HeLa cell lines carrying dCas9 and CasRx constructs were generated in the same way, except that they were seeded at 0.2106 cells in 6-well plates for reverse lipofection. Another example for a gene-dosage sensitive process is the differentiation of pluripotent stem cells into different lineages. Stable cell lines expressing the constructs shown in (a) together with Esrrb-targeting and non-targeting guides were generated as indicated. BMC Biotechnol. & Smith, A. G. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. ac Oct4 titration in mESCs (1.8XX Nanog-mCherry cell line expressing FKBP12F36V-hHDAC4-dCas9) for 3 days. Natl. GFPuv (a variant ofAequorea victoriagreen fluorescent protein) is optimized for maximal fluorescence when excited by UV light [360400 nm]. Tramier, M., Zahid, M., Mevel, J.-C., Masse, M.-J. 2). By submitting a comment you agree to abide by our Terms and Community Guidelines. Using this platform, we evaluated the performance of 6 different degron domains, each fused N-terminally to dCas9: AID29, mAID30, SMASh31, FKBP12F36V27, ecDHFR32 and ER5033. A 69, 9209 (2006). Takara Bio USA, Inc. provides kits, reagents, instruments, and services that help researchers explore questions about gene discovery, regulation, and function. Chem. Anderson, K. I., Sanderson, J., Gerwig, S. & Peychl, J. In contrast, recombinant DsRed-Express protein elutes earlier than DsRed-Monomer because of its tetrameric structure. Rev. Ideally, when you label a protein of interest, the fluorescent tag itself should not interfere with the biological function of the target protein. 10, 114 (2020). ZsGreen1 is our brightest green fluorescent protein, significantly brighter than EGFP. Stoichiometric imbalance in the receptor complex contributes to dysfunctional BMPR-II mediated signalling in pulmonary arterial hypertension. When analysing derepression upon dTAG-13 addition after 4 days of culture without the degrader, all constructs were rapidly depleted with a half time of <1h (Fig. Genome-scale CRISPR-mediated control of gene repression and activation. We have established three stably transfected HEK 293 cell lines with different levels of mCherry expression (as measured by flow cytometry). Biol. Movie of cell cycle progression visualized with Fucci probes. Such sequence was produced by PCR amplifying a 120 bp-long oligonucleotide (template) using two compatible primers to extend the sequence to its final length (157bp, including 3 additional nucleotides at each site to favour restriction enzyme activity). MathSciNet Without the addition of a functional promoter, this vector willnot express the Fluorescent Timer. Cookie Management. "Fluorescent timer": protein that changes color with time. Figure 2. tdTomato, but not GFP, can be detected in the SCID mouse cadaver phantom model. 1a). Aliquots were kept at 20C. 20 l of the mCherry Flow Cytometer Calibration Bead Suspension was thoroughly resuspended in 1ml of 1X Flow Cytometer Calibration Beads Dilution Buffer. PubMed As with our other red fluorescent proteins, DsRed-Monomer performs well when multiplexed with compatible fluorescent proteins such as AcGFP1 (Figure 5). Although phase separation has been suggested as regulatory mechanisms for a variety of cellular processes, its occurrence and functional importance in vivo is often debated59. mCherry, TdTomato, DsRed, mStrawberry, and AsRed. Thank you for visiting nature.com. =Arbitrary Fluorescence Units. Panels B and E. Transformed maize leaf and root under white light and fluorescent light. 2008). pTimer (left) is primarily intended to serve as a convenient source of the Fluorescent Timer cDNA. Sucrose density gradient ultracentrifugation yields a fractionation profile consistent with a monomeric protein (Figure 2, Panel B). S11). Pseudonative gel electrophoresis of recombinant AcGFP1 protein in comparison to an oligomeric fluorescent protein supports the same conclusion (Figure 2, Panel C). HeLa cells were transiently transfected via a lipid-based method, with mammalian expression vectors encoding mCherry fused to either human cytoplasmic actin (Panel A) or tubulin (Panel B). Cell 159, 647661 (2014). (1) describes the expression of the Cas-Repressor R and Eq. Natl Acad. Mononuclear bone marrow cells were transduced with retroviral vectors encoding DsRed-Express (Panel A), DsRed-Express2 (Panel B), or EGFP (Panel C); and fluorescent cells were sorted 87 hr later. 