Caenorhabditis elegans. By genetically inducing changes in the H3.3 distribution, we demonstrate that both H3.3K27M and pre-existing H3K27me3 act locally and antagonistically on Polycomb Repressive Complex 2 (PRC2) in a concentration-dependent manner. The heterochromatin changes result in extensive gene misregulation, and genetic screening identified upregulation of JNK as an underlying cause of the germcell aberrations. Moreover, JNK inhibition suppresses the replicative fate in human tumor-derived H3.3K27M cells, thus establishing C. elegans as a powerful model for the identification of potential drug targets for treatment of H3.3K27M tumors.Substitution of lysine 27 with methionine in histone H3.3 is a recently discovered driver mutation of pediatric high-grade gliomas. Mutant cells show decreased levels and altered distribution of H3K27 trimethylation (H3K27me3). How these chromatin changes are established genome-wide and lead to tumorigenesis remains unclear. Here we show that H3.3K27M-mediated alterations in H3K27me3 distribution result in ectopic DNA replication and cell cycle progression of germ cells in  Caenorhabditis elegans, through JNK pathway misregulation.Substitution of lysine 27 with methionine in histone H3.3 (H3.3K27M) is a driver mutation of pediatric high-grade gliomas. Here the authors show that H3.3K27M-mediated alterations in H3K27me3 distribution result in ectopic DNA replication and cell cycle progression of germ cells in  Histone H3.3 is a major variant of histone H3 that is mainly associated with regions of high nucleosome turnover3, and loss of H3.3 results in severe sterility or lethality phenotypes in most organisms5. H3.3 is highly conserved in plants, animals and fungi and is distinguished from canonical H3 by only a few key amino acids that are important for the association with the H3.3-specific histone chaperones HIRA and DAXX8.Establishing specific chromatin landscapes to regulate access to the genetic material is an effective and dynamic mechanism to control cell fate and preserve cell identity. Nucleosomes are the basic structural and functional units of this chromatin regulation. They consist of an octameric core of histone proteins that provide a structural scaffold for the organization of DNA. In addition, histones can carry epigenetic information, either through post-translational modifications (PTMs) of the N-terminal tails or through the incorporation of histone variants. These chromatin signatures are indispensable for many crucial cellular processes like DNA replication, transcription, cell division or differentiation9 and some cases of acute myeloid leukemia10. In tumor cells carrying this oncohistone, trimethylation of lysine 27 on histone H3 (H3K27me3) is strongly and globally depleted from chromatin13.Despite the importance for chromatin biology, only a few mutations in histone genes are directly associated with specific diseases. One notable example is the recently discovered replacement of lysine 27 with methionine in histone H3 or, more commonly, H3.3 that acts as a driver mutation of specific types of pediatric diffuse intrinsic pontine gliomas (DIPGs)14. H3K27 methylation is deposited, recognized and propagated by the Polycomb repressive complex 2 (PRC2), a multi-protein complex responsible for maintaining the silent state of the genes during development and cell differentiation16. PRC2-mediated H3K27me3 propagation is the result of dynamic interactions between the PRC2 complex and pre-existing H3K27me3, which allosterically activates PRC2 and facilitates the spreading of the mark to neighboring nucleosomes21.H3K27me3 is a mark characteristic for facultative heterochromatin, and is associated with transcriptionally repressed regions22. Sequestration of PRC2 can explain how H3.3K27M, present only in a small fraction of all nucleosomes, acts as a dominant negative factor to affect H3K27me3 levels genome-wide. However, retention of the mark in some genomic regions of mutant cells is inconsistent with the hypothesis that PRC2 is immobilized by H3.3K27M-containing nucleosomes, and instead suggests that part of the PRC2 pool remains active to maintain H3K27me3 levels at some PRC2 targets12. Moreover, ChIP-seq analysis showed that PRC2 is excluded from, rather than immobilized on H3.3K27M-containing nucleosomes, and residual PRC2 activity is one of the factors promoting tumorigenesis23. Regardless of the exact mechanism of PRC2 inhibition, several studies revealed H3.3K27M-induced changes of H3K27me3 levels at gene promoters and enhancers, showing that active promoters, which are enriched in H3.3 incorporation and H3K27ac, tend to lose H3K27me3 while poised enhancers tend to gain the mark24. Moreover, these studies have led to a detailed understanding of how H3K27 methylation states affect gene expression in tumor cells. However, several key aspects of how H3.3K27M mutation leads to the formation of tumors are still not understood25. First, a precise understanding of how oncohistone incorporation remodels the H3K27me3 landscape genome-wide is lacking. Second, a causal link between the chromatin changes and the cancerous cell fate at a transcriptional level has not been established. And third, the cell-type specificity of H3.3K27M-driven tumorigenesis still remains to be elucidated. The available model systems to study consequences of the H3.3K27M mutation either do not result in a replicative fate or require additional mutations  to induce tumorigenesis29.The observed depletion of H3K27me3 in H3.3K27M tumor cells was initially explained by an increased affinity of PRC2 to the K27M-containing H3.3, resulting in PRC2 trapping on the nucleosomes containing this oncohistoneC. elegans. Mutant worms display ectopic DNA replication and cell-cycle progression in the germline. We thus provide an animal model with a tumor-like phenotype induced by the oncohistone that does not require secondary mutations. Using this model system, we establish that PRC2 activity in mutant cells is affected both by H3.3K27M incorporation and the pre-existing levels of H3K27me3 in a concentration-dependent manner. Moreover, we identify the JNK pathway as an important link between the global reorganization of heterochromatin and the replicative cell fate in both nematodes and human tumor-derived cell lines.To overcome these limitations and to investigate these key outstanding questions, we introduced the H3.3K27M mutation into his-72, one of five C. elegans H3.3 genes, which is ubiquitously expressed and non-essential30. His-72/H3.3 transcript levels are 50 times lower than canonical H3 transcript levels, implying that only a fraction of all nucleosomes incorporates this H3.3 protein31. Worms carrying the H3.3K27M mutation show normal somatic development, but display almost fully penetrant sterility at 25 °C, indicative of a germ-line defect , probing for H3K27me3 enrichment and oncohistone occupancy. As previously reported for mammalian oncohistones, the K27M mutation alone does not alter the H3.3 distribution pattern Fig. 13. H3.3 ome Fig. , consistUpon changing the chaperone recognition motif from AAIG to SAVM, the resulting H3-like oncohistone shows an incorporation pattern with domains similar to H3.3, but these domains widen and spread into regions not normally occupied by H3.3 Fig. . Importa28. To test this model, we also analyzed the H3K27me3 levels in the H3-like K27M mutant, where oncohistone incorporation is increased in the domains that normally don’t contain H3.3 and on the X chromosome (domains i and iv) , and remains high in domains with low oncohistone incorporation (domains i and iv) Fig. . These oiv) Fig. . Surprisiv) Fig. , suggest20.To test whether H3K27me3 in the mutants indeed depends on both oncohistone incorporation and pre-existing (wild type) H3K27me3 levels, we performed hierarchical clustering of the ChIP-seq signal in 10 kb bins covering the entire genome. For both oncohistone mutants, the clustering was performed on three datasets: wild-type (pre-oncohistone mutation) H3K27me3 levels, oncohistone levels, and H3K27me3 levels upon oncohistone incorporation. Analysis of the H3.3K27M mutant data confirmed that oncohistone incorporation and PRC2 activity are largely antagonistic  kinase signaling or cell cycle control, and misregulation of some of them, like tumor suppressor Wilms Tumor 1, were shown to promote proliferation of the tumor-derived cells24. However, a clear understanding of how changes in gene expression result in tumorigenesis is still lacking, especially since many of the animal models require secondary mutation for the activation of cell cycle programs. The C. elegans model presented here contains the H3.3K27M mutation in an otherwise wild-type background, thus potentially allowing us to link the effect of the mutation to specific pathways that drive the cells towards their aberrant fate. We identified about 500 genes with changes in expression upon acquisition of the H3.3K27M mutation  transcription factor FOS-144, which in turn controls oocyte ovulation via IP3 signaling45. Misregulation of this regulatory cascade was shown to result in disruption of the Ca2+ homeostasis in the germ line and in an oocyte endomitosis phenotype similar to the one observed in H3.3K27M worms45.To overcome this limitation and identify key genes involved in aberrant oocyte replication within these pathways in an unbiased way, we performed a random mutagenesis screen for genetic suppressors of H3.3K27M-induced sterility Fig. . We isol2+ levels using the indicator dye Calcium Green-1 dextran, injected into the worm gonads. We found that H3.3K27M mutant worms showed elevated levels of Ca2+ in the germ line compared with wild type. This defect was partially rescued by the KGB-1S287N mutation, confirming that the replicative phenotype observed in these mutants correlates with aberrant Ca2+ signaling, a known downstream effector of the JNK pathway , a suppressor of neuronal differentiation upon JNK-dependent induction69. Platelet-derived growth factor receptor alpha (PDGFRA), which is often amplified in the K27M-derived tumors, is one of the upstream regulators of JNK70. Finally, the causative relationship between aberrant JNK activity and replicative fate has been established in many types of gliomas51. We found that inhibition of JNK suppresses proliferation of cells derived from K27M tumors more strongly than cells derived from non-K27M gliomas  was used as wild type. A list of strains used in this study, including the numbers of alleles generated, is given in Supplementary Table his-72, which is expressed in all cells at all developmental stages both in the soma and in the germ line, throughout the paper34. All genome edits were generated at the endogenous loci of his-72 and kgb-1 using CRISPR/Cas-9 as described in71. Briefly, sgRNAs and Cas9 were delivered as plasmids, and repair template as single strand oligonucleotides. To change the chaperone specificity for HIS-72, the H3.3-specific motif AAIG was replaced with the H3-specific motif SAVM. sgRNAs and repair templates, as well as PCR primers used to detect and sequence the mutations, are listed in Supplementary Table t-test. For sterility in context of RNAi, both F1 mothers and