V12, provides Drosophila cultured cells with a proliferation and survival advantage that simplifies the generation of continuous cell lines. Here, we used lineage-restricted RasV12 expression to generate continuous cell lines of muscle, glial, and epithelial cell type. Additionally, cell lines with neuronal and hemocyte characteristics were isolated by cloning from cell cultures established with broad RasV12 expression. Differentiation with the hormone ecdysone caused maturation of cells from mesoderm lines into active muscle tissue and enhanced dendritic features in neuronal-like lines. Transcriptome analysis showed expression of key cell-type-specific genes and the expected alignment with single-cell sequencing and in situ data. Overall, the technique has produced in vitro cell models with characteristics of glia, epithelium, muscle, nerve, and hemocyte. The cells and associated data are available from the Drosophila Genomic Resource Center.Expression of activated Ras, Ras Fruit flies are widely used in the life and biomedical sciences as models of animal biology. They are small in size and easy to care for in a laboratory, making them ideal for studying how the body works. There are, however, some experiments that are difficult to perform on whole flies and it would be advantageous to use populations of fruit fly cells grown in the laboratory – known as cell cultures – instead.Ras in fruit fly cells helps the cells to survive and grow in cultures, making it simpler to generate new fruit fly cell lines compared with traditional methods. However, the methods used to express activated Ras result in cell lines that can be a mixture of many different types of cell, which limits how useful they are for research.Unlike studies in humans and other mammals, which – for ethical and practical reasons –heavily rely on cell cultures, few studies have used fruit fly cell cultures. Recent work has shown that having an always active version of a gene called Ras to generate a collection of cell lines from specific types of fruit fly cells in the muscle, nervous system, blood and other parts of the body. The experiments show that selectively expressing activated Ras in an individual type of cell enables them to outcompete other cells in culture to generate a cell line consisting only of the cell type of interest.Here, Coleman-Gosser, Hu, Raghuvanshi, Stitzinger et al. aimed to use The new cell lines offer models for experiments that more closely reflect their counterparts in flies. For example, the team were able to recapitulate how fly muscles develop by treating one of the cell lines with a hormone called ecdysone, which triggered the cells to mature into active muscle cells that spontaneously contract and relax.In the future, the new cell lines could be used for various experiments including high throughput genetic screening or testing the effects of new drugs and other compounds. The method used in this work may also be used by other researchers to generate more fruit fly cell lines. The use of cell cultures has been important for studying biological processes that are not easily accessible in whole organisms . A numbeDrosophila melanogaster) cell cultures are among the most widely used invertebrate cell cultures   . The majr (DGRC) . Furtherr (DGRC) . All linr (DGRC) . Thus, tpathways , exploripathways , establipathways , charactpathways , nucleospathways  among mupathways .D. melanogaster cell lines listed in the DGRC catalog were derived from whole embryos and the remainder are from various larval imaginal discs, the larval central nervous system, larval hemocytes, or adult ovaries. The potential of cells from these different sources to differentiate into adult cell types is not known. However, temporal transcriptional profiling of the Ecdysone response of 41 cell lines  we identified lines with neuronal and hemocyte characteristics. Collectively, these cell lines provide in vitro models for five different cell types and are expected to be a valuable resource for high-throughput and biochemical approaches, which rely on large numbers of homogeneous cells.Here, we describe a second-generation version of the Ras method in which Raspression . As we sV12UAS-Ras expression was restricted to glial, tracheal epithelial, and mesodermal cells using lineage-specific Gal4 drivers (V12UAS-Ras (Drosophila Gene Expression Tool (DGET) database (https://www.flyrnai.org/tools/dget/web/), which is the bulk RNAseq data portal at Drosophila RNAi Screening Center (DRSC)  . The TM4r (DRSC) . As exper (DRSC)  and new r (DRSC) . Moreover (DRSC) ; Table 2r (DRSC)  reveals repo-Gal4 driver that recapitulates Repo expression was used to express V12UAS-Ras  line has been passaged more than 50 times. The parental Rbr6 line and three clonal derivatives  have an elongated morphology and stained positive for Repo , which encodes a potassium-dependent sodium/calcium exchanger, is upregulated in all three clones, consistent with the literature  a master regulator of tracheal identity  and is expressed in mesoderm and muscle cells  and control larvae  showed that RasV12 expression disrupted muscle development, suggesting that the prevalent amorphous GFP-positive cells observed in primary cultures were abnormal muscle cells  and twist (twi) (myoblast city (mbo), which encodes an unconventional bipartite GEF with a role in myoblast fusion  ; Table 2t fusion . The capV12UAS-Ras with the pan-neural drivers scratch-Gal4 and elav-Gal4, however none of the primary cultures resulted in continuous cell lines , slowpoke (slo), 5-hydroxytryptamine (serotonin) receptor 1A (5-HT1A), Protein C kinase 53E (Pkc53E), Diuretic hormone 31 Receptor (Dh31-R), and straightjacket (stj). In addition, comparison of the top upregulated genes in these cells to marker genes from scRNAseq data identifies a cell type of neuronal origin as the best match , Hemese (He), eater, u-shaped (ush), Cecropin A2 (CecA2), and Cecropin C (CecC). Comparison of top upregulated genes with scRNAseq data showed that the cells have a strong match to the top marker genes of hemocytes  were diploid, or near diploid , co-expression of the p35 baculovirus survival factor, or growth stimulation by downregulation of tumor suppressors (dsRNA for warts or brat). One line, also harboring a Gal80ts transgene, reached passage 25; however, the line was unstable and in early passages the cells variably lost Repo expression and changed morphologically. The one continuous glial line generated expresses a transgene that targets the tumor suppressor, V12; UAS-bratdsRNAbrat . Given a single success, it is not clear if downregulation of brat contributed to derivation of the line. Moreover, there is no evidence that these genotypic variations enhanced cell line generation with other drivers, as primary cultures expressing RasV12 without modulation or a survival factor produced lines with similar success rates for the btl-Gal4 or 24B/how-Gal4 drivers  will provide good models for investigating assay conditions that promote polarization and 3D cell interactions that could allow the cells to manifest a more complex tissue architecture. In keeping with this possibility, treating these cells with ecdysone to induce differentiation showed cell clumping suggestive of a multicellular structure .RNAseq analysis of cells from the ActGSI-3 cell clone showed a striking similarity to hemocytes, and the cells may be a good model for studying immunity . The celglial cell missing (gcm) is not differentially expressed in the three glial-lineage cell clones and gcm2 is differentially expressed in only two of the three clones. Further, trachealess (trh) is only differentially expressed in one of the three tracheal-lineage cell lines. Similarly, the muscle-specific transcription factors twist (twi), nautilus (nau), snail (sna), and Mef2 show variable expression in the muscle-lineage cell clones. It should also be noted that the expression patterns were determined for undifferentiated cells and expression levels could change after hormone exposure.The most significantly upregulated marker genes in each cell line are significantly enriched for top marker genes from expected cell types based on the single-cell RNAseq data from Fly Cell Atlas in most cases. This indicates the potential value of these cell lines as corresponding in vitro models for studying these cell types. While the cells will prove to be valuable models, it should be noted that even those showing a clear differentiated phenotype exhibit unexpected patterns of gene expression. For example, some cells in the mesodermal clone, 24B5-B8, are positive for HRP  and the Drosophila have only been performed in S2 cells, which have hemocyte-like features. The availability of new cell lines with muscle, glial, and epithelial characteristics will enable screens designed to interrogate biological processes specific to these cell types.The cells will have value for both low- and high-throughput approaches, including genetic or compound screens for which screening in the relevant cell type will result in identifying targets that are more likely to be of physiological relevance. Most of the cells have an attP-flanked cassette , which mDrosophila cell lines; however, the number corresponding to known cell types is low. This is due in part to the lack of a method for generating cell lines from specific tissues. We expect that the method described here, using restricted expression of RasV12, will be a tractable approach for investigators to generate lines of cell types of interest. Single-cell cloning followed by cell characterization (immunohistochemistry and RNAseq) also proved to be a useful method to identify cell-type-specific lines and this approach could identify additional valuable lines in the existing collection at the DGRC. In summary, we show that lineage-restricted Ras expression and cell cloning has produced a set of new cell lines that will be of immediate value for analyses in the five cell types they represent.There are hundreds of Gal4 drivers: 24B/how-Gal4, w[*]; P(w[+mW.hs]=GawB)how[24B] (BL 1767); repo-Gal4, P(GAL4)repo (BL 7415); btl-Gal4, P(GAL4-btl.S)3-2 (BL 78328); Act5C-GeneSwitch-Gal4, P(UAS-GFP.S65T)Myo31DF[T2]; P(Act5C(-FRT)GAL4.Switch.PR)3 (BL 9431). Transgenes: UAS-RasV12 (3), P(w[+mC]=UAS-Ras85D.V12)TL1 (BL 64195); UAS-RasV12 (2), P(w[+mC]=UAS-Ras85D.V12)2 (BL 64196); UAS-RasV12 with RMCE site (3), P(w[+mC]=UAS-Ras85D.V12)TL1, P(w[+mC]=attP.w[+].attP)JB89B (BL 64197); UAS-GFP nuclear, P(UAS-GFP.nls)14 (BL 4775); bratdsRNA, P(y[+t7.7] v[+t1.8]=TRiP.HMS01121)attP2 (BL 34646); UAS-p35 baculovirus death inhibitor, P(w[+mC]=UAS-p35.H)BH1 (BL 5072) and Gal80ts, w[*]; P(w[+mC]=tubP-GAL80[ts])20 (BL 7019).The following fly stocks were used to create primary cell lines: V12 lines. Some RasV12 stocks had additional alleles as noted in g. The supernatant was discarded, and the pellet was resuspended in 3-ml Schneider’s S2 medium and centrifugation step and washing with Schneider’s S2 medium was repeated twice more. The final pellet size was estimated and plated in 1 or more 12.5 cm2 T-flasks with 2–3 ml Schneider’s S2 medium. The number of flasks needed for a given pellet size can also be estimated from the volume of packed embryos with approximately 30 µl of packed embryos being sufficient for one flask.This