Standard Institute (ANSI/ATCC ASN-0002-2011 Authentication of Human Cell Lines: Standardization of STR Profiling).Chromosomal analysis was performed on the cells at passage. The cells were treated with 0.1 µg/mL colchicine for 1 h and were digested with trypsin/EDTA; slides were prepared according to standard methods. The hypotonic treatment was performed using 0.075 M potassium chloride solution for 20 min at room temperature. The slides with fixed cells were stained using trypsin-Giemsa to identify individual metaphase chromosomes. Fifty chromosomes in the split phase were randomly counted under an oil-immersion objective lens (1000× magnification) of a light microscope and 10 well-dispersed G passage specimens were selected for karyotypic analysis. Abnormal chromosomes were identified according to the International Human Cytogenetic Nomenclature System, 2016  was added, and the cell culture plate was placed at 37 °C and humidity in an incubator with 5% CO2. The culture medium was changed every 3–4 days and passaged every 1–2 weeks.The logarithmically growing cells of passage 40 were collected, trypsinized, and prepared as a single-cell suspension. The tumor cell concentration was adjusted to 1000 cells/100 μL. A volume of 100 μL of the cell suspension was taken, mixed with matrigel 1:1, inoculated into a 12-well plate, and incubated at 37 °C for 30 min to form a gel. To each well, 1 mL of organoid culture medium [RPMI-1640 supplemented with 1% Glutamax (Invitrogen), 1% penicillin/streptomycin, 1× B27 supplement (Gibco), 50 ng/mL EGF (Peprotech), 1.25 mM Gemcitabine was purchased from Jiangsu Hansoh Pharmaceutical Group Co., Ltd., China; oxaliplatin was purchased from Jiangsu Hengrui Pharmaceutical Co., Ltd., China; 5-fluorouracil (5-FU) was purchased from Tianjin Jinyao Pharmaceutical Co., Ltd., China; and paclitaxel purchased was from Jiangsu Osaikang Pharmaceutical Co., Ltd. Gemcitabine, 5-FU, and paclitaxel were dissolved in normal saline and oxaliplatin was dissolved in 5% glucose solution. The stock solutions were aliquoted as different working solutions to avoid degradation.Logarithmically growing RBE and ICC-X1 cells of passage 50 were collected separately and prepared as single-cell suspensions after trypsinization. The cells were inoculated into 96-well plates at 10,000 cells/well, and each group was inoculated into 6 wells. After cell adherence, different concentrations of antitumor drugs were added to the experimental group and equal volumes of solvent of each drug group were added to the vehicle control group. After 72 h of incubation, the complete medium was replaced with 100 μL of serum-free medium containing 10% CCK-8 reagent. The optical density was measured at 450 nm after 2 h.7 cells/mL) were collected, washed, resuspended in 0.1 mL of complete RPMI-1640 medium, and injected subcutaneously into the middle and posterior left axilla of three 6-week-old female BALB/C nude mice. Tumor diameters and body weight were measured every week after cell injection and tumor volume was calculated as ½ × length × (width2) [The cells were prepared at passage 25 to determine their tumorigenicity in BALB/C nude mice. The cultured cells (1 × 10(width2) . Tumor-bCells at passage 36 were digested and grown on sterile glass slides. At 48 h, slides were washed with PBS, fixed with 4% paraformaldehyde for 15 min, air-dried, and treated with 0.5% Triton X-100 for 20 min. For primary tumor tissues, transplanted tumors, and organoids, the specimens were immersed in a formaldehyde solution for fixation, dehydration, embedding, and sectioning. The slides were then covered with the following antibodies: mouse mAb against human cytokeratin (CK)7 , mouse mAb against human CK19 , rabbit mAb against Ki-67 , mouse mAb against human p53 clonal antibodies , mouse mAb against human E-cadherin , and mouse mAb against human vimentin . The slides were incubated with the antibodies for 60 min and washed thoroughly with PBS. Biotinylated rabbit anti-mouse IgG  was then added for 15–20 min and the slides were washed. Then, a 3,3′-diaminobenzidine solution was added to the slides for 1–5 min at room temperature. Finally, slides were rinsed with distilled water, stained with hematoxylin and eosin (H&E), and examined under a light microscope.The mRNA profiles of ICC-X1 cells were compared to that of RBE cells to better understand their molecular features and altered pathways. First, the purity, concentration, and integrity of the extracted RNA samples were determined to ensure that qualified samples are used for transcriptome sequencing. The library was constructed using qualified samples by (1) enriching eukaryotic mRNA with magnetic beads with oligo(dT); (2) adding fragmentation buffer to randomly interrupt mRNA; (3) using mRNA as a template and synthesizing, the first cDNA chain with random hexamers and the second cDNA chain by adding buffer, dNTPs, RNase H, and DNA polymerase I, and purifying the cDNA using AMPure XP beads; (4) subjecting the purified double-stranded cDNA to end repair, adding a tail, and connecting a sequencing adapter, and then using AMPure XP beads to select fragment sizes; and (5) obtaining a cDNA library by PCR enrichment. After the library was constructed, quantitative PCR was used to accurately quantify the effective concentration of the library (library effective concentration > 2 nM) to ensure