Cell Sci Research Articles

Live cell fluorescence microscopy-an end-to-end workflow for high-throughput image and data analysis.

Fluorescence microscopy images of biological samples contain valuable information but require rigorous analysis for accurate and reliable determination of changes in protein localization, fluorescence intensity, and morphology of the studied objects. Traditionally, cells for microscopy are immobilized using chemicals, which can introduce stress. Analysis often focuses only on colocalization and involves manual segmentation and measurement, which are time-consuming and can introduce bias. Our new workflow addresses these issues by gently immobilizing cells using a small agarose block on a microscope coverslip. This approach is suitable for cell-walled cells (yeast, fungi, plants, bacteria), facilitates their live imaging under conditions close to their natural environment and enables the addition of chemicals during time-lapse experiments. The primary focus of the protocol is on the presented analysis workflow, which is applicable to virtually any cell type-we describe cell segmentation using the Cellpose software followed by automated analysis of a multitude of parameters using custom-written Fiji (ImageJ) macros. The results can be easily processed using the provided R markdown scripts or available graphing software. Our method facilitates unbiased batch analysis of large datasets, improving the efficiency and accuracy of fluorescence microscopy research. The reported sample preparation protocol and Fiji macros were used in our recent publications: Microbiol Spectr (2022), DOI: 10.1128/spectrum.01961-22; Microbiol Spectr (2022), DOI: 10.1128/spectrum.02489-22; J Cell Sci (2023), DOI: 10.1242/jcs.260554.

Targeting of biomolecular condensates to the autophagy pathway.

Biomolecular condensates are membraneless compartments formed by liquid-liquid phase separation. They can phase transit into gel-like and solid states. The amount and state of biomolecular condensates must be tightly regulated to maintain normal cellular function. Autophagy targets biomolecular condensates to the lysosome for degradation or other purposes, which we term biocondensophagy. In biocondensophagy, autophagy receptors recognize biomolecular condensates and target them to the autophagosome, the vesicle carrier of autophagy. Multiple types of autophagy receptors have been identified and they are specifically involved in targeting biomolecular condensates with different phase transition states. The receptors also organize the phase transition of biomolecular condensate to facilitate biocondensophagy. Here, we briefly discuss the latest discoveries regarding how biomolecular condensates are recognized by autophagy receptors.

Expression of Concern: Leukotriene D4 activates MAPK through a Ras-independent but PKCε-dependent pathway in intestinal epithelial cells.

There are potential issues in J. Cell Sci. (2002) 115, 1883–1893 (doi:10.1242/jcs.115.9.1883).A possible duplication of the LTD4+PP1 and LTD4+ PD98059 bands in Fig 5E was highlighted to us via the PubPeer website. Owing to the period of time that has passed since the experiments were conducted, the authors have not been able to provide original data for this blot. They have conducted analyses which they feel show the two blots to be different; however, without the original data, it is difficult to conclude if the bands are duplicated, so the journal is publishing this Expression of Concern to alert readers to this issue.

Approaches to Determine Nuclear Shape in Cells During Migration Through Collagen Matrices.

Fibrillar collagen is an abundant extracellular matrix (ECM) component of interstitial tissues which supports the structure of many organs, including the skin and breast. Many different physiological processes, but also pathological processes such as metastatic cancer invasion, involve interstitial cell migration. Often, cell movement takes place through small ECM gaps and pores and depends upon the ability of the cell and its stiff nucleus to deform. Such nuclear deformation during cell migration may impact nuclear integrity, such as of chromatin or the nuclear envelope, and therefore the morphometric analysis of nuclear shapes can provide valuable insight into a broad variety of biological processes. Here, we describe a protocol on how to generate a cell-collagen model in vitro and how to use confocal microscopy for the static and dynamic visualization of labeled nuclei in single migratory cells. We developed, and here provide, two scripts that (Fidler, Nat Rev Cancer 3(6):453-458, 2003) enable the semi-automated and fast quantification of static single nuclear shape descriptors, such as aspect ratio or circularity, and the nuclear irregularity index that forms a combination of four distinct shape descriptors, as well as (Frantz et al., J Cell Sci 123 (Pt 24):4195-4200, 2010) a quantification of their changes over time. Finally, we provide quantitative measurements on nuclear shapes from cells that migrated through collagen either in the presence or the absence of an inhibitor of collagen degradation, showing the distinctive power of this approach. This pipeline can also be applied to cell migration studied in different assays, ranging from 3D microfluidics to migration in the living organism.