follows a detailed method, which has additional information , except 2 T-flasks at 25°C in Schneider’s S2 medium and were passaged at between 90% and full confluence  using Effectene Transfection Reagent (QIAGEN). After 24 hr, cells were selected with puromycin at 0.5–2.5 µg/ml for 5 days. After 2–4 weeks, colonies were isolated and expanded. For dilution cloning, cells were seeded into a 96-well plate at a concentration of 0.5–1 cell/well in 100 µl conditioned media  at 1 µg/ml separated by 24 hr in non-supplemented medium.Cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) for 15 min or 3.5% formaldehyde (Sigma) for 30 min at room temperature, and then rinsed twice with 0.1% Tween-20 in phosphate-buffered saline (PBS-T). Cells were permeabilized (0.2% Triton X-100 in PBS) for 10 min at room temperature. Cells were blocked  for 30 min at room temperature and incubated with diluted primary antibodies overnight at 4°C. Cells were washed three times with PBS-T and incubated with diluted secondary antibodies in blocking buffer for 1 hr at room temperature or overnight at 4°C. Cells were washed three times with PBS-T and mounted in VectaShield with DAPI (Vector Laboratories). For the Dcad2 antibody, cells were fixed and processed as described in 5 cells were plated in a 12-well plate. Cells were counted from triplicate wells every 3 days over a 9-day period. Doubling time was calculated using log2 cell numbers (1–2 × 10 numbers .Cells were grown to 50–90% confluence and incubated with 0.05 µg/ml KaryoMAX (Gibco-Thermo Fisher 15212012) for 3–18 hr. Cells were processed for analysis using the method in Cells in a 6-well plate (approximately 70% confluent) were transfected with 0.4 µg of an Actin5C-EGFP plasmid  using Effectene Transfection Reagent (QIAGEN). The fraction of GFP-positive cells was scored after 48 hr.Drosophila Medium (Gibco Cat # 21720001), supplemented with 10% fetal bovine serum (Cytiva Hyclone Cat SH30070.03). For Act5C-GS>Ras attP-GFP-LI-Clone 2, Act5C-GS>Ras attP-GFP-LI-Clone 3, and Act5C-GS>Ras attP-GFP-LB-Clone 6 cultures were grown in the same basal media supplemented with 10 nM of Mifepristone (Thermo Fisher Cat# H11001). Cultures were allowed to grow in T-25 flasks to become confluent before treatment with trypsin (Gibco Cat# 12604013) for 4 min to dislodge the cell monolayer from the growth surface. The cells were resuspended in 4 ml of their respective media and 1 ml of the cell suspension was collected for pelleting, followed by washing in 1× PBS, and then flash-freezing in liquid nitrogen. All cell samples were processed in triplicates.Cell cultures were grown and expanded in their respective media. All cell lines were cultured in Schneiders A260/A280 ratio >1.8 and RIN >7.Total RNA was isolated from the pellets using the TRIzol reagent  as per the manufacturer’s instructions. The isolated total RNA was subjected to further purification using the RNeasy Mini Kit  and the RNA post-cleanup was eluted in RNase-free water. The eluted total RNA was confirmed to have a Upon passing the quality control parameters, Illumina TruSeq libraries were constructed using TruSeq stranded mRNA HT kit . Paired end sequencing was performed on an Illumina NextSeq 500 with a 150-cycle high output kits .https://github.com/alexdobin/STAR; Drosophila genome and featureCounts were used to get gene counts from all samples into a count matrix for downstream analysis. A principal component analysis plot was produced using heatmaply. FPKM values were calculated using fpkm(DEseq2) using gene length output by featureCounts. The reference genome used was FB2022_05, dmel_r6.48 (FlyBase) (https://www.flyrnai.org/tools/dget/web/).Raw data processing was performed using the STAR sequence aligner (FlyBase) . Both raEach sample was compared against all other samples by using DESeq2   to deterDrosophila cell lines  at Indiana University. The lines are available for distribution to the research community.All cell lines described here have been deposited to the  Drosophila. The evidence supporting the claims of the authors is convincing, with rigorous characterization of the cell lines and incorporation of their transcriptomes into Drosophila Gene Expression Tool website for user-friendly access. These lines will be a valuable resource that complements in vivo Drosophila genetics, improving biochemistry and facilitating high-throughput screening.This valuable work describes the establishment and characterization of new cell lines derived from specific tissues of the fruit fly  public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:Drosophila research" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Erika A Bach as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Claude Desplan as the Senior Editor. The following individual involved in the review of your submission has agreed to reveal their identity: David Bilder (Reviewer #2).Thank you for submitting your article "Continuous muscle, glial, epithelial, neuronal, and hemocyte cell lines for The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:1. Increase the 'identity' of the cell lines– Provide information and possibly new data to increase the utility of Table 2– Provide information on what expected marker genes are NOT expressed in the new cell lines– Compare the new lines to existing lines, especially the workhorse S2 cells.– Provide information/commentary on growing in new cells line in large numbers or employing them in high-throughput screening.2. Improve user access to genomics data. Downloading and accessing GEO files are not user-friendly. Genomic data should be accessible to the potential users of these cell lines.– Provide tabular genome-wide data for each cell line.– Consider generating a website with these data3. Improve the images and figure legends– Provide higher magnification views to support the conclusions.– Figure 2: show Ecad staining on a non-epithelial cell line with comparable density.– Figure 3D: show more clearly the multinucleate cells.– Provide information on magnification for images.4. Methods– Mention how specific the GAL4 drivers are in embryos .– Provide a detailed description of how the new cell lines were generated .– Provide a detailed description of the culture conditions for the new cell lines.– Provide reagent tables and RRIDs for all reagents5. Improve karyotyping.Reviewer #1 (Recommendations for the authors):1. The authors should review their reference manager because not all of the references are cited in the same way. Most are last name comma first initial but A. Simcox is frequently cited.2. Under the Methods section, the authors should provide a description of how they generated the cell lines .3. Is it possible to put scale bars on the micrographs? If not, could the authors please state the magnification?Reviewer #2 (Recommendations for the authors):1) The critical assignment of the 'identity' of the cell lines would benefit from more detail and explanation. The process is a bit confusing. For instance, in Table 2, why is the dataset Whole body for some and a specific organ for others? In the latter case, why does Btl8 map to the oenocyte sample? On line 182, the authors say that the glial lines did not result in as clear a similarity as other lines, but that is not obvious from the values in Table 2. Is it possible/useful to compare the enrichment scores of Table 2 to those of well-characterized tissue-specific mammalian cell lines and their in vivo equivalents  for perspective?2). Related, the paper would benefit from an explicit critical discussion of the cell lines. For instance, what expected marker genes are NOT expressed? E.g. the tracheal-derived cell lines L8 do not express trh, L7 downregulates trh. In what ways are these not simple immortalized cells of in vivo equivalents -that may be important for people thinking about using them? The authors' simple statements e.g. line 212 do a disservice to the nuances of identity.3) The authors could make clear how these new lines compare to existing lines. For instance, S2 was generally thought to be hemocyte-like, how does it compare to Act GSI-3? What reason would researchers interested in hemocyte biology want to use one over the other? An example of this value is the comparison of transfection efficiency to S2 (line 237).4) Any experience with growing in large numbers, or in high-throughput screening format? Since these are promoted as uses, it would be very helpful to have a perspective from the authors, who include those most experienced with the variety of existing cell lines and their use in these contexts.Other points – not absolutely needed for accceptanceIn general, the figures are rather sparse, there is room for more data such as those suggested above.Any speculation as to why so many of the lines correlate with an adult, rather than embryonic, profiles when the tissue source was embryos?Figure 2: would be nice to show Ecad staining on a non-epithelial cell line with comparable density.Would be nice to mention how specific the GAL4 drivers are in embryosThe culture conditions for the cell lines, which is critical information, are buried in a section about 'RNA extraction'.Figure 3D: would be nice to show more clearly the multinucleate cells.Mef2; elav) -is it possible that the levels of active Ras were too high to be compatible with life and/or immortalization?For the lines where there was a failure to derive :The paper is clear and well-written, but I believe that the work needs to be extended to make it useful.1) The RNAseq characterization is perhaps the most important and global aspect of the utility, because someone interested in a particular gene, could check to see if it was well expressed and in which of the novel (or existing) cell lines. I did not see tabular genome-wide data that would fit this bill. The expression data is basically not presented in the manuscript. The authors are used to presenting data to the community through the DGRC, DRSC, and FlyBase. They need to make these data easy to access. It is hard to judge the GEO submission as it is still listed as private.2) The phase images are not sufficient to understand the cell biology nature of these cell lines. High magnification views that show higher content information on the cells are needed. It is nice to show the hormonally induced differentiation onset using one or a few markers, but for a resource, I would hope for more granularity. Again it is difficult to make choices of cell lines for experiments with this resolution.3) Although I do not recall reading it explicitly, I assume that the chromosome spreads were scored based on chromosome size. This is not very satisfying in the era of chromosome paints and especially DNAseq (e.g. PMID: 25262759).4) The figure legends should indicate what is being detected. "Expressed" is too vague.5) Reagent tables are much easier to read than in-text methods with names and undescribed identifiers. Essential revisions:1. Increase the 'identity' of the cell lines– Provide information and possibly new data to increase the utility of Table 2– Provide information on what expected marker genes are NOT expressed in the new cell lines– Compare the new lines to existing lines, especially the workhorse S2 cells.