28, 687704 (2008). The system employs the FKBP12F36V degron to precisely control Cas-repressor abundance by varying the concentration of the dTAG-13 ligand. Lastly, for genes repressed by NANOG or OCT4, the equation is written as: No statistical method was used to predetermine the sample size. 1e). Boiled and unboiled recombinant proteins (7.5 g) were separated by SDS-PAGE electrophoresis (12% acrylamide). No more tradeoffsperform reporter studies with both a low background and a broad dynamic range. Panel A. Recombinant DsRed-Express and DsRed-Monomer (100 g) were analyzed by FPLC gel-filtration chromatography. All systems could be homogeneously titrated (tBFP), but again striking differences in the distribution of target gene repression (EGFP) were observed (Fig. designed and cloned most of the degron-dCas9-tRFP-P2A-tBFP plasmids. ZsGreen1 was used to label human estrogen receptor-positive breast cancer cells, and fluorescent whole-body imaging on living, unanesthetized, immunocompromised nude mice identified lymph node metastases that arose from estrogen-dependent tumors grown orthotopically in mammary glands. Since \({\alpha }_{R}\) is modulated by the dTAG-13 concentration, we estimated the parameter both in the presence (\({\alpha }_{R+}\)) and in the absence (\({\alpha }_{R-}\)) of the ligand from time course data upon dTAG-13 addition and withdrawal, respectively. A two-sided t-test between each dTAG-13 condition compared to the 500nM dTAG-13 condition was performed. Acad. This finding is in agreement with previous reports analysing heterozygous Oct4 mutant cell lines8,41. Biotechnol. =Arbitrary Fluorescence Units. Two of the most commonly used fluorophores in flow cytometry, R-phycoerythrin (R-PE) and allophycocyanin (APC), are phycobiliproteins originally derived from algae. Silva, J. et al. The observed bimodal repression pattern is thus unlikely to arise from heterogeneity in repressor levels, but appears to be an inherent property of the repression mechanism. A.F.U. b Bg-subtr. 2002). The different designs compared in (bg) are shown on the right. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval. Ono, S. Mechanism of depolymerization and severing of actin filaments and its significance in cytoskeletal dynamics. Its excitation and emission maxima are 574 nm and 596 nm, respectively. & Fedoroff, N. Arabidopsis primary microRNA processing proteins HYL1 and DCL1 define a nuclear body distinct from the Cajal body. To induce repression, the dTAG-13 degrader was withdrawn for 4 days (Fig. Biol. Cell 129, 813822 (2007). The black curve represents the most likely Hill curve fitted using a non-linear least square approach (see Methods). Nat. Alternatively, engineered variants of Cas13 with reduced collateral activity have been recently reported, which could be easily used to substitute the original CasRx used in our study58. Thus, the migration of mCherry . Use pTimer-1 (right) to monitor transcription from different promoters and promoter/ enhancer combinations inserted into the MCS located upstream of the Fluorescent Timer coding sequence. The dynamics of repression upon ligand withdrawal and derepression upon ligand addition depend on the repressor dynamics, but also on the speed with which the repressed state is established and erased, respectively. =arbitrary fluorescence units. The same filament pattern was observed with DD-AcGFP1-Actin as with mCherryActin (Figure 3, Panels CD and GH). Insert and pLentiRNAGuide_001 were linearised with the BsmBI restriction enzyme and ligated using T4 DNA ligase (NEB #M0202S). Tramier, M., Zahid, M., Mevel, J.-C., Masse, M.-J. We refer to repression tunability as the ability to partially repress a target gene, resulting in stable intermediate expression levels. Since incomplete compensation can affect quantification of transfection efficiency when flow cytometry is used, we avoided use of fluorochromes with spectral overlap (1). Methods 13, 10431049 (2016). Figure 4. RFP-Trap Magnetic Particles M-270 | Proteintech - ptglab a Experimental design: degron-Cas-repressor cell lines expressing ESRRB-mCherry are transferred from medium containing high concentration of dTAG-13 (repressor degraded) to media with a range of dTAG-13 concentrations. We indeed observed induction of the TE program and loss of mESC markers, when Oct4 levels dropped below ~30% (Fig. and E.G.S. Nature 347, 449 (1990). For CasRx and to a lesser extent, for dCas9-hHDAC4, by contrast, mCherry and tBFP levels were negatively correlated, suggesting that cell-to-cell variability