scored F2 progeny were grown on bacteria expressing the desired RNAi constructs. For sterility in context of JNK inhibition, both F1 mothers and scored F2 progeny were grown on OP-50 plates containing 50 µM JNK inhibitor SP600125 . Boxplots were drawn using default parameters in R, in these and all subsequent quantifications.For sterility levels at 20 °C, 100 L4 larvae were singled, and plates were scored for presence or absence of progeny after 3 days. For sterility levels at 25 °C, L4 larvae were shifted to 25 °C, and 100 of their F1 progeny were singled as L3 larvae (before any germ-line phenotype is visible) and kept at 25 °C. F1 worms with at least one viable F2 offspring were categorized as fertile. To determine the cause of sterility , the 100 single worms were examined by light microscopy. Sterility levels comparisons were performed in at least three biological replicates, and different strains/conditions were compared using student’s 2, 10 mM EGTA pH 7.5, 0.1% NaN3). For antibody stainings, gonads were fixed in methanol for 20 min and acetone for 10 min at −20 °C. Slides were washed three times with PBS for 5 min and incubated with H3K4me3 antibody , H3K27me2me3 antibody , OLLAS antibody , H3 phosphoS10 antibody , POLD2 antibody , or RAD-51 antibody  overnight at 4 °C. Slides were washed in PBS three times for 5 min and incubated with a Cy3- or Cy5-conjugated secondary antibodies  for 1.5 h at room temperature. Samples were washed with PBS three times for 5 min, stained with DAPI, and mounted with VECTASHIELD Antifade Mounting Medium. Images were obtained using a Leica DM5000 B microscope or a Leica SP8 confocal microscope. Confocal images shown are maximum projections of 0.2–0.4 µm Z-sections of the entire nuclei, except for images showing whole gonads for which 0.8–1 µm Z-sections were taken.Worm gonads were dissected in anesthetizing buffer  and intestine nuclei (which are endoreduplicated to the DNA content of 32n).For DAPI intensity quantification, gonads were dissected as describe above, fixed in anesthetizing buffer containing 2% paraformaldehyde (PFA) for 5 min, and permeabilized in PBS containing 0.1% Triton X-100 for 5 min. Gonads were washed in PBS, stained with DAPI in PBS (1:500) and washed once more in PBS. Z-stacks of DAPI-stained gonads were merged with maximum intensity. DAPI signal was measured for wild-type oocytes, intestine nuclei and endomitotic oocytes using the Area Integrated Intensity function in Fijihis-72 and his-74, was performed on the live imaging of GFP-tagged version of the proteins from ref. 30 using the Area Integrated Intensity function in Fiji72.Quantification of different H3.3 proteins, 73 with minor modifications. Two milliliters of E. coli MG1693 (obtained from the E. coli stock center) overnight culture were added to 100 ml of M9 buffer supplemented with 1% glucose, 1.25 µg/ml thiamine, 0.5 µM thymidine, 1 mM MgSO4, and 20 µM BrdU (BD-Pharmingen 550891). Bacteria were grown for 24–36 h at 37 °C in the dark, spun down, resuspended in 1 ml of M9 buffer and used to seed M9 agar plates.Worms were labeled with BrdU on plates as described in ref. Wild-type and H3.3K27M mutant young adult worms that had been maintained for one generation at 25 °C were put on feeding plates for 4 h at 25 °C. Gonads were dissected as described above and fixed in methanol for 1 h at −20 °C. Slides were blocked in PBSB (PBS containing 0.5% BSA) for 15 min, washed in PBS, fixed with 1% PFA for 15 min and again washed in PBS. To denature DNA and expose the epitope, the slides were incubated with 2 M HCl for 20 min and then neutralized with 0.1 M sodium borate (pH 8.5) for 15 min. Samples were re-blocked in PBSB for 15 min and incubated with anti-BrdU antibody  overnight at 4 °C. The slides were washed three times in PBSB, incubated with secondary antibody  for 2 h at room temperature, washed again in PBS, counterstained with DAPI and mounted with Vectashield mounting media.® secondary antibodies appropriate for each primary antibody were incubated for 1 h at room temperature.Adult worms were washed three times with M9, resuspended in lysis buffer  and frozen in liquid nitrogen. Samples were sonicated  and spun down  to pellet debris. Protein concentrations were measured with the Bio-Rad Protein Assay . A total of 20 µg (or 2 µg for H3 loading control) was loaded SDS-page gels. Western blotting was performed according to standard procedures using the LI-COR Odyssey system. Primary antibodies  were incubated overnight at 4 °C, and IRDye74 with minor modifications. Briefly, synchronized worm populations were grown on 15-cm plates seeded with E. coli NA22. Worms were harvested as young adults, washed in M9 and resuspended in buffer A  supplemented with 0.5% NP-40 and 0.1% Tx-100. Total nuclei, which are enriched for germ-cell nuclei based on morphologic analysis, were obtained by light grinding of the worms under liquid nitrogen followed by douncing, low speed spin and washing of the nuclei. Chromatin was digested to mononucleosomes using MNase (NEB M0247S) at 37 °C using 5 µl of enzyme per 150 µl of nuclei in 1000 µl of the buffer. Each sample was divided into six parts that were incubated for 3, 5, 8, 10, 12, and 15 min, respectively, and then pooled together in order to obtain nucleosomal ladders. Following digestion, chromatin was extracted, solubilized and pre-cleared with empty beads. The soluble chromatin fraction was incubated with primary antibodies against H3K27me2/me3 (Active Motif 39535), OLLAS  or H3K36me3 (Abcam ab9050) overnight at 4 °C. Antibody-bound chromatin was captured with magnetic beads for 1 h at 4 °C . DNA from both input and ChIP samples was then extracted using phenol/chloroform. Libraries were prepared using NEBNext® Ultra™ II DNA Library Prep with sample purification beads using 100 ng of input DNA. Libraries were sequenced using the TruSeq SBS HS v3 chemistry on an Illumina HiSeq 2500 sequencer.Native ChIP was performed as described in ref. C. elegans reference genome and normalized to the total number of reads for each sample.Endomitotic oocytes or proximal parts of wild-type gonads were dissected. Samples from 20 to 25 worms were pooled and sonicated using a Covaris instrument, and libraries were prepared using TruSeq DNA-nano kit from Illumina. Whole genomes of the oocytes were sequenced using an Illumina HiSeq 2500. Reads were mapped to C. elegans reference genome WBcel215 with Novoalign software . Reads were transformed into bins of the desired size (JSON format) with a custom UNIX script. JSON files were transformed into bedgraphs annotating maximum amount of reads present in each bin with a custom R script. All files were normalized to the same number of reads. For each ChIP experiment, IP files were normalized using the input file with the corresponding bin size. Biological replicates of ChIP-seq experiments that passed the quality control were merged on the level of aligned reads (SAM files), and the downstream pipeline was performed again using the merged file and smoothed to obtain the final normalized bedgraph for each experiment. Processed data files are bedgraphs containing normalized ChIP results. Normalization was performed by input subtraction  or by division of input followed by log2 transformation (marked in the file name by _logratio). Bin sizes used in the analysis were 100 bp (marked in the file name by _100bp), 1 kb (marked in the file name by _1kb) or 10 kb (marked in the file name by _10kb). The appropriate bin size was selected for each analysis. Genome browser images were obtained using the UCSC genome browser (100 kb windows) or with Sushi Bioconductor package (whole-genome views). H3K27me3 occupancy domains were called with custom Python and R scripts, heatmaps and hierarchical clustering were performed with Morpheus software (https://software.broadinstitute.org/morpheus).Paired-end reads (length = 150 bp) were mapped to the 75. Briefly, H3.3K27M L4 worms were washed in M9 and incubated in 50 mM ethyl methanesulfonate (EMS) for 4 h at 20 °C. Worms were washed in M9 again and recovered on OP50-seeded NGM plates. Pools of 10 worms each were transferred to fresh NGM plates  and were allowed to lay eggs for 6 h after reaching the adulthood, resulting in ~100 F1 eggs each. Plates were maintained at 25 °C, and F1 adults were washed off at the second day of adulthood to retain staged F2 eggs. F2 worms were maintained at 25 °C, and 2000 of non-sterile looking worms were singled. Plates that contained progeny after three more generations at 25 °C were retained for further analysis. Mapping of the suppressor mutation was performed as described in ref. 76. Briefly, suppressor strains were backcrossed to the original H3.3K27M strain, and 30 independent F2 plates homozygous for the suppressor mutation (scored based on fertility levels at 25 °C) were pooled together for total DNA isolation. Samples were sonicated using a Covaris instrument, and the libraries were prepared using TruSeq DNA-nano kit from Illumina. Whole genomes from the suppressor strains as well as from original H3.3K27M mutant worms were sequenced using an Illumina HiSeq 2500. Reads were mapped to C. elegans reference genome, and SNPs were identified using the High-Throughput Sequencing portal of the EPFL Bioinformatics and Biostatistics core facility (hts station). Candidate causative mutations were identified as SNPs with nearly 100% presence in suppressor strains when compared with the H3.3K27M mutant strain. Presence of the mutation was confirmed by Sanger sequencing. Suppressor function of the mutation was confirmed by introducing the same mutation into H3.3K27M worms using CRISPR/Cas9 genome editing.The screen for suppressors of H3.3K27M-induced sterility was performed as described in ref. 77. Briefly, adult worms were injected with 100 µM Calcium Green-1 dextran, 10,000 MW (Molecular Probes). Worms were rested for 2 h and imaged with a fluorescence microscope. Levels of Ca2+ were estimated by measuring intensity of green fluorescence in multiple equally sized areas in each gonad using ImageJ.Measuring of gonadal calcium levels was carried out as described in ref. 78. The software identified and used human JNK1 crystal structure (PBD ID 3VUG) as a template for the structure prediction. GMQE  and QMEAN Z-score for the obtained model equal 0.74 and −3.83, respectively, indicating that the model is of good quality and high fidelity. To determine if S287 is a potential phosphorylation site we utilized serine, threonine or tyrosine phosphorylation site prediction software NetPhos3.179.The KGB-1 amino acid sequence was obtained from WormBase and entered into the SWISS-MODEL structure prediction softwarex = pre-existing (wild-type) levels of H3K27me3, y = H3.3K27M levels and z = resulting (observed in H3.3K37M mutant) H3K27me3 levels. The approximation was performed in 3-steps. First, the formula for obtaining z values based on the experimental x and y was proposed for the first 20 data points. Then that formula was applied to all of the data points and best-fit function was approximated using Solver, a Microsoft Excel add-in program. The error was optimized by maximizing the R2 correlation value and minimizing the formula error understood as