– Provide information/commentary on growing in new cells line in large numbers or employing them in high-throughput screening.In Table 2, we have added the cell-type based on in situ data. This complements the single cell RNAseq and confirms the cell type identity.We have added a section on transcription factors, not expressed in the new cell lines, which might be expected for that cell type. Also, we note that the RNAseq data is for undifferentiated cells and genes not expressed in the proliferative state may be expressed in differentiated cells. This is one reason why information on what is not expressed may not be definitive.We have included figures that show the morphology of the cells, including S2 cells . These show that none of the new lines are similar to S2, including the hemocyte-like cell line ActGSI-3. While both S2 and ActGSI-3 cells are round and grow in suspension, S2 cells grow individually, whereas ActGSI-3 form large cell rafts.6 in a single well of a 12 well plate. In this experiment S2 cells grew to a confluent density of 6.2 X 106. As all cells, except ActGSI-3, are adherent, the cells are also potentially well suited for multi-well plate assays.In Table 3, we have added data about the confluent density of the cells. These numbers show that the cells can be grown to densities of 1.4-8.1 x 102. Improve user access to genomics data. Downloading and accessing GEO files are not user-friendly. Genomic data should be accessible to the potential users of these cell lines.– Provide tabular genome-wide data for each cell line.– Consider generating a website with these dataWe added a supplementary table (Table S3) with genome-wide expression levels for each gene in each cell line .Drosophila Gene Expression Tool (DGET) database (https://www.flyrnai.org/tools/dget/web/), which is the bulk RNAseq data portal at Drosophila RNAi Screening Center (DRSC). DGET can be used to query the expression levels for the genes of interest as well as search the genes with similar expression pattern for any input gene. The expression dataset is called “Ras cell lines”.The dataset has been imported into the 3. Improve the images and figure legends– Provide higher magnification views to support the conclusions.– Figure 2: show Ecad staining on a non-epithelial cell line with comparable density.– Figure 3D: show more clearly the multinucleate cells.– Provide information on magnification for images.The new Figure 1 shows the cells at high magnification. This was a very useful suggestion because it allows comparison of cell morphologies within and between lineages. For example, the glial cells have a long spindle-like shape, and the epithelial cells are cuboidal.Figure 6 shows Btl3 and S2 cells stained for the epithelial marker E-Cadherin/Shotgun. Btl3 cells expressed Ecad at the cell periphery and S2 cells had low level cytoplasmic expression.New panels in Figure 5 show the multinucleate muscle cells.Scale bars are included in Figures 1 and 8, which show phase images of the cells.4. Methods– Mention how specific the GAL4 drivers are in embryos .– Provide a detailed description of how the new cell lines were generated .– Provide a detailed description of the culture conditions for the new cell lines.– Provide reagent tables and RRIDs for all reagentsMef2-Gal4 and 24B/how-Gal4. Only 24B/how-Gal4 produced continuous cell lines. In a new figure we show that RasV12 expression with Mef2-Gal4 in muscle cells disrupted muscle development and that in primary cultures the RasV12-expressing cells (marked with GFP) failed to attach and grow . We. Do not the know the basis for this; however, expression level is a possibility. Even after successful cell line generation it is important to use a battery of assays to determine lineage characteristics because cells change as they adapt for survival in vitro. This is exemplified by some of the 24B-Gal4 derived lines that failed to differentiate after extended passages.We find that additional factors other than specificity of expression of Gal4 are necessary for successful cell line development. This is illustrated for the muscle lineage where we tested two well characterized drivers—A detailed primary culture procedure is provided in the Methods section.Culture conditions and passaging regimes for the new cell lines are provided in the Methods section. This method also refers to Figure 8, which shows the cells at confluence and will help users determine when cells are ready for sub-culturing (passaging).All reagents and RRIDs (when available) are provided.5. Improve karyotyping.We redid the karyotypes for all the cell lines and include karyograms for each. The quality is greatly improved and equivalent to the standard for the field. The figures showing the karyotypes and the chromosomes ordered in a karyogram appear in supplemental figures for each cell line and the refined karyotypes are summarized in the revised Table 3.


It is becoming more and more apparent that they are highly active, diverse, and involved in a large variety of processes. The essential role of lysosomes is exemplified by the detrimental consequences of their malfunction, which can result in lysosomal storage disorders, neurodegenerative diseases, and cancer. Using lysosome enrichment and mass spectrometry, we investigated the lysosomal proteomes of HEK293, HeLa, HuH-7, SH-SY5Y, MEF, and NIH3T3 cells. We provide evidence on a large scale for cell type–specific differences of lysosomes, showing that levels of distinct lysosomal proteins are highly variable within one cell type, while expression of others is highly conserved across several cell lines. Using differentially stable isotope-labeled cells and bimodal distribution analysis, we furthermore identify a high confidence population of lysosomal proteins for each cell line. Multi–cell line correlation of these data reveals potential novel lysosomal proteins, and we confirm lysosomal localization for six candidates. All data are available   •Analysis of lysosome-enriched fractions/whole cell lysates from six cell lines.•High dynamic range of lysosomal proteome.•SILAC labeled background cells enable identification of enriched proteins.•Definition of high confidence potential novel lysosomal proteins.•Validation of six lysosomal candidate proteins by immunostaining. We present a comprehensive analysis of lysosome-enriched fractions from six commonly used mammalian cell lines. We show that lysosomal proteins are highly dynamic with respect to their expression and/or localization at lysosomes and provide a novel strategy for the identification of specifically enriched proteins. Based on the correlation of data from all cell lines, we identify high confidence novel lysosomal proteins and confirm six selected candidates. Lysosomes, the central lytic organelles of the cell, are responsible for the degradation of a large variety of cellular compounds and the recycling of their building blocks, fulfilling a pivotal function for cellular homeostasis. Organelles and macromolecules, which are delivered to lysosomes through endosomes, phagosomes, and different forms of autophagy, are hydrolyzed by ∼60 enzymes residing in the lysosomal lumen . Defectse.g., for the exchange of metabolites (mTORC1), whose members and functions have been investigated in a variety of studies , theiewed in . The mosiewed in , 12.e.g., in their pH value or their mobility . Given In order to be able to assess such differences on a global scale, and to identify novel lysosomal proteins, unbiased large-scale approaches play a pivotal role. The method that is currently used most frequently is mass spectrometry–based proteomics, as it allows to simultaneously identify and quantify large numbers of proteins in a given sample. A variety of studies have been performed, which investigated lysosomes and lysosomal proteins in HEK293 , 19, 20,in vitro cultivated cells  have been identified  , which ied cells , making entified , which mWhile it is common practice to assess the likelihood of localization to a specific organelle in differential centrifugation approaches by comparison with other fractions , 31, to In the current study, we investigated the lysosomal proteome of six widely used cell lines using lysosome enrichment by SPIONs and mass spectrometry–based proteomics . For thr13C615N2 lysine , and 87.8 mg/ml light or 13C615N4 arginine  at 37 °C, 100% humidity, and 5% CO2. Each cell line was passaged at least six times to ensure complete SILAC labeling.HEK293 , HeLa , HuH-7 , SH-SY5Y , MEF , and NIH3T3  cells were cultured in SILAC Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% dialyzed fetal calf serum (FCS), 100 IU/ml penicillin, 100 μg/ml streptomycin, 181.2 mg/ml light or 6 cells were seeded per 10-cm plate and cultured in DMEM with 2.5% FCS for 72 h. Subsequently, the medium was changed to DMEM with 10% FCS including 10% of SPIONs solution  for either light or heavy SILAC labeled cells (two replicates each), followed by incubation for 24 h. For HEK293 cells, plates were coated with 100 μg/ml poly-L-lysine . For HEK293 and SH-SY5Y cells, 6 × 106 cells were seeded directly in DMEM with 10% FCS and 10% SPIONs followed by incubation for 24 h. Subsequently, for all cell lines, cells were washed twice with 1× PBS, fresh medium was added, and the cells were incubated for 36 h. Cells were washed three times with ice-cold 1× PBS, scraped in 2 ml isolation buffer  was transferred to a new tube. The pellet was resuspended in 2 ml isolation buffer, the procedure was repeated, and the PNS fractions were combined. The pooled PNS fractions of the individual cell lines were passed by gravity flow through LS columns  placed in a MidiMACS Separator , and washed with 5 ml isolation buffer. Lysosomes were eluted from the columns twice with 1 ml isolation buffer each using a plunger. Lysosomal integrity and isolation efficiency were assessed using the β-hexosaminidase assay with/without the addition of 0.8% Triton X-100   . To calcFor each sample, 100 μg of protein was precipitated by ice-cold chloroform/methanol  as described . Protein18 particles  for data-dependent acquisition (DDA) or data-independent acquisition (DIA) analyses, respectively. Peptides were resuspended in 5% ACN/5% FA and loaded on the analytical column at a flow rate of 600 nl/min, 100% solvent A (0.1% FA in water). Peptide separation was performed at a flow rate of 300 nl/min with a 240-min linear gradient (DDA) or a 120-min gradient (DIA) from 5 to 35% solvent B (95% ACN/0.1% FA). Survey spectra were acquired in the Orbitrap mass analyzer with a mass range of m/z 375 to 1575 at a resolution of 60,000. For DDA analyses, tandem mass spectrometry (MS/MS) fragmentation was performed for charge states between 2 and 4 by higher-energy collisional dissociation (HCD) with 30% collision energy and data were acquired in the Orbitrap at a resolution of 30,000. The cycle time was set to 5 s and the precursor isolation width to 1.6 m/z using the quadrupole. For mass spectrometry (MS) and MS/MS scans, the automatic gain control was set to 4 × 105 and 5 × 105, respectively. For DIA analyses, survey spectra were acquired in the Orbitrap mass analyzer with a mass range of m/z 350 to 1200 at a resolution of 120,000 and a maximum injection time of 50 ms. MS/MS was performed by isolation of m/z 24.1 windows using the quadrupole and higher-energy collisional dissociation fragmentation with 