the quality of the library. After passing the library check, sequencing was performed on the Illumina platform . This project used the reference genome: Homo_sapiens.GRCh38_release95.genome.fa as a reference for sequence alignment and subsequent analysis. The reads on the StringTie alignment were used for assembly and quantification after the alignment analysis was completed.The statistical significance between the experimental and control group was determined using the Student’s t-test. The statistical significance was set at p ≤ 0.05.We successfully established a tumor cell line from a 48-year-old Chinese male patient with ICC in situ and named it ICC-X1. The cell line has now been passed down to passage 104. The clinical and pathological features of the tumor tissues are shown in Fig. The genetic characteristics of the ICC-X1 cells were analyzed using DNA fingerprinting. This analysis included 21 STR loci validated at different chromosomal locations. The results showed that the cultured cell line was 91.4% similar to the original tumor tissue at the 21 loci tested and did not correspond to any cells in the public cell bank  for gene enrichment analysis because of the large number of differentially expressed genes. We found that the upregulated cancer-related genes were mainly enriched in several signaling pathways including the TNF signaling pathway, NOD-like receptor signaling pathway, and NF-κB signaling pathway. Among these, the TNF signaling pathway and NF-κB signaling pathway exist in many important cellular processes, such as inflammatory response, proliferation and metastasis of tumor cells, and occurrence and development of various tumors. Figure Transcriptomic analysis revealed 2824 upregulated genes in the ICC-X1 cells compared with RBE cells. We used the DAVID tool v6.8 (Similarly, the transcriptomic analysis revealed 2770 downregulated genes in the ICC-X1 cells. We found that the downregulated cancer-related genes were mainly enriched in the Rap1 signaling pathway and Hippo signaling pathway among others. The primary function of the Rap1 and Hippo pathways is to restrict tissue growth and regulate cell proliferation, differentiation, and migration. When these pathways are dysregulated, abnormal cell growth and tumor formation occur. Figure The incidence of ICC in China has been increasing in recent years . The majIn this study, a new ICC cell line, ICC-X1, was successfully established from a primary culture and subculture of the primary tumor tissue derived from a Chinese patient. The cell line was genetically verified using STR analysis and was found to be similar to the primary tumor tissue. It was not cross-contaminated with other tumor cell lines in public cell banks. The cell line was further characterized through doubling time determination, chromosome analysis, drug sensitivity testing, nude mouse transplantation tumor formation experiments, and cell stemness detection.We found that the primary culture of tumor tissues having > 40% nuclear protein Ki-67 has a higher success rate. This aspect is related to the high malignancy of the tumor itself, and the rapid proliferation of tumor cells so that tumor cells are not easily competed by fibroblasts. This result was consistent with previous findings , 25.Organoids are a promising disease model that can be used to better understand biology and to test the efficacy of drugs in vitro. Few studies on ICC organoids have been reported and the culture success rate of these organoids is lower than that of gastrointestinal tumor organoids . In thisNude mice are the experimental animals of choice to study tumor molecular mechanisms, the development of antitumor drugs, and drug sensitivity. The tumor formation rates vary greatly after the cell lines are inoculated into nude mice. The tumor formation rates of breast cancer and colon cancer cell lines are higher, whereas the transplantation rates of hepatobiliary tumor cell lines are lower. The commonly used ICC cell lines, TKKK, RBE, and HCCC-9810, are unable to form xenograft tumors after inoculation in nude mice, greatly limiting in vivo tumor research . The ICCIntrahepatic cholangiocarcinoma can be divided into proliferative and inflammatory types based on differences in gene expression profiles and signaling pathways . The proTNF-α is a key cytokine involved in inflammation, immunity, cell homeostasis, and tumor progression. The role of TNF-α in cancer development is a double-edged sword. TNF-n is a cytokine that can kill tumor cells. In contrast, TNF-T can also mediate the proliferation, invasion, and metastasis of many cancer cells to promote and develop tumors . In addiBcl-2 proteins inhibit the apoptotic process by inhibiting the release of cytochrome C from mitochondria and preventing the oxidative destruction of cells. Their overexpression and phosphorylation are related to the regulation of cell proliferation, tumor formation, and multidrug resistance. It plays an extremely important role and is an effective tumor therapy target –41. TranRNA seq data revealed that many genes highly expressed by RBE relative to ICC-X1 are related to cell proliferation and cell cycle, whereas the ICC-X1 cell line does not express these genes that promote cancer cell proliferation. For example, overexpression of TM4SF4 significantly promotes the growth and colony formation of HCC cells . ICC-X1 We have established and characterized ICC-X1, a new ICC cell line derived from the primary tumor of a Chinese patient. This cell line can be used as a resource to study the pathogenesis, invasion, and metastasis mechanisms of ICC. Further, this cell line could be a potential preclinical model for drug discovery and development to diagnose and treat patients with ICC.Additional file 1. DNA fingerprinting of ICC-X1 cells.Additional file 2. The dose-response curve of ICC-X1.