Zi are the z values obtained by the function, and zi are the experimental z values. That procedure was repeated in several iterations with different base functions, and the use of an approximation based on the Taylor’s theorem resulted in the best fit:To model the dependency between oncohistone incorporation and H3K27me3 levels, a formula approximating the 1 kb logratio ChIP-seq data from wild-type and H3.3K27M mutant worms was identified, where 52. Briefly, 5000 cells per well were plated in 96-well plates (in duplicates) and incubated in presence of the drug for 72 h. Relative numbers of viable cells in each well were determined using CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega). Experiments were performed in six biological replicates, and differences between cell lines at each drug concentration were calculated using one-way ANOVA.The SF8628 cell line was purchased from Merck (SCC127), and SF9402 and SF9427 cell lines were kindly provided by Rintaro Hashizume (Northwestern University). The proliferation assays in presence of DMSO or increasing concentration of the JNK inhibitor SP600125 were performed as described in ref. 30. Briefly, the TruSeq mRNA stranded kit from Illumina was used for the library preparation with < 500 ng of total RNA as input. Reads of 50 bases were generated using the TruSeq SBS HS v3 chemistry on an Illumina HiSeq 2500 sequencer. The reads (length = 50 bp) were mapped with the TopHat v2.0.13 (default parameters) software to the C. elegans reference genome (WBcel235). The normalization and differential expression analysis between N2 and H3.3K27M mutant samples were performed with the R/Bioconductor package edgeR v.3.10.5, for the genes annotated in the reference genome. Genes with a p-value with FDR < 0.05 and fold change > 2 were considered significant. Raw and normalized counts as well as statistical measures are provided in Supplementary Data RNA was isolated from dissected gonads of N2 and H3.3K27M mutant worms grown at 25 °C using Trizol, in biological replicates for both samples. RNA quantification, library preparation and data analysis were performed as described in ref. Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Reporting SummarySource Data


The main chain of CXCL8 appears largely rigid on the picosecond time scale as evident from high order parameters (S2). However, on average, S2 are higher in the bound state. Interestingly, several residues show millisecond-microsecond (ms-μs) dynamics only in the bound state. The CXCR1 N-domain is unstructured in the free state but structured with significant dynamics in the bound state. Isothermal titration calorimetry (ITC) data indicate that both enthalpic and entropic factors contribute to affinity, suggesting that increased slow dynamics in the bound state contribute to affinity. In sum, our data indicate a critical and complex role for dynamics in driving CXCL8 monomer-CXCR1 Site-I interactions.Interleukin-8 (CXCL8), a potent neutrophil-activating chemokine, exerts its function by activating the CXCR1 receptor that belongs to class A G protein-coupled receptors (GPCRs). Receptor activation involves interactions between the CXCL8 N-terminal loop and CXCR1 N-terminal domain (N-domain) residues (Site-I) and between the CXCL8 N-terminal and CXCR1 extracellular/transmembrane residues (Site-II). CXCL8 exists in equilibrium between monomers and dimers, and it is known that the monomer binds CXCR1 with much higher affinity and that Site-I interactions are largely responsible for the differences in monomer vs. dimer affinity. Here, using backbone  Chemokines constitute the largest subfamily of cytokines that play diverse and fundamental roles from trafficking immune cells including neutrophils and organogenesis to combating infection and injury ,2. On thStudies using CXCL8 variants that are trapped either in the monomeric or dimeric state have shown that both forms bind the CXCR1 receptor, but that the monomer alone functions as the high affinity ligand ,10. The Solution nuclear magnetic resonance (NMR) spectroscopy has proven to be quite powerful for providing atomistic description of motions from picosecond to millisecond time scales ,17,18,1915N-longitudinal (R1) and 15N-transverse (R2) relaxation rates, and the heteronuclear {1H}-15N nuclear Overhauser effect (NOE) −1), and the orientation of the unique axis relative to the coordinates of the molecular structure. Fully anisotropic diffusion is defined by the diffusion rates about three orthogonal axes  and the orientation of the axis system relative to the structure. The components of the molecular diffusion tensor were estimated from the R2/R1 ratio of a select set of 46 residues (described in Methods). For the free CXCL8 monomer, an axially symmetric rotational diffusion model was required to adequately describe the 600 and 800 MHz data. The oblate axially symmetric rotational diffusion model (Dpar/Dper = 0.86 ± 0.01), relative to the isotropic model, allowed for a better fit to the 600 MHz data according to the F-statistic test . However, the fully anisotropic model relative to an axially symmetric model was not statistically significant . Similar results were obtained for the 800 MHz data, with Dpar/Dper = 0.81 ± 0.01.Isotropic molecular rotational diffusion is characterized by a single correlation time τc was calculated to be 4.7 ns for both the 600 and 800 MHz data. At 600 MHz, 56 of the 60 residues could be fitted to one of the five models. Residues A35, I40, R47, and W57 did not fit to any model. A total of 21 residues fit to model 1 with an average S2 of 0.86. Fifteen residues fit to model 2 with an average S2 of 0.80 and a τf value ranging from 10 to 90 ps. Four residues fit to model 3 with an average S2 of 0.87 and exR values ranging from 0.4 to 0.8 s−1. Nine residues fit to model 4 with an average S2 of 0.81, exR values ranging from 0.4 to 1.8 s−1, and τf values ranging from 10 to 56 ps. Of the thirteen residues that fit to models 3 and 4, only I10, S14, and V62 showed exR > 1 s−1. The necessity of an exR term suggests a conformational exchange process at these sites. Seven residues fit to model 5 with an average S2 of 0.55 and τs values ranging from 500 to 3500 ps.The relaxation data were fit to five spectral density models for describing the internal dynamics of the CXCL8 monomer with respect to axially symmetric rotational diffusion using the Modelfree and Fast Modelfree programs ,47. The exR values (<0.8 s−1) fit to different models between the two fields switching from models 3 and 4 to models 1 and 2 and vice versa, and some that fit to model 5 did not fit any of the models. Residues I10, S14, V62, and E63 showed exR >1 s−1.For data collected at 800 MHz, 54 of the 60 residues fit to one of the five motional models. A total of 34 residues fit to models 1 and 2, 13 residues to models 3 and 4, and seven residues to model 5. Some residues with small exR terms obtained from fitting the relaxation experimental data to Lipari–Szabo model-free approach must be interpreted with caution, as they may reflect anisotropy of overall tumbling or data fitting artifacts, and are best validated using CPMG relaxation dispersion experiments tailored to detect ms–μs dynamics 15N-CXCL8 upon addition of the CXCR1 N-domain were followed using 2D 1H-15N HSQC NMR spectra. Titration of 200 μM 15N-CXCL8 was conducted through addition of increasing amounts of stock solution containing 2.5 mM unlabeled CXCR1 peptide. Concentrations of protein and peptide for the titration points were 200, 199.2, 196.5, 192.3, 185.2, 175.4, 156.3, and 135.1 μM for CXCL8, and 0, 13.9, 61.9, 134.6, 259.3, 429.8, 765.6, and 1135.1 μM, with corresponding peptide/protein molar ratios of 0, 0.1, 0.3, 0.7, 1.4, 2.5, 4.9, and 8.4. The combined 15N and 1H chemical shift changes (Δδ) were determined as Δδ = [(Δδ15N/5)2 + (Δδ1HN)2]1/2, where Δδ15N and Δδ1H are the respective 15N and 1H chemical shift changes in ppm. Δδ values were fit to a 1:1 binding model to determine the protein-peptide interaction constant (DK), as previously described [DK determined from chemical shift analyses described above, line shape analyses yield the kinetics of binding (onk = offk/DK), through fitting of offk.The main chain amide chemical shift changes of escribed . Combine15N-R1 and -R2 relaxation rates, hetNOE, and relaxation dispersion experiments for the CXCL8 monomer and CXCR1 N-domain 29mer in the free and bound states were recorded at 25 °C on a Bruker 600 MHz (equipped with a quadruple resonance QCI cryoprobe) and Bruker Avance III 800 MHz (equipped with a triple resonance TXI cryoprobe) spectrometers. All relaxation data were collected as single scanned interleaved pseudo three-dimensional (3D) experiments. The spectra were processed using NMRPipe and analyzed using NMRView. Steady state heteronuclear NOE values were obtained by recording spectra with and without proton saturation. For the free and bound CXCL8 monomer, and the bound CXCR1 N-domain, a saturation time of 3 s and delay of 3 s between scans were used. In the case of the free CXCR1 N-domain, a saturation time of 5 s and a delay of 10 s were used.15N-R1 and -R2 data were acquired using 10 different relaxation delays . The hetNOE values were calculated using the hetNOE analysis routine in NMRView. 15N-T1 and T2 relaxation times were calculated from non-linear least-square fits of the changes in cross peak intensities to a two-parameter exponential decay using a suite of Mathematica notebooks [otebooks .c) were initially calculated on a per residue basis by minimizing the differences between the experimental and the calculated T1, T2, and hetNOE values using the spectral density function. The global rotational correlation times (τc) are the averages from per residue fits excluding residues with NOE < 0.65. The components of the molecular rotational diffusion tensors were estimated from the R2/R1 ratios of individual residues for the case of isotropic and axially symmetric diffusion tensors using the programs r2r1_diffusion after excluding residues with hetNOE < 0.65 and those with short T2 values and potentially undergoing conformational exchange [F-statistic and the statistical significance of the models were determined by a p-value calculation [Rotational correlation times . The relaxation dispersion data were fit to two-site chemical exchange with the parameters exk (rate of exchange), δω , and the populations of the individual states . Peak intensities extracted from the individual spectra were analyzed using the NESSY program [exk >> δω, and the slow-limit with exk << δω. Model selection was performed using AICc (Akaike’s information criteria with second order correction).CPMG relaxation dispersion profiles were obtained by recording spectra with varying CPMG pulse frequencies  using a 60 ms constant CPMG relaxation delay . Titrations were performed by injecting 1 × 0.5 µL and 19 × 2 µL aliquots of 890 μM CXCR1 N-domain peptide to 80 μM CXCL8 monomer prepared in 50 mM sodium phosphate buffer, pH 6.0. The experiments were repeated twice. The raw data were corrected using a buffer control wherein CXCR1 N-domain was titrated into the buffer. The data were analyzed using the software supplied by the manufacturer.