30% collision energy. Data were acquired in the Orbitrap at a resolution of 30,000, with a maximum injection time of 54 ms, and a fixed mass range of m/z 200 to 2000. The cycle time was set to 3 s, and the automatic gain control was set to 5 × 105 and 1 × 106 for MS and MS/MS spectra, respectively.Mass spectrometry analyses were performed using a Dionex Ultimate 3000 system coupled to an Orbitrap Fusion Lumos mass spectrometer (both Thermo Scientific). Columns were manufactured in-house as follows: 50-cm spray tips were generated from 360 μm outer diameter/100 μm inner diameter fused silica capillaries using a P-2000 laser puller (Sutter Instruments) and packed with 1.9 μm or 3 μm Reprosil AQ Cwww.matrixscience.com). For database searching, UniProt (Swiss-Prot + TrEMBL) Homo sapiens  and UniProt Mus musculus  in combination with the cRAP database (ftp://ftp.thegpm.org/fasta/cRAP/crap.fasta) including common contaminants were used. The following parameters were defined: variable modifications: oxidation of methionine, acetylation of protein N termini; fixed modification: propionamide at cysteine; mass tolerance: 10 ppm for precursor ions, 50 mmu for fragment ions; enzyme: trypsin except proline was the next amino acid; missed cleavage sites: two. Data were filtered with a false discovery rate (FDR) of 1% at the peptide level using Percolator  and M. musculus . The same search parameters as for Proteome Discoverer were applied with the exception of definition of heavy labeled arginine (Arg10) and lysine (Lys8) as fixed modification for cells receiving SPIONs in the heavy SILAC channel. DIA data were analyzed by Spectronaut (version 16.2.220903.53000). DDA and DIA raw files were used to generate hybrid spectral libraries using UniProt (Swiss-Prot) H. sapiens  or UniProt (Swiss-Prot) M. musculus  including common contaminants. For each peptide, the three to six most abundant b/y ions were selected for library generation, depending on their signal intensity. The same fixed/variable modifications were used as in Proteome Discoverer and MaxQuant analyses. Data were filtered with the following q value cutoffs: precursor = 0.01, protein (experiment) = 0.01, protein (run) = 0.05. Dynamic retention time alignment was performed based on the high-precision indexed retention time concept  in combination with Mascot 2.6.1  , Microsohttps://cran.r-project.org/web/packages/BH) with a default significance level . Principal component analysis was performed using the prcomp function of the R package stats, version 3.6.2, setting center = TRUE and scale = TRUE (https://CRAN.R-project.org/package=Hmisc). Heatmaps of abundance ratios and Pearson correlation were created with the heatmap.2 function from the R package gplots, version 3.0.3, and using hierarchical clustering (order = hclust) (http://cran.r-project.org/package=gplots). For UpSet plots, Proteome Discoverer data were joined using the full join function of the R package dplyr, version 0.7.4 . If a protein had been found in a cell line, the value was set to 1. Otherwise, if there was a missing value, the respective cell was set to 0. UpSet plots were generated with the upset function from the R package UpSetR, version 1.4.0, setting order.by = degree  and g:Profiler (https://biit.cs.ut.ee/gprofiler/) . For functional analysis of high confidence potential novel lysosomal proteins, the Gene Group Functional Profiling (g:GOSt) tool available in the g:Profiler web server (version from 2020-03–09) was used. Statistical enrichment analysis with g:GOSt was performed with GO molecular function, GO cellular component, GO biological process, and Reactome .The derived probability describes the likelihood of the observation based on the calculated model. Extreme abundance ratio values (100 and 0.01) were handled separately, as they indicate missing values in one of the distributions and are arbitrarily set by Proteome Discoverer. Therefore, they were replaced with the maximal or minimal abundance ratios observed during data analysis and the posterior probabilities of these values were set to 1 or 0, respectively. For each dataset, the FDR was calculated applying the Benjamini–Hochberg  multiplee = TRUE . Pearson= degree . For proofiler/) , 45. OveSamples for mass spectrometric analysis were generated for each cell line in four independent biological replicates. In order to address potential bias in the analysis of SILAC ratios, label switching was performed for the individual cell lines with two replicates receiving SPIONs in the light and heavy channels, respectively. In subsequent analyses, only proteins were considered with valid values in at least three biological replicates.Rab5-GFP expression vector was a kind gift from Sergio Grinstein.RNA was isolated from MEFs or NIH3T3 cells using the Nucleospin RNA plus kit . Subsequently, cDNA was generated using the RevertAid First Strand Synthesis kit  according to the manufacturer’s instructions utilizing random hexamer primers. Downstream RT-PCR amplification of lysosomal/endosomal genes was carried out with the respective set of primers . PCR proNIH3T3 cells were seeded 24 h prior to transfection, and 4.5 μg of the respective plasmid DNA was transfected using TurboFect transfection reagent . Cells were cultured for 24 h in DMEM with 10% FCS and 100 IU, ml penicillin/100 μg/ml streptomycin . Subsequently, the culture medium was replaced, fresh medium containing 500 μg/ml hygromycin B  was added to the cells, and this step was repeated after 24 h. Before analysis, all transfected cells were cultured for a minimum of 4 weeks in DMEM containing 500 μg/ml hygromycin B.LAMP2  overnight at 4 °C. The next day, all samples were repeatedly washed in 1× PBS with 0.2% saponin and incubated with the secondary antibodies donkey anti-rat Alexa Fluor 488 , donkey anti-rabbit Alexa Fluor 488 , and donkey anti-mouse Alexa Fluor 594  for 1 h at RT. After washing with 1× PBS, 0.2% saponin, coverslips were rinsed twice with H2O, and embedded in Mowiol-DABCO with DAPI  . Transiently transfected cells were analyzed with an inverted confocal laser scanning microscope  in combination with a UPLSAPO 60× oil immersion objective (NA:1.35) at 2× zoom, and stable cells were analyzed with the LSM 980 with Airy scan 2  microscope. All images of transient cells were acquired and processed with the Olympus FluoView Software, whereas images of stable cells were acquired and analyzed with the Zeiss Zen 3.2 Blue edition software.HeLa cells were cultured using cell culture medium as described above. Cells were seeded on glass coverslips 1 day prior to transfection. The transfection was carried out with 750 ng plasmid DNA using TurboFect and cells were cultured for 48 h before they were processed for immunostaining. Both transient (HeLa) and stable (NIH3T3) cells were washed three times with 1× PBS and fixated with either methanol or 4% paraformaldehyde in 1× PBS for 10 or 20 min, respectively. Subsequently, samples were rewashed three times with 1× PBS and incubated for 5 min in 1× PBS with 0.2% saponin, and for 15 min in 1× PBS with 0.02% glycine and 0.2% saponin. All samples were blocked for 1 h in 1× PBS with 0.2% saponin and 10% FCS. After blocking, cells were stained with primary antibodies against HA tag , Myc tag , and g for 10 min at 4 °C, and the supernatant was removed. Cell pellets were resuspended in RIPA lysis buffer , sonicated three times for 15 s with a Sonoplus sonicator  with 0.8 amplitude, and centrifuged at 20,000 g for 10 min at 4 °C. The supernatant was collected and the protein concentration determined using the BCA protein assay . Protein concentrations were adjusted to 2 μg/μl, samples combined with Laemmli buffer , SDHA , Calnexin , GM130 , CTSD , NPC2 , LIMP2 , ATP6V1B2 , ATP6V1D , HGSNAT , GBA , RAPTOR , MAN2B2 , NEDD4 , CSTB , KIF5B , FLOT1 , PPT1 , and β-actin . The next day, blots were washed three times with TBS-T and incubated for 1 h at RT with the respective secondary antibodies .The enrichment of lysosomes from mammalian cells typically results in high numbers of contaminating nonlysosomal proteins . In ordeFig. 1B and Figs. 1C and Fig. 1C), indicates that the percentage of lysosomes receiving SPIONs through unspecific fluid phase endocytosis in these cells is lower in comparison with the other lines.Following this strategy, we performed four biological replicates for each cell line. In order to account for systematic errors related to SILAC labeling and data analysis, we performed label switching, including SPIONs in two replicates of light and heavy labeled cells, respectively. After pooling and disruption of cells, we assessed lysosomal integrity using activity assays for the lysosomal luminal enzyme β-hexosaminidase . As we were especially interested in proteins located at the lysosome, we subsequently extracted a subset of proteins that are assigned to the lysosomal compartment in PANTHER and/or UniProt for mouse or human, irrespective of the fact if they fulfil a crucial function for this organelle .Using an optimized protocol , we analFig. 1rganelle . For theE). When considering only the putative lysosomal proteins, correlation coefficients were in general higher. The exceptions were comparisons of HeLa and NIH3T3/HEK293 cells, yielding the lowest values, indicating stronger quantitative differences between the lysosomal proteomes of these cell lines (C). Based on unsupervised hierarchical clustering, we observed a general clustering for samples that received SPIONs in the light or heavy SILAC channel, respectively (F). Since especially unspecific binding proteins are affected by the SILAC label switch, this observation underlines the strong contribution of background proteins to the individual datasets. Also for putative lysosomal proteins, we observed an effect of the SILAC labeling state of the cell line, but no general clustering occurred depending on which SILAC labeled cell line received SPIONs (D).To investigate the correlation among individual cell lines, we determined the Pearson correlation coefficients for the log2 abundance ratios of the identified proteins. On the whole protein level, NIH3T3 cells showed the lowest correlation with all other cell lines, while SH-SY5Y cells were most similar E. When cll lines C. Based ectively F. Since d SPIONs D.e.g., Western blot, quantitative PCR, or immunostaining approaches have shown highly variable expression levels across tissues, to our knowledge so far no attempts have been made to compare expression levels of lysosomal proteins between cell lines on a global scale. It remains therefore largely elusive in how far lysosomes differ between cell types.Based on the heterogeneity of tissue-specific phenotypes for individual lysosomal storage disorders , it is hA). HEK293 and NIH3T3 cells contributed with 512 and 257 proteins, respectively, the highest and lowest numbers of unique identifications. In all four human cell lines, 326 proteins were identified that were not detected in the two mouse lines, while 235 were unique for the mouse cells.To further assess differences in the lysosomal proteomes of the individual cell lines analyzed, we performed direct comparisons of the identified proteins . Of the Fig. 3versus 25% of total proteins were identified in all six cell lines, Fig. 3B). Furthermore, we detected 130 lysosomal