in repressor levels results in variable repression strength (Fig. DNA constructs were first packaged into lentiviral particles. Far red proteins such as HcRed1, mRaspberry, and mPlum are preferred forin vivoimaging since they avoid the natural green autofluorescence produced by plant and animal cells. Background-corrected MFI values of Cas-Repressors (tBFP) were scaled between 0 and 1 (min-max scaling), while the target gene (mCherry) was computed as a fold change relative to levels in the absence of repression (500nM dTAG-13). Sci. We can conclude that the sensitivity to NANOG dose is variable among target genes. (B) Alternative emission filters for the detection of EYFP/Venus with EGFP; RFP657 with mIFP; mApple with mCherry; and mCherry with tdTomato. Miyanari, Y. All trademarks are the property of Takara Bio Inc. or its affiliate(s) in the U.S. and/or other countries or their respective owners. Anyone you share the following link with will be able to read this content: Sorry, a shareable link is not currently available for this article. 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Biochem. 6f). 66, 17661776 (2000). The KRAB domain thus appears to be the most potent repressor of the three. Black dots are single replicates. An account with takarabio.com entitles you to extra features such as: Creating and saving shopping carts Keeping a list of your products of interest Saving all of your favorite pages on the site* Accessing restricted content. 6e) and also categorised them as undifferentiated, differentiated or mixed (Fig. 4, 1451 (2013). Navarro, P. et al. In this way, treatment with variable concentrations of different degrader molecules would allow simultaneous independent tuning of two endogenous genes. NOT FOR USE IN DIAGNOSTIC PROCEDURES. Although if sensitivity is an issue, then try to find an appropriate negative control and compensate the mCherry out -- this will help with detecting lower signals (it's like autofluorescence compensation to increase sensitivity against high autofluorescence). Chem. We then used qRT-PCR to quantify the expression of NANOG target genes Esrrb, Rex1 (Zfp42), Nrb01, Sox2 and Klf4, which have been reported to be activated by NANOG42, and of Xist, which is known to be repressed by NANOG43 (Fig. Detecting protein-protein interactions requires a good (high-efficiency) FRET pair: a donor with a high quantum yield (QD) and an acceptor with a high Frster radius (R0). For the NANOG titration experiment, cells were first seeded at a densityof 3104 cells/cm2 for 3 days at different dTAG-13 concentrations. Use adenoviral vectors to transduce dividing and non-dividing cells. A new configuration of the Zeiss LSM 510 for simultaneous optical separation of green and red fluorescent protein pairs. Lin, Y., Irani, N. G. & Grotewold, E. Sub-cellular trafficking of phytochemicals explored using auto-fluorescent compounds in maize cells. b Schematic overview of degron-dCas9 comparison as shown in (cg). In the absence of Shield1, DD-AcGFP1-Actin is not present (Panels B & F) despite a normal, mCherry-labeled actin filament network (Panels A & E). CasTuner is a degron and CRISPR/Cas-based toolkit for analog - Nature Study promoter activity using the Living Colors Fluorescent Timer, a fluorescent protein that shifts color from green to red over time (Terskikh et al. Figure 1. Flow cytometer calibration beads for AcGFP1/EGFP and mCherry - Takara Bio Actin is a 42-kDa protein that forms a filament system that spans the cell and is continuously self-assembling and disassembling (reviewed in Holmes et al. Natsume, T., Kiyomitsu, T., Saga, Y. Biol. reef coral, which makes it the optimal choice for use as a red fluorescent fusion tag. FPLC gel-filtration chromatography of recombinant AcGFP1 yields a single elution peak (Figure 2, Panel A). Different values were tested for \(\varDelta t\), ranging from 0 to 25h and numerical solutions were compared to the experimentally measured mCherry levels through computing the Mean Absolute Error (MAE). f Normalised 2D density plots showing tBFP and mCherry levels in populations of cells treated with different dTAG-13 concentrations (indicated on the right). The experiments were not randomised. Please see the Kit Components List to determine kit components. Metastatic lung tumor expressing ZsGreen1. We sought to create a tool for tuning endogenous gene expression that can be easily applied to any target gene. The images or other third party material in this