The study aimed to investigate the inhibitory effects of AL on the ERK signaling molecules  and the growth and proliferation of CCA cells. The viability of the three CCA cell lines CL-6, HuCCT1, and HuH28 was determined using MTT assay. The effect of Ras/ERK inhibitors on protein expression in the presence of AL extract was investigated. The protein extracted from each CCA cell following exposure to AL and/or Ras/ERK inhibitors were separated on 12.5% SDS-PAGE. The analysis of mRNA expression following 48 and 72 hours of AL exposure in comparison with 0 hours (non-exposed cells) was performed by using RT-PCR. 50 (concentration that inhibits cell growth by 50%) of AL for the CL-6, HuCCT-1 and HuH28 cell lines were 29.77±6.64, 35.45±4.96, and 35.32±6.69 µg/mL (mean+SD), respectively. The cells were exposed to AL extract at the IC50 for 0, 12, 24, 48, and 72 hours in the absence and presence of Ras/ERK inhibitors . Protein expression was determined by Western blot analysis. The results suggested the lack of significant inhibitory effect of AL on ERK at 48 and 72 hours of exposure in all CCA cell types. On the other hand, a significant inhibitory effect was observed with p-ERK expression in all CCA cell types. Cyclin D was significantly down-regulated at 72 hours of exposure in all cell types with different potencies. The expression of eIF4B was markedly inhibited in HuCCT-1 but slightly inhibited in CL-6 and HuH28 cells. Real-time PCR analysis revealed significant down-regulation of ERK following 72 hours of AL exposure in the HuCCT1 and HuH28, but not CL-6 cell. The potency of cytotoxic activity of AL (by MTT assay) was about three times higher than the standard drug 5-fluorouracil. The ICThe ERK signaling cascade and downstream molecules are potential targets of action of AL in CCA. Opisthorchis viverrini, Opisthorchis felineus, and Clonorchis sinensis) are endemic . Long-term parasitic infection, coupled with the consumption of the precarcinogen dimethyl-nitrosamine (DMN) in the fermented fish and meats, is the major risk factor of CCA in this area . Other risk factors include primary sclerosing cholangitis (PSC), hepatolithiasis, drug usage, and genetic factor . The prognosis of CCA patients is generally poor due to the rapid and aggressive cell proliferation. The most effective treatment is surgery, but the prognosis is markedly variable . The average survival time ranges from 6 to 12 months . Effective control of CCA has been limited by the lack of specific and sensitive diagnostic tools for early detection of CCA as well as effective chemotherapeutic drugs . Cholangiocarcinoma (CCA), the biliary ductal cancer, is recognized as a highly progressive cancer that originates from the transformed intra- and extrahepatic cholangiocytes or sometimes metastasis from other adjacent cancers . High prevalence rates have been reported from several countries, especially in the Mekong subregion, where liver fluke infections (Atractyloides lancea (Thunb.) DC. (AL), a tuber plant belonging to the family Asteraceae (Compositae), distributes in several Asian countries, such as China, Japan, and Korea. The dried rhizome has long been used for the treatment of several illnesses and health conditions such as rheumatic fever, digestive diseases, night-blindness syndrome, fever, and cold . The plant rhizome extract has been shown in the in vitro and in vivo models to exert anticancer, anti-inflammatory, antibacterial, and antifungal activities. Besides, it also possesses antipyretic, analgesic, and immunomodulatory activities . The anti-CCA potential of AL and its major constituents (e.g. β-eudesmol and atractylodin) have recently been reported in a series of non-clinical studies . The antiproliferative activity, inhibitory activities on cell migration and cell invasion, inducing effect on cell cycle arrest and apoptosis are well demonstrated . Nevertheless, the intracellular signaling cascades of AL action remain to be elucidated. Excessive cell proliferation is one of the most critical properties of cancerous cells. It is triggered by several stimuli, leading to the alteration of several downstream signaling molecules to promote cell proliferation. The most investigated cell proliferation cascade is Ras/Raf/ERK  signaling pathway . This cascade is stimulated by extracellular ligands such as mitogens, serum, growth factors, cytokines, stress, GPCRs (G-protein coupled receptors) ligands, and calcium ions . Mutations of the proto-oncogenes and tumor suppressor genes or inappropriate synthesis of ligands and receptors can hyperactivate many signaling pathways, leading to activation of the cell cycle machinery. Activated-ERK phosphorylates numerous downstream molecules, either enhancing cell proliferation, e.g., Myc, RSK , MNK, and Fos-Jun), or inhibiting tumor-suppressive molecules such as CKIs (cyclin-dependent kinase inhibitors). The phosphorylated