Escherichia coli that hold great promise as viable complements or alternatives to antibiotics. Cell-free protein synthesis (CFPS) is a useful production platform for toxic proteins because it eliminates the need to maintain cell viability, a common problem in cell-based production. Previously, we demonstrated that colicins produced by CFPS based on crude Escherichia coli lysates are effective in eradicating antibiotic-tolerant bacteria known as persisters. However, we also found that some colicins have poor solubility or low cell-killing activity. In this study, we improved the solubility of colicin M from 16% to nearly 100% by producing it in chaperone-enriched E. coli extracts, resulting in enhanced cell-killing activity. We also improved the cytotoxicity of colicin E3 by adding or co-expressing the E3 immunity protein during the CFPS reaction, suggesting that the E3 immunity protein enhances colicin E3 activity in addition to protecting the host strain. Finally, we confirmed our previous finding that active colicins can be rapidly synthesized by observing colicin E1 production over time in CFPS. Within three hours of CFPS incubation, colicin E1 reached its maximum production yield and maintained high cytotoxicity during longer incubations up to 20 h. Taken together, our findings indicate that colicin production can be easily optimized for improved solubility and activity using the CFPS platform.Colicins are antimicrobial proteins produced by  Multidrug-resistant bacteria can be difficult to treat and are a serious threat to society . There iEscherichia coli, can be produced using an E. coli-based CFPS system [E. coli cells [Antimicrobial peptides are short (5~100 amino acids) and can be synthesized either chemically or biologically ; howeverS system . Colicinli cells  by inhibli cells , formingli cells  and Ia [li cells ), and deli cells  or RNA  which facilitate the transcription and prevent the degradation of messenger RNAs, respectively [E. coli K361 strain for testing colicin cell-killing activity [araB or Pzt-1 promoters which are induced by L-arabinose or tetracycline, respectively  strain using a sonication method [E. coli cultivation. An overnight culture of BL21 Star (DE3) containing chaperone plasmids was diluted 1,000 times in 1.0 L of 2xYTPG medium  with appropriate antibiotics in a 2.5 L Tunair flask. Cells were grown to a turbidity at 600 nm (OD600nm) of 0.5 at 37 °C at 220 rpm. The GroES/EL chaperone system from pGro7 was induced by adding 0.5 mg/mL L-arabinose. Similarly, DnaK/DnaJ/GrpE system from pKJE7 was induced by adding 0.5 mg/mL L-arabinose. For cell cultures containing both chaperone systems from pG-KJE8, GroES/EL was induced by adding 1 ng/mL tetracycline and DnaK/DnaJ/GrpE was induced by adding 0.5 mg/mL L-arabinose. After inducing chaperones, the cells were further grown to an OD600nm of 3.0. Then, cells were harvested by centrifuging at 5,000× g at 4 °C for 15 min, washed twice to remove all the medium with cold S30 buffer , and stored at −80 °C. Thawed cells were mixed with S30 buffer (1 mL buffer per 1 g of cells) and lysed on ice using a Q125 sonicator  using 50% amplitude and three cycles of 45 s pulses at 60 s intervals. Insoluble components including cell debris were removed by two centrifugation steps at 14,000× g at 4 °C for 10 min. The crude extracts were filtered by a 0.2 μm sterile syringe filter  to remove unlysed cells completely. The total protein concentration of the extracts was approximately 50 mg/mL, as assessed by Quick-Start Bradford protein assay kit . The crude extracts were stored at −80 °C until needed.n method  as descrn method . Chaperocma gene encoding colicin M was amplified from the pJL1-cma plasmid using GAcol-F and GAcol-R, ceaC encoding colicin E3 from the pKSJ167 plasmid using ColE3-F and ColE3-R, and cea encoding colicin E1 from the pKSJ331 plasmid using ColE1-F and ColE1-R. PCR was performed using Phusion High-Fidelity DNA polymerase  at 98 °C for 30 s, with 30 cycles of denaturing at 98 °C for 10 s, annealing at 55 or 60 °C for 30 s, and extending at 72 °C for 2 min 30 s, and a final extension at 72 °C for 10 min. Three rounds of PCR were performed to insert T7 promoter and T7 terminator sequences for genes of colicin M, E1, and E3. Phosphorothioated T7Mega-F and T7Mega-R primers were used to protect T7 promoter and terminator sequences from nuclease degradation. The first PCR amplified the colicin genes, the second PCR added the T7 promoter sequence, and the third PCR added the T7 terminator sequence. DNA sequences of all colicin genes are found in Colicin genes were amplified by polymerase chain reaction (PCR) using primers found in imm was cloned into the pJL1 plasmid. First, the gene was amplified from the pKSJ167 plasmid using the primers E3Imm-F and E3Imm-R which installed a C-terminal Strep-tag (WSHPQFEK). The PCR fragment was double digested by NdeI and SalI restriction enzymes and ligated into pJL1-backbone at the same restriction sites. BL21 Star (DE3) cells were transformed with the ligated plasmid. An overnight culture of E. coli BL21 Star (DE3) harboring the pJL1-E3 imm plasmid was diluted 100 times in 250 mL of LB containing 50 μg/mL of kanamycin in a 1 L flask and incubated at 37 °C at 220 rpm until OD600nm ~ 0.5. Then, E3 immunity protein production was induced by adding 1 mM of isopropyl β-d-1-thiogalactopyranoside at 37 °C at 220 rpm for an additional 5 h. E3 immunity protein was purified by a Strep-Tactin column , and the concentration of the E3 immunity protein was measured as 520 ± 30 μg/mL.The E3 immunity gene CFPS reactions were performed to synthesize colicins according to established protocols . Chapero14C-Leu incorporation [14C-leucine  was added into triplicate CFPS reactions. After incubating at 30 °C or room temperature for specified periods of time, soluble fractions were separated by centrifuging at 12,000× g for 15 min at 4 °C. CFPS reactions in the time-course study of colicin E1 synthesis were quenched at indicated times using 833 µg/mL kanamycin and flash freezing at −80 °C. Proteins were precipitated, washed with 5% trichloroacetic acid three times and then 100% ethanol, and quantified using a liquid scintillation counting. To eliminate background radioactivity and protein synthesis, scintillation counts from no plasmid control were subtracted from the colicin samples. For colicin M, total or soluble fractions of each reaction containing 14C-leucine were visualized by running an SDS-PAGE gel, exposing the gel Storage Phosphor Screen, and acquiring an autoradiogram using a Typhoon FLA700 imager as described previously [Total and soluble protein yields were measured by determining radioactive poration . Brieflyeviously .600nm of approximately 0.7–0.9. Cells were harvested, adjusted to an OD600nm of 0.1  or 1.0  with LB medium, and then incubated with CFPS reaction products for 1 h with shaking at 37 °C. Cell viability was quantified by counting colony forming units. To generate the activity curve of colicin E3, we treated cells at high initial population (5.0 × 108 CFU/mL) with varying concentrations of colicin E3 for 1 h. Effective multiplicity (m), a measure of colicin cytotoxicity, was calculated by the equation: m = −ln(S/S0), where S indicates the surviving cell population with colicin treatment, and S0 is the untreated control cell population [Cell viability assays were performed as described previously . An overpulation .t-tests between colicin samples and no colicin controls were performed [p < 0.01), ** (p < 0.001), and *** (p < 0.0001).Two-tailed erformed . StatistE. coli cultures and then harvested and prepared extracts from these chassis strains using a sonication method [While our previous study showed that colicins E1, E2 and Ia were nearly completely soluble when produced in CFPS, we found that the majority of colicin M produced was insoluble and that only soluble colicin M (~5%) was active in killing K361 indicator cells . Based on method  followedn method . First, n method  and has n method . The sfG14C-Leu incorporation. Total colicin M yield was approximately 300 ng/μL in all extracts except the GroES/EL-containing extract which yielded approximately 500 ng/μL  between approximately 0.7–0.9, centrifuged, and resuspended in fresh LB to adjust cell populations to approximately 5.0 × 107 CFU/mL. The indicator cells were exposed to a 750 ng/mL total concentration of cell-free produced colicin M for 1 h at 37 °C with shaking at 220 rpm  in CFPS does not require the addition or co-expression of its immunity protein. In this study, we were interested in producing another colicin (E3) that is known to have a specific RNase activity  which deor cells A.7 CFU/mL for 1 h [Our results suggest that the immunity protein is necessary for colicin E3 to maintain its activity. While initially unexpected, this finding makes sense in the context of a structural study of colicin E3  which fo for 1 h . Taken tBecause CFPS reactions can be lyophilized, stored without cold-chain, and rehydrated to provide simplified and rapid access to high yields of proteins , on-dema14C-Leu scintillation counting  of 1.0 μg/mL requires an oral dosing of 250~750 mg per 12 h [90 of 0.016 μg/mL [E. coli CFPS reactions can reach 2.3 mg/mL for sfGFP [The rapid and robust production of colicin E1 reported here supports further research into the possible utility of CFPS production of colicins for on-demand protein manufacturing. Because colicins kill non-host  surface , they ma disease . Althoug disease . The FDAor sfGFP , indicatIn summary, we utilized the open nature and engineering flexibility of CFPS to improve colicin M solubility with chaperone-enriched cell extracts, enhance the activity of colicin E3 via co-expression of the immunity protein, and assess the production kinetics and stability of colicin E1 in CFPS reactions. We anticipate that the approaches presented in this study can be applied to optimize the production of other colicins or colicin-like bacteriocins in vitro, providing a useful alternative to the in vivo production of toxic proteins.


PTEN cause PTEN Hamartoma Tumor Syndrome (PHTS). Pediatric patients with PHTS frequently develop lipomas. Treatment attempts with the mTORC1 inhibitor rapamycin were unable to reverse lipoma growth. Recently, lipomas associated with PIK3CA-related overgrowth syndrome were successfully treated with the novel PI3K inhibitor alpelisib. Here, we tested whether alpelisib has growth-restrictive effects and induces cell death in lipoma cells. We used PTEN-haploinsufficient lipoma cells from three patients and treated them with alpelisib alone or in combination with rapamycin. We tested the effect of alpelisib on viability, proliferation, cell death, induction of senescence, adipocyte differentiation, and signaling at 1–100 µM alpelisib. Alpelisib alone or in combination with rapamycin reduced proliferation in a concentration- and time-dependent manner. No cell death but an induction of senescence was detected after alpelisib incubation for 72 h. Alpelisib treatment led to a reduced phosphorylation of AKT, mTOR, and ribosomal protein S6. Rapamycin treatment alone led to increased AKT phosphorylation. This effect could be reversed by combining rapamycin with alpelisib. Alpelisib reduced the size of lipoma spheroids by attenuating adipocyte differentiation. Since alpelisib was well tolerated in first clinical trials, this drug alone or in combination with rapamycin is a potential new treatment option for PHTS-related adipose tissue overgrowth.Germline mutations in the tumor suppressor gene  PTEN show a wide variety of phenotypes related to cellular overgrowth. There are several syndromes associated with PTEN mutations, including Cowden syndrome, Proteus syndrome, and Bannayan–Riley–Ruvalcaba syndrome (BRRS), all subsumed under the term PTEN Hamartoma Tumor Syndrome (PHTS) [2) to phosphatidylinositol -trisphosphate (PIP3). PIP3 is the key molecule to activate further downstream signaling components, e.g., the pro-survival molecule AKT. PTEN acts as a lipid phosphatase on PIP3, catalyzing the conversion to PIP2, and therefore is a negative regulator of the AKT/mTOR signaling cascade [Patients with germline mutations in the tumor suppressor gene e (PHTS) . Symptome (PHTS) . Lipomate (PHTS) ,4, but ce (PHTS) . PI3K li cascade . mTORC1  cascade . This loRecently, patients with lipomatous tumors associated with a related spectrum of syndromes caused by mosaic activating PI3K mutations  were successfully treated with the novel PI3K inhibitor alpelisib BYL-719) . The siz . The siWe treated five different primary lipoma cell cultures with alpelisib concentrations ranging from 1 to 50 µM and measured cell viability  using the WST-1 assay after 72 h for alpelisib alone a or in cp < 0.0001). At 10 µM alpelisib, cell viability was significantly decreased in all cell cultures. A combination of 10 µM alpelisib and 10 nM rapamycin further decreased cell viability compared to single treatment (p < 0.001). IC50 values for 72 h WST-1 assays were calculated to be 9.09 µM (LipPD1), 6.31 µM (LipPD2), 18.33 µM (LipPD3), 6.7 µM (Lip3), and 15.74 µM (Lip4). A summary of all IC50 values is given in p < 0.0001) and time-dependent