article are included in the articles Creative Commons license, unless indicated otherwise in a credit line to the material. smallest) bandwidth required to obtain a kernel density estimation of the experimental data with a single mode. Although DsRed-Monomer is somewhat less bright than DsRed-Express, it is nevertheless an excellent choice for fluorescence microscopy imaging and flow cytometry. Our mission is to develop high-quality innovative tools and services to accelerate discovery. We offer a wide array of premade lentivirus particles for whole-cell labeling or for labeling subcellular compartments, such as mitochondria, golgi, membranes, etc. Res. Each bead suspension contains six distinct populations of beads that vary in the number of attached AcGFP1 or mCherry molecules, which gives each population a distinct fluorescence intensity. NOT FOR USE IN DIAGNOSTIC PROCEDURES. Similarly, the precise quantity of the pluripotency factor NANOG is critical for the control of naive and primed pluripotent states both in vitro and in vivo9,10. wrote the paper with inputs from R.A.F.G. When the cells were analysed using FACS tubes, at least 30,000 events were recorded. Cell Genom. The MFI values and fold changes reported are always after subtraction of background autofluorescence. by sensing a morphogen gradient)2. In the first steps, we aimed at identifying suitable degron and repressor domains. Existing visualization methods use markers such as fluorescently labeled actin, anti-actin antibodies, or fluorescently labeled phalloidin (a protein that specifically binds to actin filaments). The observed NANOG upregulation is seemingly at odds with the fact that OCT4 binds together with SOX2 to the Nanog promoter, thereby promoting its expression41,45. Flow Cytometry: An Overview - PMC - National Center for Biotechnology The flow-cytometry experiments were performed in 96-well plates seeding 10,000 cells per well and passaged 1:5 after 2 days for longer treatments. & Mitchison, T. J. Actin microfilament dynamics in locomoting cells. Curr. Shortly after its synthesis, the Fluorescent Timer begins emitting green fluorescence but as time passes, the fluorophore undergoes additional changes that shift its fluorescence to longer wavelengths. Cell 82, 22012214 (2022). Its open reading frame has been human codon-optimized to increase the translational efficiency of the AcGFP1 mRNA, which results in higher expression in mammalian cells. HDAC4 deacetylase associates with and represses the MEF2 transcription factor. TA183030S. Song, L., Han, M.-H., Lesicka, J. mESCs=mouse embryonic stem cells; EpiSCs=epiblast-like stem cells; PE=primitive endoderm; TE=trophectoderm. of 3 biological replicates together with the best fit with a Hill function (black line) is shown. & Mitchison, T. J. Actin microfilament dynamics in locomoting cells. Johnstone, C. N. et al. B., Micheelsen, M. A., Sneppen, K. & Thon, G. Theoretical analysis of epigenetic cell memory by nucleosome modification. designed and cloned all other plasmids, performed all the experiments and analysed the data. Transduction was performed in the absence of the targetable repressor protein (high dTAG-13). & Gorr, S. U. Oligomerization of green fluorescent protein in the secretory pathway of endocrine cells. }:seVM[S>pukVU*zKF5Sj)r.A)c9? Complex transcriptional modulation with orthogonal and inducible dCas9 regulators. 2006). A noncytotoxic DsRed variant for whole-cell labeling. Cells were fixed using 4% paraformaldehyde and imaged 36 hr posttransfection with a 40X objective on a Zeiss Axioskop microscope using the 575/50, 610, 640/50 filter set. The authors declare no competing interests. The fusion protein rapidly accumulates to detectable levels, in as few as 1520 minutes after adding Shield1 to the culture media (Banaszynski et al. For Esrrb, the shortest translated transcript isoform that contains all constitutive exons was used in the query (ENSMUST00000167891.1) and the 3 highest ranking guide sequences were selected. The mean and s.d. J. The oligomeric structure of proteins is preserved during SDS-PAGE analysis if samples are kept at 4C and not boiled prior to loading on a gel. Winnard, P. T., Kluth, J. Choose plasmids coding orange or yellow fluorescent proteins for use as tags or reporters. This color change provides a way to visualize the timeframe of promoter activity, indicating where in an organism the promoter is active and also when it becomes inactive. PDF MACS Comp Bead