Myc stimulates cell proliferation by activating numerous genes, including those encoding CyclinD/E, CDK4/6 (cyclin-dependent kinase4/6), and E2F-family transcription factors . Multiple kinases in the mitogen-, stress-activated kinase (MSK), and ribosomal S6 kinase (RSK) are also phosphorylated by ERK, and these kinases, in turn, phosphorylate the transcription factors that regulate cell cycle progression . MSKs play a crucial role in regulating translation following mitogenic stimulation by phosphorylating the translation initiation factor eIF4E . RSK regulates gene translation by phosphorylating eIF4B, which increases its interaction with the translation initiation factor eIF3. Moreover, eIF4B is also activated directly by the phosphorylated ERK. Activation of the translation process by these mechanisms is essential for cell growth and proliferation . In CCA, ERK signaling cascade has been documented as important cancer surviving pathway . In this study, the effects of AL rhizome extract on cell growth and proliferation associated with ERK signaling pathway were investigated in the three CCA cell lines, i.e., CL-6, HuCCT1, and HuHT28 cells. The effects of ERK/Ras inhibitors on modulating AL action was also examined.Preparation of ethanolic AL rhizome crude extract® No. 1 filter paper , evaporated in the rotary evaporator , and stored at -20ºC until use.The crude ethanolic extract of AL was prepared as previously described . Briefly, the rhizomes were washed with tap water, cut into small pieces, and dried in the hot air oven (50 ºC) for 2-3 days. The dried AL rhizomes were powdered using an electric grinder, and 100 g of the powder was transferred to a stoppered flask containing 95% ethanol and incubated at room temperature (25-30 ºC) for seven days. The extract was filtered through WhatmanCell culture 2. The cells were cultured until reaching approximately 80% confluence and detached from primary culture flasks by treating with 0.25% trypsin-EDTA (Gibco™) for 1-2 minutes. The cells were stained with 0.4% trypan blue , and cell number was counted using a hemocytometer.The CCA cell lines used in the study were CL-6, HuCCT1, and HuH28. CL-6 cell was obtained directly from Thai patients and was established and kindly provided by Associate Professor Adisak Wongkajornsilp, Department of Pharmacology, Faculty of Medicine Sirisaj Hospital, Mahidol University, Thailand. HuCCT1 cell was obtained from the Japanese Collection of Research Bioresources Cell Bank . HuH28 cell was obtained from the JCRB cell bank. All were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% Antibiotic-Antimycotic  at 37ºC under 5% COConcentration-response analysis4 cells/well) and incubated at 37ºC under 5% CO2 for 24 hours. Cell quality was examined under an inverted light microscope. 5-Fluorouracil (5-FU) was used as a reference compound . The extract and 5-FU were added to each well, and the plate was further incubated for 48 hours. Cell viability was determined by adding 10 µL of MTT substrate and incubated at 37ºC under 5% CO2 for 4 hours. The cells were dissolved in DMSO, and the absorbance was measured at the wavelength of 570 nm. The experiment was performed in three independent assays, triplicate each. The IC50 (concentration that inhibits cell growth by 50%) was calculated using the concentration-response analysis software CalcuSynTM . The viability of the three CCA cell lines following exposure to various concentrations of AL extract  was determined using MTT assay . In brief, the cells were seeded onto a 96-well microtiter plate (1×10Effect of incubation time on CCA cell growth in the presence of AL exposure4 cells/well) and incubated at 37ºC under 5% CO2 for 24 hours. AL extract at the IC50 concentration was added to each well, and cell viability at 0, 12, 24, 48, and 72 hours was measured by MTT assay as described above. Non-exposed cells served as control of normal cell growth. The experiment was performed in three independent assays, triplicate each. CL-6, HuCCT1, and HuH28 were plated onto 96-well microtiter plates . Total proteins were extracted using RIPA buffer  containing protease inhibitors , and the concentrations were measured by BCA assay . The AL-exposed and non-exposed cells  were cultured in 25 cm6 cells) was seeded onto a 25 cm2 culture flask and incubated at 37 ºC under 5% CO2 for 24 hours. The cells were exposed to AL in the presence and absence of the ERK inhibitor (XMD8-92), Ras inhibitor  as follows: control cells ; AL (2 x IC50) alone; XMD 8-92 (0.5 µM) alone; salirasib (0.5 µM) alone; AL (2 x IC50) + XMD 8-92 (0.5 µM); and AL (2 x IC50) + salirasib (0.5 µM). At the end of 72 hours of exposure , proteins were extracted using RIPA buffer and concentrations measured by BCA assay. The experiments were performed in three independent assays, triplicate each.The effect of Ras/ERK inhibitors on protein expression in the presence of AL extract was further investigated in the three CCA cell lines. Each cell line . The membranes were stained with 0.1% Ponceau S dye and washed three times with TBS (pH 7.5) to remove the remaining dye. Non-specific binding was blocked using 4% BSA  in TBS (pH 7.5) for one hour at room temperature with agitation. Primary antibodies used in the experiments were 1:1,000 anti-ERK1/2 , 1:1,000 anti-phospho-ERK1/2 , 1:2,000 anti-cyclin D1 [EPR2241] - C-terminal , 1:2,000 