proteins that were unique to human cells , while 42 proteins were unique to mouse cells (17 detected in both cell lines). Overall, while the majority of proteins were reproducibly detected across cell lines, each comparison of the individual proteomes resulted in unique subpopulations. This indicates distinct features of the lysosomes of the respective cells, which are possibly related to their individual functions/characteristics.For the putative lysosomal proteins identified across all datasets , we observed a similar behavior, however, with a markedly increased reproducibility compared with the whole dataset  heterogeneity. Furthermore, every cell requires a certain lysosomal “core proteome” to be able to deal with the turnover of ubiquitous cellular components. We argued that it is therefore highly unlikely that proteins belonging to this class are not expressed at all. It should be more reasonable that the expression levels of individual proteins belonging to this group are adapted to the specific characteristics of an individual cell type, reflecting its unique composition and function .A and S5A), presenting a “core feature” of lysosomes. This is probably also related to the fact that the presence and correct stoichiometry of the V-ATPase are essential for lysosomal function. Subsequently, we filtered for proteins with iBAQ values in at least two cell lines and three biological replicates each, calculated the median of the V-ATPase-normalized value for individual proteins, and grouped them into 32 classes based on their function and/or localization . Lysosomes enriched from HuH-7 and HeLa cells contained the highest amounts , while values in the other cell types were roughly similar. The observed differences were mainly due to the strong overrepresentation of hydrolases in HuH-7 cells, transporters in HeLa cells, and membrane proteins in both of them. Only lysosome-associated ubiquitin ligases and proteins related to mTORC1, which were most abundant in SH-SY5Y and HEK293 cells, respectively, were the highest in abundance in cell lines other than HeLa and HuH-7 (B).Initially, we calculated the sum of V-ATPase-normalized iBAQ values in the individual cell lines to estimate how many lysosomal proteins are present relative to the V-ATPase B. Lysosond HuH-7 B.TCIRG1 and ATP6AP1/ATP6AP2 (A). For proteins involved in the translocation of small molecules across the lysosomal membrane (A), we observed a dynamic range of three orders of magnitude with the cholesterol transporter SCARB2 and the Cl-/H+ exchanger CLCN6 showing the highest and lowest median expression levels, respectively (difference of ∼1400 fold).Subsequently, we investigated the expression levels of individual lysosomal proteins in the different cell lines. For the V-ATPase complex itself, we detected a conserved stoichiometry for proteins belonging to both the V0 and the V1 part with the exception of the associated proteins /ATP6AP2 A. For prmembrane A, we obs2+ channels MCOLN1, TPCN1, and TPCN2, for example, were expressed at very similar levels in all cell lines, while the amino acid- and oligopeptide-transporting members of the solute carrier (SLC) family showed both similar and highly variable expression patterns. SLC3A2 and SLC7A5, which are known to heterodimerize . Overall, amino acid/oligopeptide transporters were expressed especially by HeLa cells at high levels , implying a high importance of amino acids derived from lysosomal protein degradation for their metabolism. This is in line with studies reporting the dependence of cancer cell metabolism on amino acids originating from lysosomal degradation of proteins . The Ragulator members LAMTOR1, LAMTOR2, LAMTOR3, and LAMTOR4 were similarly expressed in all cell lines, while LAMTOR5 was ∼100-fold more abundant in HEK293 compared with SH-SY5Y cells. For all proteins of this group, we observed, in general, high expression levels in HEK293, HeLa, and HuH-7 cells, with the exception of RHEB, which showed for HEK293 cells an inverse behavior compared with SLC38A9. In accordance with the role of FLCN in the mTORC1-dependent phosphorylation and cytosolic retention of the lysosomal transcription factors TFEB and TFE3  or TFEB/TFE3 (only MEFs) in the lysosome-enriched fractions.For other proteins related to patterns B. The Raand TFE3 , 55, we B) and the highest abundances for LAMP1 and LAMP2. Proteins involved in lysosomal positioning (mainly components of the BORC complex (ARL8B and DTNBP1 (C). For lysosome-associated ubiquitin ligases, on the other hand, no trend with respect to certain cell lines was detectable (D). For hydrolases, the dynamic range of expression was in general smaller than for proteins located at/in the lysosomal membrane, while proteins related to lipid metabolism (E) were less variable compared to proteases (F) and those involved in the hydrolysis of glycosidic bonds (C and S5G). For the latter, we observed, for example, a highly conserved abundance across cell lines for GBA, while other proteins, such as NEU1 or MAN2B2, presented with variabilities of up to ∼140-fold. For the majority of hydrolases, HuH-7 cells showed the highest expression levels, indicating a higher throughput of substrates in their lysosomes, which is probably related to the liver’s function as the main recycling organ of the body.For membrane and membrane-associated proteins, we observed a similar dynamic range as for transporters B and the complex ) were prd DTNBP1 C. For lytectable D. For hytabolism E were leroteases F and thoic bonds C and S5GThe observed differences in  protein abundance could originate from differential protein expression, differential association/localization with lysosomes, and/or variabilities in lysosome enrichment efficiency. We, therefore, further investigated lysosomal protein abundance in whole cell lysates from all six cell lines to investigate which of these possibilities are most likely . We utilIn general, we observed a similar range of dynamic protein expression within individual cell lines, spanning >3 orders of magnitude between the lowest and highest expressed lysosomal proteins. When considering variability of expression levels for individual proteins across the six cell lines, we observed less pronounced differences  and S6. A and B). This could be related to signal suppression effects for peptides originating from low abundant lysosomal proteins in highly complex samples  for transporters and exchangers, also proteins that were of higher relative abundance in whole cell lysates . We calculated the log2-transformed ratios of SPIONs/control, median-normalized the values for individual replicates to compensate for differences in lysosome enrichment efficiency, and performed a bimodal distribution analysis. For each cell line, we estimated the mixture of two univariate normal distributions and assigned a posterior probability of lysosomal localization to each protein using an expectation-maximization algorithm , we hypothesized that the frequency of identification of an individual protein in multiple cell lines could be used as an indicator for the likelihood of lysosomal localization. We tested this hypothesis by correlation of the percentage of proteins belonging to a certain GO category with their frequency of identification across the individual datasets of the six cell lines . At the same time, we were able to deplete proteins from the cytoskeleton, nucleus, and mitochondria, while the percentage of cytoplasmic and Golgi apparatus proteins remained stable. For proteins assigned to endosomes, we observed the highest increase (∼3-fold) after such of lysosomal origin, indicating a specific enrichment, probably due to SPIONs, which did not completely transfer to the lysosomal compartment during the chase period. The increase in percentage of lysosomal proteins relative to the whole dataset confirmed that true positive lysosomal proteins are more likely to be reproducibly detected across several cell lines.Despite the increase in abundance for putative lysosomal proteins, their percentage in relation to the whole dataset peaked at only 11% (SH-SY5Y cells). As we detected putative lysosomal proteins more reproducibly across the individual cell lines B, we hypNext, we compared our dataset with results from previously published studies that identified potentially novel lysosomal proteins with other approaches. We considered datasets based on proximity biotinylation with BirA∗ , densityC). Finally, we removed previously reported putative lysosomal proteins from the high confidence lysosomal identifications in our dataset, resulting in a list of potentially novel lysosomal proteins. With respect to the complete list of proteins identified in at least one of the cell lines, only 2% were reproducibly identified in ≥5 cell lines, presenting high confidence potential novel lysosomal proteins , which was shown to maintain protein homeostasis through attenuation of ER stress (SLC12A9 (Solute carrier family 12 member 9), which belongs to the SLC12 family of electroneutral transporters facilitating the symport of Na+/K+ with Cl- (SLC31A1 (High affinity copper uptake protein 1), which belongs to the SLC31 family of copper transporters (TMEM63B (CSC1-like protein 2), an osmosensitive Ca2+ permeable channel (TSPAN3 (Tetraspanin-3), a member of the tetraspanin superfamily, which has been involved in trafficking of membrane proteins (NDFIP2 (NEDD4 family-interacting protein 2), which was reported to be involved in controlling the activity of WW-HECT domain E3 ubiquitin ligases, in particular NEDD4  colocalized with LAMP2, thereby confirming their lysosomal localization. For NDFIP2, however, we only observed a partial colocalization with LAMP2 while colocalization with the early endosomal marker Rab5 was much more pronounced. Subsequently, we further generated stable NIH3T3 cell lines for TM7SF3, NDFIP2, SLC31A1, and SLC12A9 and investigated these by coimmunostaining and Western blotting (LAMP2 (A) with median Manders' coefficients between 0.7 and 0.8 and Pearson's coefficients between 0.6 and 0.8 (B), indicating their predominantly lysosomal localization. Based on these experiments, we were able to show lysosomal localization for all six candidates in cells either transiently or stably overexpressing the respective protein, presenting for four of them a first proof with a method orthogonal to large-scale proteomics studies.For each candidate, we transiently transfected HeLa cells with an expression vector containing an HA- or MYC-tagged version of the respective cDNA and investigated lysosomal localization by coimmunostaining with an antibody directed against the respective tag and the lysosomal marker protein LAMP2 . In thesblotting . For allg . Surprisingly, the V-ATPase-normalized iBAQ levels of both β-hexosaminidase subunits HEXA and HEXB (C) did not match the pattern of enzymatic activity for all cell types, as both proteins were significantly less abundant in MEF than in HeLa and HuH-7, which all showed similar enzymatic activity for whole cell lysates (B). This implies that MEF cells contain a higher number of lysosomes with lower individual HEXA/HEXB levels. This could be the reason that they performed best in terms of lysosome enrichment efficiency, as more lysosomes can receive SPIONs in these cells (C).This study