Kit, anti-REA - Miltenyi Biotec Although more complex circuits have been designed to improve quantitative control of gene expression (tunability)15, recent technological developments, such as conditional destabilising domains (degrons) and Cas9-based approaches seem to be more promising for tuning protein abundance16,17,18. Niwa, H., Miyazaki, J. MATH Its high solubility leads to low cytotoxicity. Monomeric fluorescent proteins such as DsRed-Monomer, mCherry, and mStrawberry are often ideal for fusions, as they tend to be the least likely to disrupt protein function. All degron-Cas-repressors were efficiently degraded with the exception of the C-terminal hHDAC4 construct (dCas9-hHDAC4), where tBFP levels were only reduced by ~56% (Fig. 2006). Picard, D., Suslova, E. & Briand, P.-A. 8, 15178 (2017). You can detect the Fluorescent Timer with the Living Colors DsRed Polyclonal Antibody. This relative tRFP/tBFP ratio was then expressed as a percentage. Cancer Res. Its t0.5 for maturation at 37C is approximately 2.5 hours. The calculated molecular weight of AcGFP1 is 26.9 kDa. Miyazaki, Y., Imoto, H., Chen, L. & Wandless, T. J. Destabilizing domains derived from the human estrogen receptor. 5g). In summary, we have generated a set of degron-controlled Cas-repressor constructs that can efficiently repress a target in a strictly inducible manner. PubMed Once the repressor was upregulated, we observed immediate target gene repression when using dCas9, hHDAC4-dCas9 and CasRx (t=0h), while a small delay was detected for the two KRAB-mediated systems (t=36h, Fig. To understand whether target repression and derepression is immediate or follows changes in repressor levels with a delay, we used the ODE model to simulate target expression with the estimated parameters, either assuming an immediate (\(\varDelta t\)=0) or a delayed (\(\varDelta t\)>0) effect of the repressor on the target. We show that sensitivity and also non-linearity of the response is variable between targets. Choose from our lentiviral, retroviral or adenoviral vectors. For both KRAB- and hHDAC4-mediated repression, the repressor is tethered to the Esrrb promoter region via dCas9. 134, 39423945 (2012). It is however technically challenging, since it requires the ability to quantitatively modulate protein abundance. For the dTAG withdrawal time course, where all repressors reached their maximal level in <25h, the mean MFI of the 25h150h time points was set to 1 and the mean MFI at time 0h0. Nanog is the gateway to the pluripotent ground state. AcGFP1 is a monomeric protein. We then calculated the tRFP/tBFP ratio and normalised it to the mean of the tRFP/tBFP ratio for the no-degron control cell line, averaged across all replicates treated with maximal ligand concentration and mock-treated samples. a Experimental scheme: STAG2-EGFP HeLa Kyoto cells stably expressing either (degron-)KRAB-dCas9, hHDAC4-dCas9 or CasRx together with the respective guide RNAs targeting STAG2 were grown in high dTAG-13 (500nM) media and then titrated with a range of dTAG-13 concentrations. The Molecular Equivalent of Soluble Fluorophore (MESF) value for each peak is determined by correlating the fluorescence intensity of each respective bead population with the amount of soluble AcGFP1 or mCherry yielding the same fluorescence intensity. The two active X chromosomes in female ESCs block exit from the pluripotent state by modulating the ESC signaling network. For the KRAB-repressor, we created an additional construct, where dCas9 and KRAB are not directly fused, but tethered by the ABA-inducible PYL1/ABI dimerisation system (here referred to as KRAB-Split-dCas9)35, with which we have achieved potent repression in the past36. Flow cytometer analysis of mCherry Flow Cytometer Calibration Beads. When expressed in mammalian cells, the protein is highly soluble and homogeneously distributed within the cytosol, with no detectable aggregation (Figure 3). Biochem. The dotted line shows the 99th percentile of non-fluorescent control cells. e Same as in (c) but for mCherry. Panels C and F. Kernels from a segregating transformed line under white light and fluorescent light. 