anti-eIF4B (Abcam), and 1:1,000 anti-β-actin . Each antibody was diluted in TBS (pH 7.5) containing 1% BSA and added to the membranes and incubated overnight at 4 ºC with gentle shaking. The membranes were washed with TBS (pH 7.5) containing 0.05% Tween™-20 three times. Secondary antibodies, i.e., 1:30,000 goat anti-rabbit IgG H&L AP-conjugated (Abcam) and 1: 30,000 goat anti-mouse IgG H&L AP-conjugated (Abcam) were added onto the membranes and incubated at room temperature for one hour with gentle shaking. The membranes were washed and immersed in detection buffer (pH 9.5) for 5 min. BCIP/NBT substrate  was added, and the membranes were incubated at room temperature in the dark without shaking until the positive signal was observed. Densitometric analysis of the positive bands was performed using ImageJ software (imagej.nih.gov).The protein (10 µg) extracted from each CCA cell following exposure to AL and/or Ras/ERK inhibitors were separated on 12.5% SDS-PAGE . Separated protein bands were transferred to the nitrocellulose membrane  using a semi-dry transfer  were seeded onto a 6-well culture plate and incubated at 37 ºC under 5% CO2 for 24 hours. The cells were exposed to AL extract in 50% ethanol at the IC50 concentration for 48 and 72 hours. At the end of each incubation time, culture medium was removed, and the cells were washed three times with cold PBS (pH 7.4). TRIzolTM reagent  was added, and total RNA content was isolated according to the manufacturer’s protocol. The RNA was converted to complementary DNA (cDNA) using a cDNA synthesis kit  with Oligo (dT) 18 primer. The cDNA concentration was quantified using NanoDropTM 2000 spectrophotometers . An aliquot of 100 ng cDNA was used as a template for RT-PCR analysis PCR using iTaq™ Universal SYBR® Green Supermix (Bio-rad Laboratories Inc.) in a CFX96™ Real-Time PCR machine . The mRNA expression of ERK1/2 was determined and normalized with GAPDH  for semi-quantification of the PCR products. The primers used were ERK1/2 (F) 5´-TCAAGCCTTCCAACCTC-3´, ERK1/2 (R) 5´- GCAGCCCACAGACCAAA-3´, GAPDH (F) 5´-AGCCACATCGCTCAGACA-3´, and GAPDH (R) 5´- TGGACTCCACGACGTACT-3´. The PCR condition consisted of denaturation at 95°C for 1 min and amplification for 40 cycles at the following conditions: 95°C for 30 sec, 50°C for 30 sec, and 72°C for 2 min, and followed by final heating at 72°C for 3 min. The fold change of the gene expression level was determined using the 2∆∆CT method . The experiments were performed in three independent assays, triplicate each. The analysis of mRNA expression following 48 and 72 hours of AL exposure in comparison with 0 hours (non-exposed cells) was performed using RT-PCR. Each CCA cell . Data are expressed as mean±SD values. Comparison of more than two quantitative data sets was performed using ANOVA. Comparison of the two quantitative data sets was performed using an independent t-test or paired t-test where appropriate. The statistical significance was set at α = 0.05. Antiproliferative activity of AL extract against CCA cell lines 50 (concentration that inhibits cell growth by 50%) values (mean ± SD) of AL extract for the CL-6, HuCCT1, and HuH28 were 29.77± 6.64, 35.45±4.96, and 35.32±6.69 µg/mL, respectively. The corresponding IC50 values of the reference drug (5-FU) were 94.05±10.74, 93.26±6.35, and 104.01±11.42 µg/mL, respectively. The ICInhibitory effect of AL extract on CCA cells growth The growth of CL-6 and HuCCT1 cells showed similar patterns during the 72 hours, characterized by the rapid growth phase during the first 48 hours, followed thereafter, by the relatively slow growth phase. In the presence of AL extract, the slow growth phase was observed during the first 24 hours but significantly inhibited after 24 hours. The growth of both CCA cells was markedly inhibited (60-90%) at 72 hours of exposure . The HuHInhibitory effect of AL extract on the expression of downstream ERK-stimulated proteins The inhibitory effects of AL on the level of p-ERK1/2, cyclin D1, and eIF4B proteins are shown in Inhibitory effect of AL extract on the expression of downstream ERK-stimulated proteins in the presence of Ras/ERK inhibitors The inhibitory effects of AL extract, XMD8-92 (ERK inhibitor), and salirasib (Ras inhibitor) on p-ERK1/2 level in CL-6, HuCCT1, and HuH28 cells are shown in The expression of cyclin D1 was significantly down-regulated after exposing all CCA cell lines to salirasib. On the other hand, its expression was down-regulated after exposing HuH28 and CL-6 cell lines to AL or XMD8-92. For eIF4B, the expression was down-regulated in CL-6, and HuCCT1 cells following exposure to AL alone, XMD8-92 alone, salirasib alone, and in particular AL in combination with XMD8-92 or salirasib. In HuH28 cells, the expression was significantly down-regulated only when exposing to AL extract alone, and AL extract in combination with XMD8-92 or salirasib.Effect of AL extract on EKR mRNA expression Results of the semi-quantitative RT-PCR analysis showed that the mRNA expression of ERK normalized with GAPDH in the AL-exposed and non-exposed CL-6 cells were comparable at 48- and 72-hours exposing periods. The expression in HuCCT1 and HuH28 cells were significantly down-regulated after 72 hours of exposure .50 values for the CL-6, HuCCT1, and HuH28 were in agreement with that previously reported in CCA from both ethanolic crude extract and