manner (p < 0.0001). We observed a further decrease in cell viability for a combined treatment of alpelisib and rapamycin. To examine whether combining alpelisib and rapamycin has synergistic or additive anti-lipoma activity, LipPD1, LipPD2, and LipPD3 cells were treated with alpelisib and rapamycin across a range of concentrations. Analysis of the combined effect by the Chou and Talalay method [We noted a concentration-dependent decrease in cell viability for all cell cultures , 10.06 µM (LipPD2), and 15.79 µM (LipPD3) , for 10 µM to 0.55 ± 0.06 fold (p = 0.018), and for 100 µM to 0.22 ± 0.1 fold (p = 0.017) in a concentration-dependent manner (p = 0.0098). Ki-67 immunofluorescence staining in LipPD2 and LipPD3 cells after 72 h also showed a reduced fraction of proliferation marker positive cells  . The frave cells .We measured apoptosis induction in lipoma cells after 72 h alpelisib treatment by annexin V/PI staining via flow cytometry a, and cep = 0.0471) after a combined treatment with alpelisib and rapamycin. To confirm our findings, we performed LDH cytotoxicity assays for LipPD1, LipPD2, and LipPD3 cells after 24 h and 72 h of 50 µM alpelisib treatment . For LipPD1 cells, we did not observe apoptosis after 72 h 10 nM rapamycin treatment alone, while the total fraction of apoptotic and dead cells was slightly increased , while rapamycin enhanced AKT phosphorylation (p = 0.192). This effect of rapamycin was repressed when combined with alpelisib (p = 0.279). Activation of mTOR was reduced in 10 µM (p = 0.099) and 50 µM (p = 0.066) alpelisib-treated cells, as well as in 10 nM rapamycin-treated cells (p = 0.18). Phosphorylation of S6 was significantly reduced for all tested alpelisib and rapamycin concentrations. Western blots for LipPD2 (p = 0.0074) and for 100 µM to 0.01 ± 0.006 fold (p = 0.0169). pS6 immunofluorescence staining in LipPD2 and LipPD3 cells after 72 h showed similar results  analysis for the proliferation marker zyme PGK . All thrPCNA mRNA was downregulated after 24 h alpelisib treatment (to 0.67 ± 0.08 fold). Similarly, we observed a downregulation of GLUT1 mRNA (to 0.60 ± 0.11 fold) and PGK mRNA (to 0.67 ± 0.06 fold).To test whether alpelisib would attenuate adipogenesis, we differentiated LipPD1 cells in 2D culture and 3D spheroid models to assess lipid accumulation a–b, diffperoxisome proliferator-activated receptor γ (PPARγ) mRNA downregulated after 10 days in differentiation medium with 10 µM alpelisib to 0.55 ± 0.06 fold. Adiponectin mRNA was downregulated to 0.54 ± 0.04 fold and adipocyte protein 2 (aP2) mRNA to 0.54 ± 0.03 fold. Fatty acid synthase (FASN) mRNA was downregulated as well to 0.58 ± 0.09 fold, p = 0.064 (p = 0.0063 at day 4).We observed reduced lipid accumulation after alpelisib treatment of LipPD1 cells in adipocyte differentiation medium for 10 days. The fraction of adipocytes was reduced from 54.8 ± 7% to 29.9 ± 7%. Further, we investigated the mRNA expression of differentiation markers with or without 10 µM alpelisib in adipogenic medium and detected  = 0.064 . To confTo check whether the lipoma cells maintained their mesenchymal stem cell phenotype or underwent senescence during alpelisib treatment, we performed a β-galactosidase senescence staining a–b and mp = 0.236) and LipPD3 cells  (CDKN2A (p16) and stem cell markers CD44 and Thy-1 (CD90) with or without 10 µM alpelisib in adipogenic medium. p16 mRNA was upregulated after 10 days in differentiation medium with 10 µM alpelisib (to 5.4 ± 2.05 fold). CD44 mRNA was upregulated to 1.96 ± 0.32 fold. In contrast, CD90 mRNA was reduced to 0.71 ± 0.05 fold. Similar trends in the regulation of adiponectin, p16, CD44, and CD90 were observed in LipPD2 and LipPD3 cells  . Since wD3 cells .PTEN can be a severe complication in pediatric patients. So far, the only treatment option is surgical resection, which is not feasible in every patient and has to be repeated because of lipoma recurrence. In this research work, we tested effects of the novel PI3K inhibitor alpelisib on PTEN-haploinsufficient lipoma cells  from pediatric patients. Proliferation of these preadipocyte-like cells is enhanced, potentially due to a constitutive overactivation of the PI3K-pathway [Lipomatosis associated with germline mutations in -pathway . The PI3-pathway . In adip-pathway . We askeWhen we treated lipoma cells with alpelisib, we observed a concentration-dependent decrease in cell viability. This decrease was in line with a reduced cell number after alpelisib treatment. The fraction of proliferation marker Ki-67 positive cells was decreased in a concentration-dependent manner and the proliferation marker PCNA was downregulated on the mRNA level. While these findings indicate that alpelisib negatively influences cell proliferation of PTEN-haploinsufficient lipoma cells, apoptosis could not be induced during 72 h alpelisib treatment. Indeed, we did not detect any alpelisib-induced cytotoxicity in any of the cell lines tested. The number of dead/apoptotic cells was also only slightly elevated when we combined the mTOR inhibitor rapamycin with alpelisib. Nevertheless, this combination was more effective than treatment with the same concentrations of either drug alone in terms of inhibition of proliferation. Supporting this finding, we detected a synergistic effect of rapamycin and alpelisib treatment on cell viability. In line with the findings of Keam et al., in head and neck cancer cell lines, we detected a reduction of proliferation using alpelisib at similar concentrations .PTEN mutations, this leads to an activation of the whole pathway. PI3K inhibitors limit this pathway activation on the same level as PTEN, which might provide advantages over an inhibition further downstream.In the majority of cancers with mutated PI3K, the PI3Kα isoform is activated . AlpelisGLUT1 and the glycolysis enzyme PGK. Both were significantly reduced, indicating changes in glucose metabolism on the transcriptional level during alpelisib treatment, along with a reduction of proliferating cell nuclear antigen (PCNA) expression, a marker for proliferating cells. In patients, this might be advantageous to mainly target cells with a transformed metabolic profile, and therefore selectively inhibit malignancies.Many cancer cells are known to rely on aerobic glycolysis as their primary source of energy generation. The PI3K pathway plays a major role in regulating this metabolic process . To furtPPARγ, adiponectin, aP2, and FASN were downregulated.To test alpelisib in a physiological model of lipoma adipocyte differentiation, we used 3D cultures of lipoma cells. Size of lipoma cell spheroids in 3D culture increased during 10 days in differentiation medium, as shown before , which iCDKN2A (p16) by alpelisib, both in differentiated and undifferentiated lipoma cells. Moreover, alpelisib enhanced β-galactosidase staining of lipoma cells. These findings indicate that alpelisib induced senescence both in PTEN haploinsufficient preadipocytes and differentiated adipocytes. In a mouse model for PROS syndrome, no induction of senescence or apoptosis was detected [We finally tested whether the attenuated adipocyte differentiation observed in alpelisib-treated lipoma cells was caused by preservation of stem cell characteristics. When checking gene expression of CD44 and CD90, markers present on adipose-derived mesenchymal stem cells, we found gene expression differences in differentiated lipoma cells that were alpelisib-treated compared to controls. We found that the stem cell marker CD90 was downregulated. CD90 downregulation is associated with senescence in mesenchymal stem cells . In contdetected .PI3K mutations in PROS patients [Alpelisib was successfully used to treat overgrowth of different tissues associated with activating patients . Since apatients ,11,12. Apatients . We therWe used cells of the stromal vascular fraction isolated from lipomas of three different pediatric PHTS and two PROS patients . Mutations were detected by Sanger sequencing , while tFor differentiation, we plated 13,000 lipoma cells/cm² in culture medium and changed medium to differentiation medium  after 2®, cell-repellent surface, Greiner Bio-One, Kremsmünster, Austria) in 100 µl differentiation medium and incubated for 24 h. After 24 h, spheroids were incubated with 10 µM alpelisib or solvent control in differentiation medium for 10 days. Half of the medium was replaced every 72 h. Microscope images were taken daily using the EVOS FL Auto 2 Cell Imaging System . Image analysis to determine the spheroid size was performed using ImageJ [A modified method according to Klingelhutz et al. was used for scaffold-free 3D cultures of lipoma cells . To formg ImageJ .For proliferation assays, cells were seeded at a density of 3000 cells/well (24 h) or 1500 cells/well (48–144 h) on 96-well plates and treated with different concentrations of alpelisib and/or rapamycin . Medium was replaced every 72 h. Cell viability measures were performed using the Cell Proliferation Reagent WST-1  according to the manufacturer’s protocol. After WST-1 assay, cells were fixed and nuclei were stained with Hoechst 33342  for 5 min at a concentration of 10 µg/ml in DPBS. Hoechst fluorescence was detected at 455 nm.For apoptosis detection, cells were seeded at a density of 1750 cells/cm² and treated with alpelisib/alpelisib+rapamycin for 72 h. Then, 50 µM perifosine  was used as a positive control. The FITC-Annexin V Apoptosis Detection Kit I  was used for annexinV–fluorescein isothiocyanate and propidium iodide staining of the trypsinized cells. Apoptosis was detected by flow cytometry.For LDH cytotoxicity assays, cells were seeded at a density of 3000 cells/well on 96-well plates in culture medium. Cells were treated for 72 h with 50 µM alpelisib, 10 nM rapamycin, or a combination of both. LDH release was detected using the CyQUANT™ LDH Cytotoxicity Assay  according to the manufacturer’s protocol.For Western blot analysis, lipoma cells were seeded at a density of 10,000 cells/cm² in culture medium. Cells were incubated with different alpelisib and/or rapamycin concentrations for 24 h. Proteins were extracted and immunoblotting was performed as described elsewhere [For immunofluorescence staining, cells were fixed in 4% PFA after 48 h of alpelisib treatment. Cells were permeabilized and blocked in IF-buffer (DPBS + 5% BSA + 0.3% Tween20) for 1 h at RT and stained with pS6 or Ki-67 primary antibodies over night at 4 °C . Cells wGlucose transporter 1 (GLUT1), phosphoglycerate kinase (PGK), and Proliferating cell nuclear antigen (PCNA) mRNA was normalized to the housekeeper Tata box binding protein (TBP) mRNA. PPARγ, adiponectin, aP2, FASN, p16, CD44, and CD90 mRNA were normalized to the housekeepers β-actin or HPRT mRNA.For transcriptional analysis, we seeded 5000 cells/cm² in culture medium and extracted mRNA after 24 h of treatment with 10 µM alpelisib or solvent control. For transcriptional analysis on differentiation, cells were seeded at a density of 13,000 cells/cm². Cells were kept in differentiation medium with or without 10 µM alpelisib for 10 days before RNA was extracted. RNA extraction, reverse transcription, and qPCR were performed as previously described . Table 3To detect senescence, we seeded 2000 cells/well on 96-well plates in culture medium. After 72 h of treatment with 10 µM alpelisib or solvent control, we performed fixation and senescent cell staining using the Senescence β-Galactosidase Staining Kit  according to the manufacturer’s protocol. We used 60 µl β-galactosidase staining solution per well and after overnight incubation, we added 50 µL Hoechst in DPBS (1 ng/mL) and took microscope images using the EVOS FL Auto 2 Cell Imaging System (pictures of 5% of the well area). We counted nuclei (DAPI channel) with the Celleste Image Analysis Software (Thermo Fisher Scientific) and β-galactosidase positive cells  using ImageJ .t-test. For multiple comparisons, we used one- or two-way analysis of variance (ANOVA), followed by a post-hoc Tukey’s multiple comparisons test (one-way ANOVA) or Dunnett’s multiple comparisons test (two-way ANOVA). To determine the significance of fold changes, we used one-sample t-tests of the log(fold change) and compared to the hypothetical value 0 [50 values, we used GraphPad Prism 6  vs. response, least squares fit, standard slope model). Isobologram analysis [Means of at least three independent experiments were statistically analyzed using GraphPad Prism 6 software . For comparison of two conditions, means of independent experiments were compared via unpaired Student’s  value 0 . To calcanalysis  was doneLipomatosis in pediatric PHTS patients is a rare condition, but nevertheless, can affect individuals in a life-threatening manner due to limited treatment options. In this research project, we tested the PI3K inhibitor alpelisib on PTEN-haploinsufficient lipoma cells from PHTS patients. We found that alpelisib inhibited cellular growth and reduced the size of 3D lipoma cell cultures, although alpelisib did not induce apoptosis. In contrast to rapamycin, alpelisib inhibited activation of AKT, promising to overcome drug resistance mechanisms occurring during rapamycin treatment in PHTS patients .