presents a systematic comparison of lysosome-enriched fractions from different cell lines. Already during cell lysis and lysosome enrichment, differences between the individual cell types became apparent, as we observed varying yields of β-hexosaminidase activity and lysosomal intact ratios in the input fraction preceding lysosome enrichment, as well as different recovery rates in the eluate fraction , B and Cand HEXB C did not lysates B. This ise cells C.via Fenton reactions, which would result in increased levels of protein oxidation. Investigation of data published previously by our group , implying that no striking fundamental differences exist in the lysosomal composition between the cell lines. When we investigated protein identification across all cell lines, we detected a highly reproducible core proteome of 2173 proteins, of which 308 have been previously reported to be lysosomal  proteins . Interestingly, this observation was only partially replicated on a whole cell lysate level (A). While both subunits also presented with a high variability in this dataset, they were more abundant than others. This implies that, in comparison with other subunits of the complex, a lower percentage of the total pool of these proteins is located at lysosomes.In order to compensate for variability between samples, we performed a replicate-wise normalization on a set of V-ATPase subunits that were detected in all analyses of lysosome-enriched fractions with ≥10 peptides. Even though it was shown that the cytosolic V1 part of the V-ATPase is able to dissociate from the V0 part integrated in the lysosomal membrane , we did undances A. Interete level A. While BLOC-one-related complex (BORC), which is involved in microtubule-mediated lysosomal transport  and mTORC1 members (B) in the different cell lines. For individual members of Ragulator, it was quite surprising to us to find lysosome-localized LAMTOR5 at highly variable levels (∼100-fold dynamic range), clearly exceeding the dynamic range observed for the other subunits. It was shown that loss of LAMTOR2 induces degradation of the other LAMTOR proteins .For  surface , were prith BORC , 78. Alosubunits C and mTO members B in the proteins . While tCLCN7 and its β-subunit OSTM1, which facilitate Cl- import into lysosomes , indicating probable other roles for GLMP in certain cell types. Intriguingly, also on a whole cell proteome level we observed highly conserved levels of MFSD1 in all cell lines and high variability of GLMP (A and S6B). Finally, for the complex of the lysosomal cobalamin transporter ABCD4 and the lysosomal membrane protein LMBRD1, which was shown to facilitate the transport of ABCD4 from the ER to lysosomes (ABCD4 (5- to 26-fold) throughout all cell lines. This indicates that LMBRD1 may be important for the transport of ABCD4, but not for its stability or function, as a roughly similar abundance would have to be maintained in this case.For complexes consisting of lysosomal transmembrane transporters and their accessory subunits, we found roughly equal stoichiometries in lysosome-enriched fractions, confirming their direct relationship. This was, for example, the case for ysosomes . For theysosomes . For MFSnit GLMP , however of GLMP A and S6Bysosomes , we obseA possible reason for the observed heterogeneity among cell lines could be transcriptional control of lysosomal protein expression. This could be either due to the activity of TFEB and its regulation by different upstream kinases  or the aOverall, the iBAQ data from both lysosome-enriched samples and whole cell lysates should provide a valuable resource to the community. They present an estimate of abundance for lysosomal proteins in different cell types and allow to estimate the lysosome-localized population for individual proteins. This could allow, for example, to investigate lysosomal heterogeneity in response to certain stimuli or upon alteration of transcription factor activity. Also, for the conceptualization of experiments, this should allow to choose appropriate model systems, as overexpression of the same protein may result in one cell line in a 2-fold higher abundance, while it will result in a 200-fold increase in another. This applies especially to certain metabolic pathways in the individual cell lines, as, for example, we observed extremely high relative levels of most lysosomal enzymes in HuH-7 cells, while concentrations of channels, transporters, and exchangers were rather average.e.g., due to proteolytic processing, it does not strongly influence the iBAQ value if high intensity peptides from another region are present or if peptides from this region are not utilized at all. If, however, the epitope that is recognized by a specific antibody is affected, the ability of the antibody to detect the respective protein will be heavily impaired. The same is true for posttranslational modifications, such as glycosylation, which are known to be present at many lysosomal luminal protein domains  and 85% (lysosome-enriched samples) of individual trends matched. The remaining cases, in which we failed to observe a correlation, could be due to several reasons. For the iBAQ analyses, all peptides across the protein sequence are considered. If a certain region of a given protein cannot be covered for a distinct cell line,  domains , 89, or  domains , 91. It A). When combining the data of all six cell lines, almost 4500 proteins were categorized as potentially novel lysosomal, representing >20% of known human/mouse proteins.To facilitate the identification of previously unknown lysosomal proteins from our datasets, we applied a novel strategy based on the inclusion of differentially SILAC-labeled background cells (which did not receive SPIONs) and bimodal distribution analysis of the resulting samples. While we were able to significantly deplete unspecifically binding proteins from our datasets and to define a population that was specific for the presence of SPIONs , we wereFig. 7p-value cutoff, can be identified in the individual bimodal distribution analyses of the cell lines , and also for such that were previously identified to be present in lysosome-enriched fractions  with the dataset identifier PXD020600. MS/MS spectrum identifications from MaxQuant searches were deposited at MS-Viewer (https://msviewer.ucsf.edu/) with the search keys c0vxdeqfnr  and qwtwxcxfbm (combined mouse datasets from MEF and NIH3T3 cells).The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium he PRIDE  partner S-Viewer  and are This article contains The authors declare no competing interests.


SF3B1 mutation can help unravel their contribution in patients’ worse prognosis. The cytotoxic effects and delayed leukemic infiltration induced by H3B-8800 support the potential use of SF3B1 inhibitors as a novel therapy in CLL.Understanding the pathogenic mechanisms of  SF3B1 are observed in 15% of patients with chronic lymphocytic leukemia (CLL) and are associated with poor prognosis, but their pathogenic mechanisms remain poorly understood. Using deep RNA-sequencing data from 298 CLL tumor samples and isogenic SF3B1 WT and K700E-mutated CLL cell lines, we characterize targets and pre-mRNA sequence features associated with the selection of cryptic 3′ splice sites upon SF3B1 mutation, including an event in the MAP3K7 gene relevant for activation of NF-κB signaling. Using the H3B-8800 splicing modulator, we show, for the first time in CLL, cytotoxic effects in vitro in primary CLL samples and in SF3B1-mutated isogenic CLL cell lines, accompanied by major splicing changes and delayed leukemic infiltration in a CLL xenotransplant mouse model. H3B-8800 displayed preferential lethality towards SF3B1-mutated cells and synergism with the BCL2 inhibitor venetoclax, supporting the potential use of SF3B1 inhibitors as a novel therapeutic strategy in CLL.Splicing factor 3B subunit 1 (SF3B1) is involved in pre-mRNA branch site recognition and is the target of antitumor-splicing inhibitors. Mutations in  SF3B1 mutations identified in cancer cells promote the usage of cryptic 3’ss located 10–30 nucleotides upstream of the canonical 3’ss. Each 3’ss has been reported to use its own BP adenosine (SF3B1 mutations have been detected in various cancer types including myelodysplastic syndromes (in 28–65% of the cases)  is a core component of the spliceosome, the cellular machinery in charge of removing introns from precursor mRNAs (pre-mRNA splicing). The spliceosome recognizes specific sequences at the intron boundaries, namely, the 5′ splice site (5’ss) and the 3′ splice site (3’ss), the latter comprising a branch point (BP) adenosine located 18–35 nucleotides (nt) upstream of the conserved AG dinucleotide at 3′ end of the intron and a polypyrimidine tract (Py tract) located between the BP and AG. Alternative usage of splice sites, namely, alternative splicing (AS), importantly contributes to protein diversity . SF3B1 rdenosine . SF3B1 me cases) , chronice cases) , uveal me cases) , and othe cases)  and havee cases) . In CLL,reatment , and a hreatment . SF3B1 mrognosis . Patientrognosis . Even parognosis , emphasiInterestingly, several antitumor drugs targeting SF3B1 have been identified and developed in the last decade . Among tSF3B1 mutations in CLL patients and in an isogenic CLL MEC1 cell line bearing the most frequent SF3B1 cancer mutation (K700E). We also characterize the cytotoxic effect of H3B-8800 both in vitro and in vivo and we explore a novel therapeutic approach combining H3B-8800 with venetoclax, a commonly used drug in CLL treatment.Here, we characterize the changes in alternative splicing induced by SF3B1 mutations in CLL, we developed the first isogenic MEC1 cell lines harboring (or not) the most prevalent SF3B1 mutation in CLL, K700E. MEC1 cells were edited using CRISPR/Cas9 and single-stranded oligodeoxynucleotides (ssODN) harboring two stabilizing phosphorothioate linkages at their 5′ and 3′ ends SF3B1 mut 3′ ends , validat 3′ ends  and digi 3′ ends , confirmmutation . Of releT clones .SF3B1 mutation on splicing in CLL, we first used RNA-seq data from tumor B cells from 298 patients diagnosed with CLL from the International Cancer Genome Consortium (ICGC) (vast-tools (SF3B1 mutation cancer cell fraction (CCF)  formed a different cluster, with the exception of two samples harboring SF3B1 mutations outside of the domain involved in branch point recognition  with high CCF (≥50%), excluding samples with mutations E862K and M757T in SF3B1, in other splicing factors or in other genes related to RNA metabolism . PCA and hierarchical clustering in SF3B1-mutated samples did not reveal global transcriptomic changes associated with specific mutations, although the comparison between 8 SF3B1 K700E samples and 3 SF3B1 H662D mutations did identify 136 differential alternative splicing events , and 162 differentially expressed genes, suggesting specific molecular effects of individual mutations (To investigate the impact of m (ICGC)  and B cest-tools  and the st-tools  Fig 2A)SF3B1 muton (CCF) . Althoug samples . In addi samples . PCA of l status . For furBiological and clinical information of the patients from the International Cancer Genome Consortium with RNA-seq data.Table S1. SF3B1K700E and SF3B1H662D samples from the patients’ cohort (vast-tools).Differentially spliced