bioactive constituents . In all CCA cell lines, significant cell growth inhibition was observed at 48 and 72 hours of exposure. The time-dependent activity of AL extract suggested its action on cell growth early before (0-24 hours) as well as after (24-72 hours) cell division. This may suggest that the dose regimens of the AL extract should be adequately prolonged to eliminate all CCA cells. The previous study in animal models demonstrated a significant reduction of CCA tumor mass following chronic dosing . The results of the present study confirmed the potent inhibitory activity of AL extract (about 3-fold of 5-FU) on the proliferation of different types of CCA cell lines of the intrahepatic and extrahepatic origins. The observed ICRas/Raf/ERK pathway is the primary pathway involved in cell proliferation control. ERK is one of the most critical molecules responsible for several cell behaviors, especially cell growth and cell proliferation . In cancer cells, excessive ERK activation leads to uncontrolled cell division and abnormal cell proliferation . Our results suggested that the antiproliferative activity of the AL extract was through ERK cascade. The levels of p-ERK in all the three CCA cell lines were significantly reduced after 24-48 hours of exposure to AL. This is the period of highest cell proliferation . p-ERK normally activates several downstream molecules involved in cell growth, particularly transcription and initiation factors including cyclin D1 and eIF4B. Cyclin D1 regulates cell division in the G1/S phase transition, which can be stimulated by several molecules, including Myc that is upwardly activated by p-ERK . Low level of p-ERK results in the reduction of Myc and finally, the cyclin D1 level. AL was shown to reduce p-ERK and cyclin D1 production in all of the three CCA cell lines. This resulted in the termination of cell division which coincided with its inhibitory effect on CCA cell proliferation. eIF4B is another initiation factor that can be directly stimulated by ERK or indirectly activated through the ERK-RSK pathway. It is incorporated with eIF4A and binds to the eIF3 complex, leading to the recruitment of the small ribosomal subunit (40s) to the mRNA, which in turn, results in protein synthesis necessary for cell growth . AL significantly reduced the level of eIF4B in HuCCT1, but not in other cell lines where a trend of decreasing expression was observed during the 12-72 hours of exposure. Altogether, the findings suggested that the action of AL on the signaling molecules in the ERK cascade may be different in various cell types depending on CCA cell origins. The three CCA cell lines used in the experiments exhibit different pathological, biochemical, and genetic characteristics. The CL-6 cell line is a highly proliferative intrahepatic CCA cell line, whereas the HuCCT1 cell line is a moderately differentiated intrahepatic CCA cell. The HuH28 is a slow growth CCA cell that is originated from the gallbladder. The present study is the first study that investigated the modulatory effects of ERK/Ras inhibitors, XMD8-92, and salirasib, on protein and mRNA expression of p-ERK, cyclin D1, and eIF4B in CCA cell lines. Salirasib, the Ras inhibitor, has been used to investigate the potential molecular targets of various compounds in cancer, especially those with KRAS mutation . XMD8-92 effectively inhibits ERK undefinably pathway . In this study, salirasib or XMD8-92 alone was shown to inhibit p-ERK and cyclin D1, but not eIF4B. The combination of either inhibitor with AL, however, produced complete suppression of the expression of p-ERK and eIF4B proteins. The significant down-regulation of cyclin D1 level in all three CCA cell lines exposing to salirasib alone suggested that salirasib could affect cyclin D1 expression in CCA cells as reported in other cancer cells . In contrast, the cyclin D1 levels in the cells exposed to AL in combination with salirasib were comparable with that of the untreated cells. This result suggested the antagonistic effect between AL and salirasib. The underlying mechanism needs further investigation. The inhibitory effect of AL extract on the expression of ERK was confirmed by the results of mRNA expression using semi-quantitative RT-PCR. The effect was prominent at 72 h of exposure in all cell lines, but statistical significance was observed only in HuCCT1 and HuH28 cell lines. It was noted however that ERK mRNA expression in CL-6 cell was decreased at 72 h when compared with non-treated control cells. In conclusion, the results of the study confirm that AL exerts significant inhibition of the proliferation of all CCA cell types through intracellular ERK cascade, which is potentiated by ERR/Ras inhibitors. A thorough understanding of the molecular mechanism of AL, especially cell growth and proliferation, is essential for the further development of this plant for CCA control.PM and KN were involved in the design of the experimental study. PM, LP, PC and PM performed the experiments and data analysis. PM drafted the manuscript. KN revised the manuscript. All authors reviewed and approved the final manuscript for submission. All meet the ICMJE criteria for authorship.All data used to support the findings of this study are available from the corresponding author upon request.The authors declare that there is no conflict of interest regarding the publication of this paper.