Here, we established single cell-digital gene expression (1cell-DGE), a method of scRNA-seq that uses micromanipulation to extract the contents of individual living cells in intact tissue while recording their positional information. With 1cell-DGE, we could detect differentially expressed genes (DEGs) during the reprogramming of leaf cells of the moss Physcomitrella patens, identifying 6382 DEGs between cells at 0 and 24 h after excision. Furthermore, we identified a subpopulation of reprogramming cells based on their pseudotimes, which were calculated using transcriptome profiles at 24 h. 1cell-DGE with microcapillary manipulation can be used to analyze the gene expression of individual cells without detaching them from their tightly associated tissues, enabling us to retain positional information and investigate cell–cell interactions.Next-generation sequencing technologies have made it possible to carry out transcriptome analysis at the single-cell level. Single-cell RNA-sequencing (scRNA-seq) data provide insights into cellular dynamics, including intercellular heterogeneity as well as inter- and intra-cellular fluctuations in gene expression that cannot be studied using populations of cells. The utilization of scRNA-seq is, however, restricted to cell types that can be isolated from their original tissues, and it can be difficult to obtain precise positional information for these cells  Whole plants can be regenerated from tissue samples such as branch cuttings or detached leaves via a callus , a mass Arabidopsis thaliana (Arabidopsis) revealed a transition of cell identity during root regeneration (A number of single-cell RNA-sequencing (scRNA-seq) methods utilizing next generation sequencing (NGS) have been developed to prepare cDNA libraries from isolated single cells containing trace amounts of RNA ,6. Hundrneration . scRNA-sPhyscomitrella patens (Physcomitrella) is a basal land plant with a simple body plan, including leaves formed of a single cell layer (WUSCHEL-related homeobox 13 (PpWOX13) genes are upregulated during reprogramming and required for the tip growth characteristic of the chloronema apical stem cells   to the cPhyscomitrella patens Gransden 2004 , and 2.6% of a mix containing 2.5 mM of each dNTP . The capillary was attached to the capillary holder so that the beveled tip faced down without any air bubbles.A set of oil hydraulic micromanipulators  and motor-driven manipulators MM-89 (Narishige) were equipped onto an inverted fluorescent microscope IX-70  with a fluorescence filter unit . To simultaneously observe the tip of the microcapillary and the GFP fluorescence in the nuclei of the leaf cells, the fluorescence microscopy was performed under dim bright-field illumination. The 1.0-mm capillary holder was connected to a microinjector  via a silicone tube filled with mineral oil , which was in turn attached to the MMO-220A micromanipulator. The parameters of the 1.0-mm glass capillaries were as follows: inner diameter: 20 μm, pipette form: straight, beveled angle of tip: 40°, and pipette length: 55 mm . The bottom of the glass capillary contained a small amount of cell content extraction mix2 comprising 13% 10 × PCR buffer II , 7.8% 25 mM MgCl2 (25 mM), 0.225 μl DTT (0.1 M), 0.09 μl RNasin Plus RNase inhibitor (Promega), 0.09 μl of a mix containing 2.5 mM of each dNTP (Takara Bio), and 0.1 μl of 20 000-fold diluted ERCC RNA spike-in mix (Thermo Fisher Scientific). After a brief centrifugation, the samples were primed with an incubation on a thermal cycler at 70°C for 90 s, 35°C for 15 s, and cooled to 4°C. The tubes were kept on ice before reverse transcription. The RT oligos used in this work are listed in The attached microcapillary was gently filled with mineral oil under a microscope using the CellTram vario. After adjusting the tip position of the glass capillary to the center of the observation field, a dish containing an excised leaf was set on the microscope and the tip of the capillary was used to extract the nucleus and some surrounding cytoplasm from the target cell. The cell contents were immediately transferred into a 0.2-ml PCR tube containing 1.25 μl RT oligos (0.05 μM) and 2.35 μl cell content extraction mix1, containing 0.45 μl 10× PCR buffer II (Thermo Fisher Scientific), 0.27 μl MgClFor the reverse transcription, a 0.9 μl RT mix containing 0.33 μl SuperScriptIII reverse transcriptase (Thermo Fisher Scientific), 0.05 μl RNasin plus RNase Inhibitor (Promega), and 0.07 μl T4 gene 32 protein  was added to each primed RNA solution. After pipetting gently and centrifuging briefly, the tubes were incubated on a thermal cycler at 50°C for 30 min, 70°C for 10 min, then cooled to 4°C.To digest the excess RT oligos, the samples were mixed with 0.8 μl nuclease-free water , 0.1 μl 10× exonuclease I buffer, and 0.1 μl of 20 U/μl exonuclease I (New England Biolabs). After pipetting gently to mix and a brief centrifugation, the tubes were incubated on a thermal cycler using the following conditions: 4°C for 30 s, 37°C for 30 min, 80°C for 20 min with lid heating at 90°C, and cooled to 4°C. The tubes were then transferred onto ice for at least 1 min.2 (25 mM), 0.18 μl dATP (100 mM) (New England Biolabs), 0.3 μl of 15 U/μl terminal deoxynucleotidyl transferase (Thermo Fisher Scientific), and 0.12 μl of 5 U/μl RNaseH (New England Biolabs). A 6-μl aliquot of this poly(dA) mix was added to each tube after the exonuclease I treatment. After pipetting to mix and a brief centrifugation, the samples were incubated on a thermal cycler using the following conditions: 4°C for 30 s, 37°C for 1.5 min, 70°C for 10 min with lid heating at 80°C, and cooled to 4°C.The poly(dA) mix for poly(dA) tailing with RNaseH was as follows: 4.44 μl nuclease-free water (Qiagen), 0.6 μl 10 × PCR buffer II (Thermo Fisher Scientific), 0.36 μl MgCl2 (New England Biolabs), 7.6 μl of each dNTP (2.5 mM) (Takara Bio), 0.76 μl NUP3 primer (100 μM), and 1.76 μl Q5 Hot Start High-Fidelity DNA polymerase (2 U/μl) (New England Biolabs). A 76-μl volume of PCR mix1 was added into each tube after the poly(dA) tailing, pipetted to mix and briefly centrifuged, then the mixtures were divided into 21-μl aliquots which were transferred into four new 0.2-ml PCR tubes. After centrifuging briefly, the tubes were incubated on a thermal cycler in the following conditions: 95°C for 3 min, 98°C for 20 s, 50°C for 2 min, 72°C for 10 min, then cooled to 4°C.For the second-strand synthesis, the following PCR mix1 was prepared: 50.68 μl nuclease-free water (Qiagen), 15.2 μl 5× Q5 reaction buffer with MgCl2 (New England Biolabs), 1.9 μl of each dNTP (2.5 mM) (Takara Bio), 0.19 μl BTEP7v2 primer (100 μM), and 0.38 μl Q5 Hot Start High-Fidelity DNA polymerase (2 U/μl) (New England Biolabs). A 19-μl volume of PCR mix2 was added to each tube after the second-strand synthesis, pipetted to mix and briefly centrifuged. The tubes were incubated on a thermal cycler using the following conditions: an initial denaturation at 95°C for 3 min; followed by 22 cycles of 98°C for 10 s, 60°C for 30 s, and 72°C for 6 min, which extended by 6 s at 72°C in each cycle; and stored at 4°C.For the cDNA amplification, PCR mix2 was prepared, containing 12.73 μl nuclease-free water (Qiagen), 3.8 μl 5× Q5 reaction buffer with MgClAfter the PCR amplification, the cDNA libraries were purified using a Purelink PCR purification kit with Binding Buffer High-Cutoff (Thermo Fisher Scientific), according to the manufacturer's instructions. To check availability of each sample, the quantity and quality of the cDNA libraries were measured using a Bioanalyzer 2100 with a High Sensitivity DNA kit . Each cDNA library solution was placed in a 1.5-ml DNA Lo-bind tube (Eppendorf) and adjusted to a volume of 35 μl with elution buffer (EB) containing 10 mM Tris–HCl (pH 8.0). To remove the byproducts in the cDNA libraries, a 0.55× volume of SPRIselect beads  were added to each cDNA library solution, which adhered the appropriately sized cDNAs. The tubes were placed on a Magna stand  for 3 min, and the beads collected at the bottom of the tube. The supernatants were gently removed by aspiration, after which the beads were rinsed twice with 80% ethanol. After air-drying for 10 min, the beads were resuspended in 50 μl EB then left to stand on the Magna stand for 3 min. The resulting supernatants were recovered into new 1.5-ml DNA Lo-bind tubes. Purification with the SPRIselect beads was carried out at least three times. The quantity and quality of the purified cDNA libraries were measured using a Bioanalyzer 2100 with a High Sensitivity DNA kit (Agilent Technologies), and the purified cDNA libraries were stored at –30°C until required. The oligo DNAs used in this work are listed in In order to fragment the cDNAs to construct the NGS libraries, 2.5 nmol each of four or five purified cDNA libraries were combined, and the volume of the resulting solution was increased to 75 μl with EB. The mixtures were transferred into microTUBE AFA Fiber Pre-Snap-Cap tubes  and care was taken to prevent any air bubbles. cDNA shearing with a target peak of ∼400 bp was carried out using an acoustic solubilizer, Covaris S2 (Covaris), under the following conditions; bath temperature: 4–8°C, degassing mode: continuous, power mode: frequency sweeping, duty cycle: 10%, intensity: 3, cycles/burst: 200, and time: 90 s. After this treatment, the fragmented cDNAs were transferred to new 1.5-ml DNA Lo-bind tubes (Eppendorf) and purified with a MinElute PCR purification kit (Qiagen). The quality of the fragmented cDNA was measured using a Bioanalyzer 2100 with a High Sensitivity DNA kit (Agilent Technologies).To recover the fragmented cDNAs tagged with biotin, 20 μl of streptavidin-linked beads, Dynabeads MyOne C1 (Thermo Fisher Scientific), were rinsed twice with 2 × BWT buffer containing 10 mM Tris–HCl (pH 7.5), 1 mM EDTA, 2 M NaCl, and 0.02% Tween-20, then suspended in an equal volume of the fragmented cDNA solution. The solutions were left to stand for 10 min to bind the biotinylated cDNA fragments, then placed on a Magna stand for 30 s. The supernatants were discarded and the beads were rinsed three times with 1× BWT buffer and resuspended in 25 μl EBT buffer, which contained 10 mM Tris–HCl (pH 8.5) and 0.02% Tween-20.For the end repair, a 25-μl mixture containing 5 μl 10 × NEBnext End Repair reaction buffer (New England Biolabs) and 2.5 μl of NEBnext End Repair Enzyme Mix (New England Biolabs) was added to the mixture of beads and cDNA fragments. The solution was mixed by gently pipetting and centrifuging briefly, then