events between Table S2. SF3B1K700E and SF3B1H662D samples from the patients’ cohort.Differentially expressed genes between Table S3. SF3B1 mutations, additional RNA-seq analyses included RNAs from MEC1 SF3B1K700E heterozygous and WT isogenic cell lines described above. The fraction of differentially spliced events  between SF3B1 WT and SF3B1-mutant samples was enriched in Alt3’ss events both in CLL samples or MEC1 isogenic cell lines compared with the distribution of detected AS events (P < 0.0001)  . Both inormation .SF3B1WT and SF3B1-mutant samples from the patients’ cohort (vast-tools).Differentially spliced events between Table S4. SF3B1WT and SF3B1K700E MEC1 cell lines (vast-tools).Differentially spliced events between Table S5. SF3B1-mutated cases) splicing events, we developed JuncExplorer, a software designed to detect and quantify the usage of annotated and non-annotated splice junctions . Using this tool, events with a single pair of 3’ss choices (compared with events involving 3 or more 3’ss choices) were the most frequently mis-regulated type of Alt3’ss between SF3B1WT and mutated samples, both in patients and in cell lines,  in SF3B1WT samples was >5  or ≤5 (cryptic Alt3’ss), respectively. A change in 3’ss usage |ΔPSI| ≥15 upon SF3B1 mutation was required for classification as either alternative or cryptic Alt3’ss. We defined a total of 595 alternative Alt3’ss events and 268 cryptic Alt3’ss events in cell lines and 38 alternative Alt3’ss and 15 cryptic Alt3’ss in patients’ samples. Although most non-canonical AGs were located upstream and near of the canonical 3’ss   and cryptic  . Activateam Far)  represen0E cells  to analy0E cells . Alterna0E cells  and S4A.achinery . In summSF3B1WT and SF3B1-mutant samples from the patients’ cohort (Juncexplorer).Differentially spliced events between Table S6. SF3B1WT and SF3B1K700E MEC1 cell lines (JuncExplorer).Differentially spliced events between Table S7. SF3B1WT and SF3B1-mutated chronic lymphocytic leukemia samples with a single pair 3’ss plotted in Fig 4A (JuncExplorer).Differentially spliced events between Table S8. SF3B1WT and SF3B1K700E MEC1 cell lines with a single pair 3’ss plotted in Fig 4A (JuncExplorer).Differentially spliced events between Table S9. SF3B1WT and SF3B1K700E cells ordered depending on the Alt3’ss location.Alt3’ss events validated by RT–PCR in RNA samples from MEC1 Table S10. SF3B1 mutation requires the use of alternative branch point sequences. To investigate this question, we carried out mutagenesis analyses using expression plasmids (minigenes) in which the 3’ss region of an Adenovirus Major Late intron was replaced by 3’ss regions of ZNF561 , MAP3K7  or TARBP1 (displaying a NAGNAG event)  led to activation of the non-canonical 3’ss in SF3B1WT cells, whereas mutation of the 3’-most BP (BP1 mutant) prevented activation of the non-canonical 3’ss in SF3B1K700E cells  reduced the use of the non-canonical 3’ss in both cell types, whereas mutation of the 5’-most BP (BP2 mutant) did not significantly alter the choice of 3’ss in either of the two cell lines. Additional mutation of other putative branch sites were included leading to a further decrease in the usage of the alternative AG in both cell types, without affecting canonical AG usage  prevented its increased relative use in SF3B1K700E cells, whereas the 5’-most BP does not seem to have an impact on NAGNAG use ) with a ΔPSI = 50.93 showed also the lowest average PSI (2.87) in SF3B1WT patient samples . This Alt3’ss event was validated by RT-qPCR , the cryptic ZDHHC16 isoform was only detected in mutated samples with CCF >12 (except for cases when SF3B1 was mutated in E862K or M757T) and three samples with CCF < 12  involved in the activation of MAPKK were differentially spliced in SF3B1-mutated patients’ samples (Table S12). Recently, MAP3K7 AS has been postulated to promote NF-κB–driven tumorigenesis (MAP3K7 gene itself displaying strong mis-regulation upon SF3F1 mutation (ΔPSI = 60 in SF3B1WT versus SF3B1K700E CLL patients’ samples and ΔPSI = 70 in SF3B1WT versus SF3B1K700E MEC1 CLL cell lines). This 3’ss is located upstream of the canonical 3’ss of exon 5, which encodes part of the kinase domain, and its usage results in the disruption of the coding sequence and degradation of the corresponding transcript by NMD   . Measureificance .SF3B1-mutated chronic lymphocytic leukemia patient samples and SF3B1K700E MEC1 cell lines.Selected enriched Gene Ontology terms (enrichGO R package) for biological processes in genes displaying differentially spliced events in Table S12. SF3B1K700E and SF3B1WT MEC1 CLL cell lines. Cytotoxicity was measured after incubation of these cell lines with 1–100 nM H3B-8800 for 48 h. H3B-8800 induced cytotoxicity in a dose-dependent manner in both SF3B1WT and SF3B1K700E cell lines, this effect being significantly enhanced in SF3B1K700E cells at doses higher than 25 nM (mean viability at 75 nM was 52.1% ± 12.3% for SF3B1K700E cells and of 71.8% ± 10.8% for SF3B1WT cells) (We first assessed the cytotoxic effect of the splicing modulator H3B-8800 in the isogenic T cells) .SF3B1 mutations in samples with low SF3B1-mutant CCF  and in SF3B1wt CLL cells (81% ± 11.9%)  or human follicular dendritic cell-like (HK) cell lines. We observed that in the presence of HS-5 or HK cells, H3B-8800 induced lethality in both ry cells  and MEC1ry cells  with a pll lines . These dSF3B1K700E and SF3B1WT cell lines. Cells were intravenously inoculated via the tail in NOD–SCID interleukin-2 receptor gamma (IL2Rγ)null (NSG) mice, forming xenografts of isogenic MEC1 cells with SF3B1WT or SF3B1K700E . After 10 d, upon detection of bioluminescence, mice began to be treated orally with vehicle or H3B-8800 (6 mg/Kg) daily for 10 d and luciferase activity was captured at days 3, 6, and 10 by bioluminescence in vivo imaging. We observed lower bioluminescence signal or leukemic infiltration in SF3B1K700E MEC1 cell line compared with SF3B1WT MEC1 cell line and a decrease of this signal after H3B-8800 treatment in both of them (+ B lymphocytes by flow cytometry) were 54% ± 13.9% and 41% ± 17.4% for the SF3B1WT and SF3B1K700E groups treated with vehicle, respectively  for 6 h. RNA was isolated and deep sequenced using Illumina pair-ended sequencing technology and the datasets analyzed using vast-tools software. We observed that H3B-8800 induced extensive intron retention and exon skipping changes in both cell lines, with nearly 8,000 introns detected as more retained and more than 10,000 exons as more skipped upon treatment  with introns that were spliced more efficiently (n = 62) and with introns that did not change (n = 675) upon drug treatment. We observed that (i) retained introns and their flanking exons are shorter than the ones from non-changing introns, (ii) their 5’ss and 3’ss are weaker , and ii. A repreSF3B1-mutated cells to H3B-8800 might be explained by synergistic or antagonistic effects of drug treatment and SF3B1 mutation on particular splicing alterations. Out of 252 events associated with SF3B1 mutation, 56 were reversed by H3B-8800 treatment, whereas 68 were exacerbated (|ΔPSI| > 10)  . For exaSF3B1K700E and H3B-8800–treated SF3B1K700E cells  (SF3B1WT (ΔPSI = −37.6) and SF3B1K700E (ΔPSI = −34) cell lines (SF3B1WT or SF3B1K700E MEC1 cell lines with venetoclax (0–250 nM), a selective inhibitor of the anti-apoptotic protein BCL2, and H3B-8800 (0–75 nM) for 48 h. The results showed synergistic effects between the two drugs in the two cell lines . Altogether, these results demonstrate that the combination of H3B-8800 with venetoclax results in enhanced cytotoxic effect.RNA-seq analysis of H3B-8800–treated MEC1 cell lines identified 26 AS events in genes involved in apoptosis that were differentially spliced (|ΔPSI| ≥ 15) between 0E cells . 23/26 oclature)  which swclature)  and has clature) . H3B-880ll lines . Similarll lines . As BCL2ll lines , we incull lines . These dsoftware  Fig 9F)SF3B1K700SF3B1 is one of the most recurrently mutated genes, present in about 15% of CLL patients. These mutations—typically subclonal and subject to clonal evolution—have been linked to specific biological features of the disease such as unmutated imumoglobulin (Ig) heavy-chain variable region (IGHV) CLL subgroup, the intermediate CLL epigenetic subgroup, stereotyped BCR subset 2, R110-mutated IGLV3-21 subgroup, or 11q deletion  SF3B1 mutation using CRISPR/Cas9-genome editing. Although several SF3B1-mutant isogenic cell lines were previously generated from B-cell precursor leukemia cells, myeloid leukemia cells, murine embryonic stem cells, or induced pluripotent stem cells  and the distance (≥/<50 nt) to the canonical 3’ss. Interestingly, we observed, both in CLL primary cells and CLL cell lines that cryptic 3’ss can be located not only upstream but also downstream of the canonical 3’ss and that the distance between the two 3’ss, both in upstream and downstream cases, can be longer than 50 nucleotides and therefore is not as constrained as previously thought.Because the first reports of n cancer , importan cancer  and—lessn cancer  or closen cancer . Our anaK700E. The effects of mutating predicted branch points in minigene constructs were consistent with the use of different branch points by the alternative 3’ss and the canonical 3’ss, as previously described , aberrant expression of an alternatively spliced isoform of TMEM14C identified patients with good responses  and isogenic SF3B1K700E MEC1 cell lines as compared with WT cells. The cytotoxic effect of H3B-8800 was observed even in the presence of protective stroma. Other splicing inhibitors such as sudemycin D6 or E7107 also display stronger cytotoxic effects on splicing factor–mutant cancer cells, including mutations in SF3B1 and other splicing factors such as U2AF1  are dependent on their WT allele for splicing function and cell survival  splicing modulation . Also, cDifferent SF3B-binding molecules display common and differential effects on splicing regulation . In geneSF3B1 mutation compared with previous reports, (3) provides evidence that the SF3B1 inhibitor H3B-8800 displays therapeutic effects in mouse models of CLL, particularly when used on CLL cells harboring SF3B1 mutations, and (4) provides evidence of synergism between a SF3B1-targeting drug and venetoclax.In summary, the work presented here reports (1) the first generation of isogenic genome–edited CLL cell lines differing only by the presence or absence of a single amino acid substitution in SF3B1, (2) the identification of a wider variety of 3′ splice site activation events upon SF3B1 mutations in PBMCs were analyzed in previous sequencing studies (5 cells/ml in IMDM with GlutaMAX (GIBCO) supplemented with 10% (vol/vol) heat-inactivated FBS and 1% (vol/vol) penicillin–streptomycin 10,000 U/ml (GIBCO) in a humidified atmosphere at 37°C containing 5% carbon dioxide. The follicular dendritic cell line HK, kindly provided by Dr. Yong Sung Choi  (PBMCs from CLL patients (with ≥90% tumor B cells) and healthy donors were isolated, cryopreserved, and stored within the hematopathology collection registered at the Biobank . Clinical and biological data of each patient are detailed in Table S1. Written informed consent was obtained from all patients. The study was approved by the Hospital Clínic of Barcelona Ethics Committee.  