N6-methyladenosine (m6A) is a highly prevalent mRNA modification that promotes degradation of transcripts encoding proteins that have roles in cell development, differentiation, and other pathways. METTL3 is the major methyltransferase that catalyzes the formation of m6A in mRNA. As 30% to 80% of m6A can remain in mRNA after METTL3 depletion by CRISPR/Cas9-based methods, other enzymes are thought to catalyze a sizable fraction of m6A. Here, we reexamined the source of m6A in the mRNA transcriptome. We characterized mouse embryonic stem cell lines that continue to have m6A in their mRNA after Mettl3 knockout. We show that these cells express alternatively spliced Mettl3 transcript isoforms that bypass the CRISPR/Cas9 mutations and produce functionally active methyltransferases. We similarly show that other reported METTL3 knockout cell lines express altered METTL3 proteins. We find that gene dependency datasets show that most cell lines fail to proliferate after METTL3 deletion, suggesting that reported METTL3 knockout cell lines express altered METTL3 proteins rather than have full knockout. Finally, we reassessed METTL3’s role in synthesizing m6A using an exon 4 deletion of Mettl3 and found that METTL3 is responsible for >95% of m6A in mRNA. Overall, these studies suggest that METTL3 is responsible for the vast majority of m6A in the transcriptome, and that remaining m6A in putative METTL3 knockout cell lines is due to the expression of altered but functional METTL3 isoforms. The modification m6A remains in mRNA after METTL3 depletion, suggesting that other m6A methyltransferases exist. This study investigates METTL3 knockouts, finding that they often escape knockout by expressing functional METTL3 hypomorphs, and demonstrating that METTL3 is indeed responsible for most m6A in mRNA. N6-methyladenosine (m6A) is the most abundant internal mRNA modification ATP (Perkin Elmer BLU002Z250UC) for 30 min at 37°C; γ-phosphate was then removed from excess [γ-32P]ATP with 0.5 U apyrase (New England Biolabs M0398) in 1× apyrase buffer for 10 min at 30°C. RNA was then digested to single nucleotides using 2 U Nuclease P1  for 1 h at 60°C.meviously . m6A was4OH in the first dimension, then in 70:15:15 (v/v/v) isopropanol:water:HCl in the second dimension. Radioactively labeled nucleotides were detected using a phosphor storage screen and Amersham Biosciences Typhoon 9400 Variable Mode Imager. m6A levels were quantified using Image Lab software (BioRad).2 μl of the digested RNA is spotted on PEI-cellulose TLC plates (Millipore Sigma #105579) 0.5 μl at a time. Plates were developed in 5:3 (v/v) isobutyric acid:0.5 M NHMETTL3 and plotted them to identify cell lines that were dependent on METTL3. Gene dependency probability scores greater than 0.5 indicate that a cell line is dependent on the gene. To account for off-target effects, we extracted the gene dependency scores of 1,054 cell lines on METTL3, METTL14, and WTAP. We identified cell lines that were dependent on all of these genes (gene dependency probability scores >0.5).Gene dependency probability scores were obtained from the DepMap Public 21Q4 CRISPR gene dependency dataset –47. We e6A, we extracted the gene dependency scores of 1,054 cell lines on METTL3, METTL14, and WTAP. We identified cell lines that were independent of each of these genes (gene dependency probability scores <0.5). We next identified cell lines that were independent of all 3 genes to obtain a subset of cell lines that may be independent of m6A. The full list of predicted m6A-independent cell lines can be found in To identify cell lines that may be independent of mMettl3flox/flox mice were generated based on construction from the Knockout Mouse Project Repository (KOMP) and were described by Cheng and colleagues [loxP sites were inserted spanning the fourth exon of Mettl3.lleagues . loxP siMettl3 KO MEFs, embryos from Mettl3flox/flox mice were collected on day 13.5, mechanically separated, trypsinized, and plated. After 3 passages, cells were transduced with SV40 large T-antigen and passaged until they reached senescence. Cells that escaped senescence and became immortalized were then infected with Cre-ERT2 lentivirus. Successfully infected cells were selected with puromycin. Single-cell colonies were then isolated and expanded.To generate tamoxifen-inducible Mettl3 knockout, MEFs were plated at 100,000 cells/well in a 12-well plate. The next day, when the MEFs were at 50% confluency, cells were treated with 4-hydroxytamoxifen (500 nM) or ethanol (negative control) for 48 h. Cells continued to be grown and passaged normally after 4-hydroxytamoxifen treatment. Cell lysate was collected 5 days after tamoxifen treatment for western blot. RNA was collected 8 days after tamoxifen treatment for m6A measurements.To induce the Mettl3 KO, tamoxifen-inducible Mettl3 KO MEFs were plated at 5,000 cells/well in a 12-well plate. The next day, cells were treated with 4-hydroxytamoxifen (500 nM) or ethanol (negative control). Treatment continued for the duration of the experiment, and then, 0, 2, 4, 6, and 8 days after 4-hydroxytamoxifen treatment, cells were washed with PBS and then incubated in 1:1 (v/v) high-glucose DMEM without phenol red (Gibco #21063029) and MTT -2,5-diphenyltetrazolium bromide) reagent (5 mg/ml MTT (Abcam ab146345) in PBS) for 3 h at 37°C. The produced