353, 493500 (2007). When phycobiliproteins are exposed to organic . Res. Cells were passaged every 23 days at a 1:5 or 1:6 dilution, respectively. Optimized for high expression and high titers. Commun. We thank Jan-Michael Peters (IMP Vienna) for sharing the STAG2-EGFP HeLa cell line. Fucci cell cycle reporter vectors deliver fluorescent, ubiquitination-based, cell-cycle indicators that allow you to identify cells in various phases of the cell cycle. Bar = 1 cm. Biotechnol. The following four pairs have been reported in the literature to be suitable for FRET: *mOrange has been discontinued. Nat. The approach is therefore relatively simple and cost-effective, allowing the study of how the quantity of different genes relates to a phenotype, thus preserving the scalability that characterise CRISPR-based technologies. The DD-ZsGreen1 Reporter System is available in plasmid and lentiviral formats. 5a,b). DsRed-Monomer preserves key spectral features of other DsRed variants, in particular, DsRed-Express. NANOG and OCT4 might bind their target genes with varying affinity, such that a subset would remain occupied even at lower concentrations. Dev. c Density plots of tBFP levels in cells containing Esrrb-targeting guides measured by flow cytometry. Musacchio, A. This region of MODC contains a PEST domain that targets the pFP-DR fusion protein for degradation, giving it a substantially shorter half-life (Li et al. While derepression was immediate for dCas9 alone, a delay of 1618h was observed for the KRAB systems, which was substantially smaller (6h) for hHDAC4-dCas9 (Fig. As with other fluorescent proteins, the pFP-DR vectors are human codon-optimized and retain the same spectral properties as our standard fluorescent proteins. Molecular coupling of Xist regulation and pluripotency. CAS PubMed 22, 156772 (2004). 2e,f). 2004; Wang et al. For experiments with a flow cytometry readout, cell treatment and analysis was usually performed in 96-well plates. Genolet, O., Monaco, A. The peak with the highest fluorescent . A defined Oct4 level governs cell state transitions of pluripotency entry and differentiation into all embryonic lineages. 4d, top). Nanog safeguards pluripotency and mediates germline development. At least 10,000 events were recorded. Development 142, 39964009 (2015). We titrated Oct4 using 5 dTAG-13 concentrations (Fig. The ecDHFR degron domain was amplified from CAG-DDdCas9VP192-T2A-EGFP-ires-puro (Addgene plasmid # 69534; RRID:Addgene_69534, a gift from Timo Otonkoski). 2cg and Supplementary Fig. The lowest intensity represents the autofluorescence signal of cells not expressing the fluorescent protein (AcGFP1 or mCherry), The remaining five peaks are evenly distributed over the remaining scale of the green or red fluorescence detection channel, Works with any flow cytometer with a 488-nm laser line (AcGFP1) or a 561-nm laser line (mCherry), Calibrate your flow cytometer prior to analyzing cells expressing AcGFP1 or mCherry fluorescent protein, Since the spectral properties and brightness of AcGFP1 and EGFP are very similar, these beads may be used to calibrate flow cytometers prior to using EGFP-expressing cells. Penicillin-Streptomycin was kept for 2 passages. Nature 450, 12301234 (2007). Nat. Biotechnol. It is a very bright cyan, making it a good reporter and useful in multicolor analyses. The KRAB-Split-dCas9 plasmid was generated by inserting the FKBP12F36V domain C-terminally to dCas9 into the plasmid pSLQ2818 pPB: CAG-PYL1-KRAB-IRES-Puro-WPRE-SV40PA PGK-ABI-tagBFP-SpdCas9 (Addgene plasmid # 84241; a gift from Stanley Qi), after exchanging the Puromycin resistance with a resistance for Blasticidin. 24, 15562 (2006). In one xenograft mouse model of metastatic breast cancer, tdTomato was easily detected as deep as 1 cm below the surface, and extremely small lesions could be detected and tracked over time (Figure 2; Winnard, Kluth, and Raman 2006). Wessels, H.-H. et al. Initially, the Timer fluorescent protein emits green fluorescence, but as time passes, the fluorophore undergoes additional changes that shift its fluorescence to longer wavelengths. Panels A and D. Maize callus under white light and fluorescent light. Correspondence to 7c and Supplementary Fig. 2a). For each system we estimated two different parameters: the time required to reach half of the final repressor level (t1/2) and the delay between repressor up- or downregulation and effects on target gene expression (t). For example, the DsRed-Monomer-Actin fusion protein correctly incorporates into the actin filament system of the cytoskeletal network, ruffling edges, and filipodia. 2b). Science 351, 720724 (2016). Asunaprevir (ASN, MedChem Express #HY-14434) was dissolved in DMSO to 10mM stock solution and stored at 20C.
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