incubated at room temperature with shaking at 400 rpm for 30 min. The tubes were stood on a Magna stand for 30 s and the supernatants were discarded. The beads were then rinsed twice with EBT buffer while on the Magna stand, after which the stand was removed to enable the beads to be resuspended in 21 μl EBT buffer.For the dA-tailing, a commercial reagent kit, NEBNext dA-tailing Module (New England Biolabs), was utilized. A 4-μl aliquot of a mixture containing 2.5 μl 10 × NEBnext dA-Tailing reaction buffer (New England Biolabs) and 1.5 μl of Klenow Fragment (3′ -> 5′ exo-) (New England Biolabs) was added to the solution of beads with end-repaired cDNA fragments. The mixtures were pipetted gently to mix and briefly centrifuged before being incubated at 37°C with shaking at 400 rpm for 30 min. To remove the reaction mix, the tubes were stood on a Magna stand for 30 s and the supernatants were discarded. The beads were rinsed twice with EBT buffer and resuspended in 25 μl EBT buffer.To ligate the adapters to the cDNA, a 25-μl mixture containing 5 μl 10 × T4 DNA ligase buffer (New England Biolabs), 1.5 μl RP1 adaptor v2 (100 μM), and 5 μl of 400 U/μl T4 DNA ligase (New England Biolabs) was added to the solution containing the beads and dA-tailed cDNA fragments. The solutions were mixed by gently pipetting then centrifuged briefly, after which they were incubated at 20°C with shaking at 400 rpm for 20 min. A 5-μl aliquot of 1 U/μl USER enzyme mix (New England Biolabs) was added to each tube, pipetted gently to mix, then incubated at 37°C with shaking at 400 rpm for 60 min. To remove the reaction mix, the tubes were stood on a Magna stand for 30 s and the supernatants were discarded. The beads were rinsed twice with EBT buffer and resuspended in 25 μl EBT buffer.To fill the 5′ overhang in the cDNA, a 5-μl mixture containing 3 μl 10× NEB buffer 2 (New England Biolabs), 1 μl of a mix containing 2.5 mM of each dNTP (Takara Bio), and 1 μl of 10 U/μl DNA pol I (New England Biolabs) was added to the solution of adaptor-ligated cDNA fragments and beads. After pipetting gently to mix and centrifuging briefly, the tubes were incubated at 37°C with shaking at 400 rpm for 30 min. To remove the reaction mix, the tubes were stood on a Magna stand for 30 s and the supernatants were discarded. The beads were then rinsed twice with EBT buffer and resuspended in 25 μl EBT buffer.For the library enrichment, a PCR mix3 was prepared containing 50 μl nuclease-free water (Qiagen), 20 μl 5 × KAPAHiFi reaction buffer , 3 μl a mix containing 10 mM of each dNTP (KAPA Biosystems), 3 μl P5RP1 primer (100 μM), 3 μl EP7v2 primer (100 μM), and 2 μl of 1 U/μl KAPAHiFi Hot Start DNA polymerase . The 25-μl mixture of 5′-end-filled cDNAs and the beads was transferred into a new 0.2-ml PCR tube and mixed with 75 μl of PCR mix 3 and centrifuged briefly. The beads were resuspended by pipetting, after which the tubes were immediately set on a thermal cycler and a PCR was performed using the following conditions: an initial denaturation at 95°C for 2 min; followed by 10 cycles of 98°C for 20 s, 63°C for 30 s, and 72°C for 30 s; with a final extension at 72°C for 5 min, after which the samples were stored at 4°C. The enriched libraries were purified with a MinElute PCR purification kit (Qiagen) and eluted with 28 μl EB, according to the manufacturer's instructions.The NGS libraries were next subjected to size selection, where fragments measuring 300 bp to 800 bp were electrophoretically recovered from the enriched NGS libraries using BluePippin  with a 1.5% dye-free agarose gel cassette and the internal standard R2, according to the manufacturer's instructions. Size-selected NGS libraries were purified with a MinElute PCR purification kit (Qiagen) and eluted in 28 μl EB, according to the manufacturer's instruction. The quantity and quality of the NGS libraries were determined using a Bioanalyzer 2100 with a High Sensitivity DNA kit (Agilent Technologies). The oligo DNAs used in this work are listed in NGG: pENTR::NGG (5.2 kb), PpCYCD;1 (AJ428953): pJET::PpCYCD;1 (4.5 kb), PpEF1α (XM_001753007): pJET::PpEF1α (4.9 kb) and PpTUA1 (AB096718): pphb6e07 (4.9 kb). Using the molecular weight of the plasmids, the copy numbers of the transcripts were calculated as follows: weight in Daltons (g/mol) = (bp size of plasmids) (615[Da/bp]). Hence, (g/mol)/Avogadro's number = g/molecule = copy numbers. This calculation produced copy numbers equivalent to the double-stranded DNA. These experiments were carried out and evaluated using three sets of experimental replicates. Missing values were substituted for a value one-tenth of the minimum value of each transcript level.Each 20-μl qPCR mixture contained 2 μl of the cDNA templates, LightCycler 480 SYBR Green I Master mix (Roche), and 0.5 μM of each primer, which are listed in A 10-μl aliquot of 10 nM sequence libraries consisting of either 32 cells facing the cut edge of the leaf at 0 h after its excision or 34 cells facing the cut at 24 h after leaf excision was denatured and loaded into a lane of the flow cell on a HiSeq1500 sequencer , following the manufacturer's instructions. The SBS condition in HighOutput v4 was 126 bp of Read1 and 20 bp of index read. The output data in the BCL files were converted to fastq files of read1 and the index read using bcl2fastq package v1.8.4 (Illumina), and the NGS data were deposited in DDBJ (accessions DRA006455 and DRA006456).https://github.com/tomoakin/UMI_SC). The 10-bp UMI sequences were inserted into the name of read1, then the sequences were demultiplexed using 6 bp or 8 bp within the trimmed index read for each sample. The read1 sequences were then trimmed and filtered with the trimmomatic package (v0.36) .For the sequences with 18 bp of index reads, 8 bp were the multiplex index and 10 bp were the UMI. Therefore, 2 bp of 20 bp of index reads were trimmed from the 3′ end of the reads in the fastq files, using the cutadapt package . To coun (v0.36)  using th (v0.36)  and expo (v0.36)  , ELONGATION FACTOR 1α (PpEF1α), and TUBULIN α1 (PpTUA1)  and PpTUA1 (s = 7.9431), which are known to be upregulated during reprogramming (NGG (s = 2.8616) and PpEF1α (s = 1.9492) were mostly stable in all contents, as expected. The variation in the PpCYCD;1 and PpTUA1 transcripts suggests that the isolated single cells include those in different stages of reprogramming as well as those not undergoing reprogramming, as previously observed  . The tragramming ,21, variobserved . These robserved .10 molecules) for sequencing on Illumina next-generation sequencers, we amplified the single-cell cDNAs using PCR. Two methods of attaching an adaptor to the 3′ ends of the first-strand cDNAs were evaluated: template switching , even if the UMIs were not unified (R2 = 0.9743). On the other hand, the read counts in the 20-pg libraries tended to vary if the UMIs were not unified (R2 = 0.8610); however, the UMI counts of each gene were strongly correlated between duplicated samples, to a similar level as those of the 5-μg libraries (R2 = 0.9677). We found similar tendencies for replicates of the External RNA Controls Consortium (ERCC) RNA spike-in mix, which were added to the total RNA samples as an external control ..R2 = 0.9 control .To apply the methods described above to individual Physcomitrella leaf cells, we made cDNA libraries from single cells facing the cut of an excised leaf after 0 and 24 h Figure . The celAfter quantification and qualification, these batches of NGS libraries were equally mixed and sequenced on an Illumina HiSeq sequencer set to generate 126-bp single-end reads and 18-bp index reads, including 8 bp of multiplex index and 10 bp of UMI. A total of 384 993 923 reads were obtained and cleaned by trimming and filtering to remove inaccurate sequences and ribosomal sequences, respectively. As a result, 98.1% (377 495 213) of the reads were sorted to their respective samples using the multiplex index sequences, generating 2.8–8.5 million reads per sample for 66 samples . MappingGLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE and α-TUBULIN), were found to fluctuate substantially at the single-cell level  in the 1cell-DGE data taken at 0 h and 24 h after leaf excision, we carried out a statistical analysis after normalization using the iterative differentially expressed gene exclusion strategy (iDEGES) method . A totalWe performed a gene ontology (GO) term enrichment analysis for the 1978 of the total 2382 DEGs at 0 h and 3648 of the total 4000 DEGs at 24 h that were putatively homologous to annotated Arabidopsis genes Figure . Using GIn previous studies, transcriptome analyses of whole excised leaves during reprogramming were performed using 5′DGE ,21. We tWe then checked the expression levels of the top 10 DEGs detected by the statistical significance of their differences at 0 and 24 h using a q-value Figure . Pp3c23_In order to take full advantage of single-cell transcriptome data, it is possible to calculate the pseudotime, a hypothetical time scale estimating the transition between cell states during development and differentiation based on similar gene expression profiles ,36. FirsPpCSP1, PpCSP2 and PpCYCD;1 , but found a moderate correlation between the number of detected genes and the pseudotime   has previously been successfully performed with cDNA derived from the contents of single cells extracted from plant leaf cells using glass microcapillaries ; therefoAnother issue was how to prepare cDNA from a small amount of RNA without excessive amplification bias which depends on the length, nucleotide contents, and sequences of the cDNAs . Using cThe trace amount of first-strand cDNA generated from the RNA of single cells necessitates their amplification before they can be sequenced. To overcome an amplification bias, we introduced sequences of 6 or more random nucleotides, UMIs, to the cDNAs to enable their later discrimination Figure  23). We. We23). Physcomitrella protonema tissues. The 5-μg samples showed relatively low unified rates of 6.95% and 8.34%, which indicated that the starting mRNA materials were sufficient for the preparation of cDNAs with different UMIs (2) between the read counts and the UMI counts .The package UMI_SC is an open source available in the GitHub repository , PpEF1α (XM_001753007), and PpTUA1 (AB096718); pphb6e07 are available from the corresponding author Mitsuyasu Hasebe (mhasebe@nibb.ac.jp) at the National Institute for Basic Biology (NIBB), Japan.The wild-type moss den 2004  and the den 2004 , as wellSupplementary DataClick here for additional data file.