studies . MEC1 (Andation) , was culSF3B1K700E MEC1 cell line are shown in Table S14. Annealing of crRNA and Alt-R CRISPR–Cas9 tracrRNA, ATTO 550 (IDT) was carried out at 95°C for 5 min, then the assembly of the crRNA:tracRNA duplex (10 μM) and the Cas9 protein (10 μM) was performed and mixed with ssODN (10 μM) and Alt-R Cas9 Electroporation Enhancer (IDT). MEC1 cell lines were electroporated using the Neon Transfection System (Thermo Fisher Scientific) with a 20 ms pulse at 1,600 V and seeded. Serial dilution in single-cell cultures was performed for 3 wk and then clones were screened using Taqα1 restriction enzyme, recognizing the c.2106G>A from the PAM sequence (6) or QIAamp DNA Mini Kit (>1 × 106) (QIAGEN); 50 ng DNA was used to quantify SF3B1 K700E mutation by digital PCR (Bio-Rad).ssODN complementary to the non-target strand  with twosequence . 30,000 SF3B1K700E MEC1 cell line.Oligonucleotide sequences used for the generation and characterization of Table S14. Cell viability was measured by flow cytometry using Annexin V (FITC) and propidium iodide (Invitrogen). For the MTT assay, 10,000 cells/well were seeded and incubated for 48 h. Then, thiazolyl blue tetrazolium bromide 98% at a concentration of 0.5 mg/ml in PBS was added to the cells and were incubated for 2 h until the presence of violet crystal was observed at the optical microscope (Leica DMI1). The stop solution (isopropanol and HCl 24:1) was added and the cells were carefully resuspended before reading the plates at the TECAN spectrophotometer at wavelengths of 570 and 670 nm.SF3B1WT or SF3B1K700E MEC1 cell lines were treated with 75 nM H3B-8800 or DMSO for 6 h, and RNA extraction was performed using the RNeasy Mini kit (QIAGEN). Stranded mRNA–seq libraries were prepared and two samples/lane were sequenced, following 2 × 125 nt paired-ended protocol at the CRG Genomics Core Facility on a HiSeq 2500 v4 (Illumina Inc). Triplicates were sequenced for each condition in separate lines. All the samples included in the comparison passed the FastQC quality control from MultiQC  (https://github.com/vastgroup/vast-tools). For each event, minimum 10 reads coverage was required.Splicing analyses were carried out using the toolset Vertebrate Alternative Splicing and Transcription Tools   with high CCF (≥50%) were analyzed, excluding samples with mutations E862K and M757T in SF3B1, in other splicing factors or in other RNA metabolism–related genes . We made two comparisons considering the distribution of the patients in PC1 for all the AS events detected (SF3B1-mutated samples versus 136 samples SF3B1WT samples) and the samples with PC1 < 0 (7 SF3B1-mutated samples versus 89 samples SF3B1WT samples). AS events needed to have at least 10 reads in 5 of SF3B1-mutated samples and 20 of the SF3B1WT samples to take them into consideration for the comparison and a |ΔPSI| higher than 15% and range of 5 between SF3B1 mutated and SF3B1WT samples. To detect differentially spliced events between two conditions (SF3B1 mutation or H3B-8800 treatment), we required a |ΔPSI| of at least 15% between the merged replicates under each condition and a minimum range of 5 between the PSI values under the two conditions in MEC1 cell lines.225 detected  because detected . We compJuncExplorer (https://gitlab.com/aghr/juncexplorer). JuncExplorer allows the identification and quantification of annotated and non-annotated (de-novo) splice junctions from RNA-seq reads mapped with a splice-aware mapper, for example, STAR . PSI values are calculated by converting the read numbers into relative frequencies per case for each of its splice sites for each of the 5’ and 3’ss. Alt 3’ss cases were considered when a unique GT donor site (5’) had several AG acceptor sites (3’) within a single intron. To detect differentially spliced events between two conditions (SF3B1 mutation or H3B-8800 treatment), we required a |ΔPSI| of at least 15% and range of 5 between the merged replicates under each condition. Cryptic 3’ss correspond to alternative 3’ss not detected in SF3B1 WT samples and detected in SF3B1-mutated samples. Downstream analysis of AS events was performed using Matt (https://gitlab.com/aghr/matt)   with spehr/matt) , and BP hr/matt) .SF3B1WT cells, Matt software (SF3B1WT control group) upon drug treatment.For the sequence features analysis associated with AS changes induced by the drug in software  was usedsoftware . The rethttps://vastdb.crg.eu/wiki/Downloads) (Prediction of impact on protein sequence was obtained from VastDB version 3 (wnloads) . First, ggplot and ggpubr packages in R (v3.6.3).Pearson coefficient correlation plots were obtained with Cutadapt version 2.4 discarding all reads with length shorter than 15. The adapter-trimmed reads were mapped with STAR 2.7.1.a (STAR (--quantMode GeneCounts) using uniquely mapping reads only. DESeq2 version 1.14.1 . Statistical significance was defined with adjusted P-value < 0.05 and the positive false discovery rate with Q-value < 0.05.Differentially expressed genes or genes bearing differentially spliced (|ΔPSI| ≥ 15 and range of 5) AS events  between SF3B1WT and SF3B1K700E MEC1 cell lines were performed using Maxwell simplyRNA kit (Promega), Illustra RNAspin Mini RNA Isolation Kit , or RNeasy Mini kit (QIAGEN), following the manufacturers’ instructions. 50 ng of total RNA were reverse transcribed with SuperScript III (Invitrogen), and PCR reactions were carried out using GoTaq enzyme (Promega). PCR products were separated by electrophoresis on 6% acrylamide gels, analyzed with GelDocTM XR+ (Bio-Rad) and quantified with ImageJ v1.53a. Primers used are listed in Table S15. TARBP1 PCR products were analyzed by fragment analysis in an ABI 3130 Genetic Analyzer (Thermo Fisher Scientific) using GeneMapper software v6.0 (Thermo Fisher Scientific).RNA extraction and DNAse treatment in Primers used for the validation of the different Alt3’ss patterns.Table S15. RPLPO as an endogenous housekeeping gene. mRNA expression levels were provided as arbitrary quantitative PCR units, taking as a calibrator the median of the WT samples. In the case of ZDHHC16 cryptic isoform, which was not detected in the WT samples, the median of the SF3B1-mutated samples was taken as reference.Total RNA from CLL primary samples was extracted using the TRIzol reagent (Invitrogen), and 1 μg of total RNA was then reverse transcribed to cDNA. Aberrant and canonical isoforms of MAP3K7 and ZDHHC16 were quantified in StepOnePlus Real-Time PCR System (Applied Biosystems) using specific primers (Table S16). The relative expression of each gene was analyzed by the comparative cycle threshold (Ct) method (ΔΔCt) using Primers used for the qPCR validations.Table S16. pCMV 57 Δi AdML plasmid harboring two exons and an intron from the adenovirus major late promoter transcript, flanked by PT1 and PT2 sequences that allow detection of transcripts from the expression vector  by RT–PCSF3B1WT and SF3B1K700E cell lines were transfected with 50 ng of minigene DNA plasmids and Alt-R Cas9 Electroporation Enhancer (IDT) using Neon Transfection System (Thermo Fisher Scientific) with a 20 ms pulse at 1,600 V. 24 h later, cells were collected for RNA extraction and validated by RT–PCR. The PCR products from the TARBP1 gene were analyzed by fragment analysis as described above. PCR products of other minigenes were separated by electrophoresis on standard native polyacrylamide gels as described above.MEC1 6) were lysed for 30 min in Tris–Triton-X Lysis buffer  supplemented with protease and phosphatase inhibitors (Sigma-Aldrich). Lysates were centrifuged at 15,000g for 15 min at 4°C, and protein concentration assessed by Bradford assay (Bio-Rad). Total extracts were separated by 10% SDS–PAGE and transferred to an Immobilon-P PVDF membrane (Merck Millipore). Primary and secondary antibodies used for Western blot analyses are shown in Table S17. Chemiluminescence was detected by using ECL substrate (Thermo Fisher Scientific) or Amersham ECL Select Western Blotting Detection Reagent (Cytiva) on a mini-LAS4000 Fujifilm device  and quantified by Multi Gauge software (FUJIFILM).Cells  in nuclear extracts obtained using nuclear extract kit as recommended by the manufacturers.+ (BD) positivity. Synergism between H3B-8800 and venetoclax was calculated using SynergyFinder 2.0  and incubated at different concentrations for 48 h with H3B-8800 (kindly provided by H3B Biomedicine) and/or venetoclax . Cytotoxicity was measured by Annexin V (Pacific blue/FITC) and PI-mediated (Invitrogen) and flow cytometry (Attune Acoustic Focusing Cytometer from Applied Biosystems for primary cells and BD LSRFortessa Flow Cytometer for the cell lines). For co-culture experiments, HS-5 and HK cells were seeded on day 1 at a density of 20,000 cell/well and 10,000 cells/well, respectively. On day 2, 20,000 MEC1 cells/well or 200,000 primary cell/well were added and incubated with 50–75 nM H3B-8800 for 48 h. Cytotoxicity was assessed by flow cytometry as described above, after selection of MEC1 cells by CD19nder 2.0 .HEK 293T cells were transfected using jetPEI with pLV430G-oFL-T2A-eGFP, a lentivirus expressing the luciferase gene cloned in an EGFP-expressing vector , and viral stocks were collected from the culture supernatant 48 h post-transfection. MEC1 cell lines were infected, and GFP-positive cells were sorted by flow cytometry 7 d afterwards.SF3B1K700E (in 17 mice) or GFP–SF3B1K700K (in 16 mice) MEC1 CLL cell lines were injected intravenously on 6–8 wk old NOD-SCID interleukin-2 receptor gamma (IL2Rγ)null (NSG) mice. Leukemic infiltration was followed by bioluminescence using a fluorescence/luminescence imaging system AEQUORIA (Hamamatsu Photonics K.K.). 10 d after the injection, once leukemic infiltration was detected by bioluminiscence, H3B-8800 (6 mg/Kg) or vehicle control  was administrated orally and daily for 10 d until the mice were euthanized 24 h after receiving the last dose. The experimental design was approved by the Animal Testing Ethic Committee of the University of Barcelona and Generalitat de Catalunya . Peripheral blood was extracted from the submaxillary vein. Bone marrow was obtained from femurs by cutting the neck and next to the condyles, and then injecting IMDM with a syringe and a 25G needle to facilitate bone marrow extraction. Peripheral blood, bone marrow, spleen, and liver samples were first processed with ACK lysing buffer (Lonza), and then, leukemic infiltration was measured using CD19+ (Invitrogen) to detect the tumoral B lymphocytes in a BD LSRFortessa Flow Cytometer. Mouse spleens were fixed in 10% formaldehyde, dehydrated by ethanol after 24 h, and embedded in dissolved paraffin. The specimens were finally stained by hematoxylin–eosin (HE) and CD79a (Roche) immunohistochemical staining and then analyzed in an Olympus BX53 optical microscope.10 millions of GFP–Statistical tests were performed as indicated in figure legends with R (v3.6.3) or GraphPad Prism (v8.4.0).SF3B1WT and SF3B1K700E MEC1 cell lines treated with H3B-8800 are available in the following database: Gene Expression Omnibus GSE190087. RNA-seq data from patient samples were accessed from the previously available (http://www.ebi.ac.uk/ega/), which is hosted at the European Bioinformatics Institute (EBI), under accession number EGAS00000000092.RNA-seq data from vailable  database