formazan crystals were dissolved in 1.5× volume MTT solvent  and incubated for 15 min at room temperature while shaking on an orbital shaker. Samples without cells were included as background controls. Absorbance was read at 570 nM to estimate the number of cells.To measure the changes in cell proliferation upon S1 FigA) Exon2 Mettl3 KO mESCs express a novel protein that is immunoreactive to several anti-METTL3 antibodies. We found that exon2 Mettl3 KO mESCs express new proteins that are reactive to an anti-METTL3 pAb. To confirm that these new proteins may be shortened versions of METTL3, we performed a second WB with a different anti-METTL3 mAb. Similar to the first WB, we found that full-length METTL3  was lost in both KO cell lines. In the KO cell lines, the same bands that were immunoreactive to the polyclonal anti-METTL3 antibody were also immunoreactive to the anti-METTL3 mAb at approximately 50 kDa in Mettl3 KO mESC-a and approximately 55 kDa in Mettl3 KO mESC-b, respectively (arrowheads). This further validates the possibility that the Mettl3 KO mESCs express smaller versions of METTL3 proteins. 30 μg per lane. (B) Exon4 Mettl3 KO mESCs do not express METTL3 protein. An independently run replicate of the WB from Mettl3 KO mESCs do not express any proteins immunoreactive to anti-METTL3 antibodies. 30 μg per lane. (C) Exon4 Mettl3 KO mESCs do not express METTL3 protein. To confirm that exon4 Mettl3 KO mESCs do not express METTL3, we performed a second WB with a different anti-METTL3 mAb. We confirmed that exon4 Mettl3 KO mESCs indeed do not express any detectable METTL3 protein. 30 μg per lane. mAb, monoclonal antibody; mESC, mouse embryonic stem cell; pAb, polyclonal antibody; WB, western blot; WT, wild-type.((TIFF)Click here for additional data file.S2 Fig6A rescue after transfection of exon4 Mettl3 KO mESCs with WT METTL3 Click here for additional data file.S3 FigMettl3 mRNAs expressed in the exon2 Mettl3 KO mESCs are translated, we performed polysome profiling. The polysomes were separated into 4 fractions based on their ribosome load: Fraction 1 , Fraction 2 (low polysomes), Fraction 3 (medium polysomes), and Fraction 4 (high polysomes). RT-PCR of Mettl3 using primers against the 5′ and 3′ end of Mettl3 revealed Mettl3 mRNAs (arrowheads) in the medium and high polysome fractions in the WT mESCs, as well as the exon2 Mettl3 KO mESCs. Sequencing of the PCR products revealed Mettl3 mRNAs depicted in blue. They include mRNA-3 that encodes METTL3-a.ii in exon2 Mettl3 KO mESC-a, and mRNA-7 that encodes METTL3-b.ii in exon 2 Mettl3 KO mESC-b Click here for additional data file.S4 FigMettl3 KO mESCs with the METTL3-specific inhibitor STM2457 does not reduce m6A levels. Treatment of the parental WT mESC cell line with 30 μM of STM2457 lead to 65.4% reduction in m6A levels. On the other hand, STM2457 treatment did not lead to a significant change in m6A levels in exon4 Mettl3 KO mESCs, indicating that the small amount of remaining m6A was not produced by METTL3. Error bars indicate standard error (n = 3). * = p-value < 0.5, ** = p-value < 0.01, *** = p-value < 0.005, n.s. = not significant. Underlying data can be found in 6A, N6-methyladenosine; m7GMP, 7-methylguanosine monophosphate; mESC, mouse embryonic stem cell; WT, wild-type.Treatment of exon4 (TIFF)Click here for additional data file.S5 FigA) METTL3 KO A549 cells have persistent m6A. METTL3 KO A549 cells were previously reported [6A remains in these cells, we measured the levels of m6A using 2D-TLC that measures m6A specifically in the GA context [6A to only m6A in mRNAs, where they are found in a DRACH context [6A level in the METTL3 KO A549 cells was very similar to m6A levels in WT A549 cells. This suggests that either the knockout of METTL3 was incomplete or that A549 cells may express a non-METTL3 m6A methyltransferase that is responsible for the majority of m6A in mRNAs (n = 1). Underlying data can be found in 6A, N6-methyladenosine; WT, wild-type.Click here for additional data file.S6 Fig6A methyltransferase complex for proliferation. Using the DepMap CRISPR loss-of-function screening dataset, we found that 801 of 1,054 tested cell lines require METTL3 for proliferation Click here for additional data file.S7 FigA) Mettl3 conditional knockout MEFs do not express METTL3 protein. To confirm that Mettl3 is the sole m6A writer in cell lines other than mESCs, we produced a tamoxifen-inducible Mettl3 conditional knockout MEF cell line. We used a WB to confirm the loss of METTL3 using a second anti-METTL3 mAb. Five days after 4OHT treatment (500 nM), we observe loss of the WT METTL3 protein. 30 μg per lane. (B) Mettl3 knockout in MEFs leads to a decrease in cellular proliferation. Using an MTT assay, we measured cell proliferation after 4OHT-induced METTL3 knockout over 8 days. Proliferation of Mettl3 KO MEFs began to slow down compared to WT MEFs after 6 days of 4OHT treatment. Error bars indicate standard error (n = 3). * = p-value < 0.5, ** = p-value < 0.01, *** = p-value < 0.005, n.s. = not significant. Underlying data can be found in 6A, N6-methyladenosine; MEF, mouse embryonic fibroblast; mESC, mouse embryonic stem cell; WB, western blot; 4OHT, 4-hydroxytamoxifen.((TIFF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(CSV)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S1 Raw images(PDF)Click here for additional data file.