The corresponding biological consequences and lipidomic research on these positional monounsaturated fatty acid (MUFA) isomers are under development. Furthermore, sapienic acid can bring to the de novo synthesis of the n-10 polyunsaturated fatty acid (PUFA) sebaleic acid , but such transformations in cancer cells are not known. The model of Caco-2 cell line was used to monitor sapienic acid supplementation (150 and 300 μM) and provide evidence of the formation of n-10 fatty acids as well as their incorporation at levels of membrane phospholipids and triglycerides. Comparison with palmitoleic and palmitic acids evidenced that lipid remodelling was influenced by the type of fatty acid and positional isomer, with an increase of 8cis-18:1, n-10 PUFA and a decrease of saturated fats in case of sapienic acid. Cholesteryl esters were formed only in cases with sapienic acid. Sapienic acid was the less toxic among the tested fatty acids, showing the highest EC50s and inducing death only in 75% of cells at the highest concentration tested. Two-photon fluorescent microscopy with Laurdan as a fluorescent dye provided information on membrane fluidity, highlighting that sapienic acid increases the distribution of fluid regions, probably connected with the formation of 8cis-18:1 and the n-10 PUFA in cell lipidome. Our results bring evidence for MUFA positional isomers and de novo PUFA synthesis for developing lipidomic analysis and cancer research.Palmitic acid metabolism involves delta-9 and delta-6 desaturase enzymes forming palmitoleic acid (9 Laurdan, 2-dimethylamino(6-lauroyl)naphthalene ; other reagents used were from Carlo Erba, Milan, Italy.Sapienic acid and the corresponding methyl ester, 8Silica gel analytical and preparative thin-layer chromatography (TLC) was performed on Merck silica gel 60 plates, 0.25 mm thickness, and spots were detected by spraying the plate with cerium ammonium sulfate/ammonium molybdate reagent.cis-18:1 and 5cis,8cis-18:2 data are given in the S1 protocol reported in Fatty acid methyl esters (FAME) were analysed by GC  equipped with a 60 m × 0.25 mm × 0.25 µm (50%-cyanopropyl)-methylpolysiloxane column , and a flame ionization detector with the following oven program: Temperature started from 165 °C, held for 3 min, followed by an increase of 1 °C/min up to 195 °C, held for 40 min, followed by a second increase of 10 °C/min up to 240 °C, and held for 10 min. A constant pressure mode (29 psi) was chosen with helium as carrier gas. Methyl esters were identified by comparison with the retention times of authentic samples. Calibration procedures for quantitative analyses followed reported procedures , using aDimethyl disulphide adducts of FAME were analysed by GC-MS  equipped with a 15 m × 0.25 mm × 0.25 µm TG-SQC 5% phenyl methyl polysiloxane column, with helium as carrier gas, coupled to a mass selective detector (Thermo Scientific ISQ) with the following oven program: Temperature started at 80 °C, maintained for 2 min, increased at a rate of 15 °C/min up to 140 °C, increased at a rate of 5 °C/min up to 280 °C and held for 10 min.Viability was evaluated by measuring absorbance at 490 nm by a microtiter plate reader Multiskan EX . Phase contrast microscopy was carried out with a Nikon Eclipse TS100  microscope equipped with a digital camera. Flow cytometry analysis was performed on a FACSAria BD Analyser using FACSDiva software .Laurdan intensity images were obtained with an inverted confocal microscope  using a 63× oil immersion objective (NA 1.4) under excitation at 800 nm with a mode-locked Titanium-Sapphire laser . Internal photon multiplier tubes collected images in an eight bit, unsigned images at a 400 Hz scan speed. Laurdan intensity images were recorded simultaneously with emission in the range of 400–460 nm and 470–530 nm and imaging was performed at room temperature. A stack consisting of 10 z-sections every 2 µM was acquired for each field of view. Frequency represents the normalized number of pixels per cell having a specific GP value. The analysis was performed on n = 80 cells per sample before and after 4 h of the specific treatments.® Number: HTB-37™), derived from a human colorectal adenocarcinoma were from the departmental cell collection. Cells were cultured at 37 °C in humidified atmosphere at 5% CO2 in RPMI 1640 supplemented with 10% heat-inactivated FCS, 2 mM l-glutamine, 100 units/mL penicillin, 0.1 mg/mL streptomycin (hereafter referred to as complete medium). Cultures were maintained in the log phase of growth with a viability >95%. Cells were checked for the absence of mycoplasma infection.Caco-2 cells (ATCC3 cells/well) were seeded onto 96-well microtiter plates in 100 µL of complete medium. After 24 h, the medium was removed from each well and the fatty acids, dissolved in ethanol, adjusted at the final concentration, ranging from 100 to 300 µM in complete medium, were immediately added to cells . Control samples were carried out in presence of ethanol at the same concentration in free fatty acid samples. Moreover, no variations in cell viability were observed in control cells incubated in the presence or in the absence of ethanol. Viability was determined after the indicated times by adding 20 µL/well of CellTiter 96 Aqueous One Solution Cell Proliferation Assay, as described in [Caco-2 cells (2 × 10ribed in . The abs50 (fatty acid effective concentration to reduce cell viability by 50% after 48 h) and ET50 (time required to reduce cell viability by 50%) were calculated using linear regression analysis.Time-course experiments were performed using the first concentration of each fatty acid causing a cell viability reduction ≥75% after 48 h from the fatty acid supplementation . Cells were exposed to fatty acids for times ranging from 2 to 96 h. The EC5/3 mL) were seeded in 25 cm2 flasks and, after 24 h, treated with fatty acids. After treatment, the cells were centrifuged at 500× g for 5 min, washed in 2 mL complete medium, centrifuged again and stained according to the manufacturer’s instructions. Within 30 min, the cells were analysed through flow cytometry on a FACSAria BD Analyser using FACSDiva software version 4.1, 2004 . The morphological analysis of the treated cells was conducted through phase contrast microscopy directly in 96-well plates using a Nikon Eclipse TS100 microscope.Cell viability was also evaluated at 24 h in Caco-2 cells pretreated with Z-VAD (100 µM) or Nec-1 (100 µM), added to cells 3 h before fatty acid supplementation. AnnexinV and Propidium iodide positivity were detected through flow cytometry, using the AnnexinV-EGFP Apoptosis Kit, as described in . The cel3/well) were seeded onto 96-well microtiter plates in 100 μL of complete medium. After 24 h, cells were supplemented with 150 µM palmitic acid, 250 µM palmitoleic acid and 300 µM sapienic acid. After incubation for the indicated time, the medium was removed from each well and replaced with 50 µL of complete medium and 50 µL of Caspase-Glo™ 3/7 [The activity of caspase 3/7 was assessed by the Caspase-Glo™ 3/7 luminescent assay. The cells (2 × 10Glo™ 3/7 . Lumines6/20 mL) were seeded in 75 cm2 flasks and, after 24 h, the medium was supplemented with 150 or 300 µM palmitic acid, 250 µM palmitoleic acid or 300 µM sapienic acid. After 180 min, cells were harvested with a cell scraper, collected by centrifugation at 500× g for 5 min and washed twice in PBS. Cell pellets were lysed with 100 µL of Cell Lytic-M supplemented with the Protease Inhibitor Cocktail (1:100), Phosphatase Inhibitor Cocktail 1 (1:100) and sodium-orthovanadate (1:500) (Sigma-Aldrich). After 45 min at 0 °C and vortexing every 5 min, insoluble material (nuclear pellet plus membranes) was removed by centrifugation at 12,000× g for 30 min at 4 °C. Protein supernatant (cell lysate) was collected and stored at −80 °C. Protein content was quantified by spectrophotometer using Bradford’s method  and 80 µg/lane of protein were separated by SDS-PAGE (10% gel) and blotted to nitrocellulose membrane (150 V for 90 min). Non-specific antibody binding sites were blocked by incubation with blocking buffer, TRIS buffered saline, 0.1% Tween 20 (TBS/T) with 5% w/v non-fat dry milk, for 1 h at room temperature. After five washes with TBS/T, membranes were incubated overnight at 4 °C with anti-phospho-cPLA2 (Ser 505) or phospho-p38 mAbs  diluted in TBS/T with 5% bovine serum albumin, according to the manufacturer’s instructions. After a further five washes with TBS/T, membranes were incubated for 1 h at room temperature with horseradish peroxidase–conjugated anti-rabbit antibody (Sigma-Aldrich) diluted in blocking buffer. After a further five washes, proteins were detected by incubating the membrane with Immobilon Western detection reagent . The anti-phospho antibodies were then stripped for 30 min in 25 mM glycine-HCl pH 2, 1% SDS (w/v), and, after blocking with non-fat milk, the membrane was re-incubated with antibodies recognising total cPLA2 or total p38  to account for equal loading. Proteins were detected as above. The relative levels of expression of different proteins were determined using ImageJ software [Cells (3×10software .6 millions of cells) in a 1.5 mL vial was added with tridistilled water (2 mL) and 2:1 chloroform/methanol (4 times × 4 mL of 2:1 chloroform/methanol mixture) to extract lipids according to the Folch method [2SO4 evaporated to dryness, gave the total lipid extract that was checked by thin layer chromatography for lipid class separation as previously reported [n-hexane (3 times × 2 mL of n-hexane), dried on anhydrous Na2SO4, evaporated to dryness and analysed by GC in comparison with standard references. Detailed fatty acid compositions of each lipid class identified in the experiments with 150 and 300 µM palmitic, palmitoleic and sapienic acids supplementations are listed as µg/mL in A constant amount of cells (counting 6×10h method . The orgreported . In somen-hexane (50 μL), 100 μL of dimethyl disulfide and 2 drops of a 6% solution of iodine in diethyl ether were added. The reaction was stirred at room temperature for 30 min, under argon atmosphere. Then 1 mL of n-hexane and 1 mL of a 5% aqueous solution of sodium thiosulphate were consecutively added. The organic phase was isolated, dried over anhydrous Na2SO4, and concentrated under gentle stream of nitrogen, before the GC-MS analysis naphthalene) stock solution per milliliter of RPMI 1640. The stock solution concentration was 1 mM in dimethyl sulfoxide (DMSO) and was renewed every three weeks. After 30 min equilibration in the cell incubator, the medium was supplemented with 150 µM palmitic acid, 150 µM palmitoleic acid, and 150 µM sapienic acid. Imaging was performed before treatment and after 4 h. As a normalized ratio of the intensity at the two emission wavelengths regions, the generalized polarization (GP) provides a measure of membrane order, in the range between −1  and +1 (gel). The GP, defined asI(400–460) and I(470–530)) by using the program Ratiometric Image processor [G was obtained from the GP values of solutions of Laurdan in DMSO. G factor had approximately 2% variation across the imaging area. GP images (as eight-bit unsigned images) were pseudo-coloured in ImageJ software . Background values (defined as intensities below 7% of the maximum intensity) were set to zero.Cells were seeded in uncoated Petri dish  at 10rocessor ,44. The p ≤ 0.05). Data were analysed by ANOVA and ANCOVA with Bonferroni’s correction, followed by a comparison with Dunnett’s test. Analysis of acquired images for the microscopy experiments were performed with ImageJ. For GP data, mean ± SD values were determined and utilized for two-tailed Student’s t-test analysis. GP were averaged over n = 80 cells per sample. Fatty acids results are given as mean ± SEM (standard error of the mean). Statistical analysis was performed using GraphPad Prism 5.0 software . We used non-parametric unpaired t-test two-tailed with 95% confidence interval.Results by cell experiments are given as mean values ± SD. Statistical analyses of data from cell viability, caspase experiments and western blot analysis were conducted using the XLSTAT-Pro software, version 6.1.9  with a 95% confidence interval (n-10 C18 MUFA and a n-10 PUFA, increasing the unsaturated fatty acid levels in the cell lipidome, contributing to an increased fluidity and improving cell survival. These aspects must be investigated in detail in different cancer cells and conditions. The results here shown for Caco-2 cells open novel perspectives for lipidomic studies in health and diseases.Our work offers the first evidence that sapienic and palmitoleic acids are two positional hexadecenoic fatty acid isomers with a distinct metabolism in human Caco-2 cells, the former being rapidly converted to