Expression of Concern: RyR1 and RyR3 isoforms provide distinct intracellular Ca2+ signals in HEK 293 cells.

There was an error published in Fig. 6 in J. Cell Sci. (2002) 115, 2497-2504 (doi:10.1242/jcs.115.12.2497).There is a duplication of a segment of signal in Fig. 6A. According to the authors, this was due to an error in the process of assembling the trace. They made every effort to find the original data, but, as the work dates back more than twenty years, they were unable to recover the original files.The authors state that this error does not significantly impact the interpretation of the article, the data reliability or the reader's understanding of the paper and that the scholarly integrity of the article remains intact. “Data reported in Fig. 6A and B aimed to illustrate variability of spontaneous Ca2+ signals in two RyR3-expressing cells; spontaneous Ca2+ release events are also reported in Fig. 5 and duplication of signal in Fig. 6Ab does not impact on the main interpretation of the article which was that HEK293 cells expressing RyR3 display spontaneous Ca2+ release events and those expressing RyR1 do not. This suggested that, at least in these cells, isoform-specific functional properties may contribute to generation of intracellular Ca2+ signals.”Without the original data, it is difficult to determine how the issues arose, so the journal is publishing this note to alert readers to our concerns, with the agreement of the authors.

Notch-dependent Abl signaling regulates cell motility during ommatidial rotation in Drosophila.

A collective cell motility event that occurs during Drosophila eye development, ommatidial rotation (OR), serves as a paradigm for signaling-pathway-regulated directed movement of cell clusters. OR is instructed by the EGFR and Notch pathways and Frizzled/planar cell polarity (Fz/PCP) signaling, all of which are associated with photoreceptor R3 and R4 specification. Here, we show that Abl kinase negatively regulates OR through its activity in the R3/R4 pair. Abl is localized to apical junctional regions in R4, but not in R3, during OR, and this apical localization requires Notch signaling. We demonstrate that Abl and Notch interact genetically during OR, and Abl co-immunoprecipitates in complexes with Notch in eye discs. Perturbations of Abl interfere with adherens junctional organization of ommatidial preclusters, which mediate the OR process. Together, our data suggest that Abl kinase acts directly downstream of Notch in R4 to fine-tune OR via its effect on adherens junctions.

Mechanotransduction in the pathogenesis of non-alcoholic fatty liver disease.

Mechanobiology is a domain of interdisciplinary research that aims to explore the impact of physical force, applied externally or internally, on cell and tissue function, including development, growth, and differentiation. Mechanotransduction is a term that describes how cells sense physical forces (such as compression, stretch, and shear stress), convert them into biochemical signals, and mount adaptive responses integrated by the nucleus. There is accumulating evidence that mechanical forces extensively inform the biological behaviour of liver cells in health and disease. Recent research has elucidated many cellular and molecular mechanisms involved in this process including the pleiotropic control and diverse effects of the paralogous transcription co-activators YAP/TAZ, which play a prominent role in mechanotransduction. The liver sinusoids represent a unique microenvironment in which cells are exposed to mechanical cues originating in the cytoskeleton and at interfaces with adjacent cells, the extracellular matrix, and vascular or interstitial fluids. In non-alcoholic fatty liver disease (NAFLD), hepatocellular lipid accumulation and ballooning, activation of inflammatory responses, dysfunction of liver sinusoidal endothelial cells, and transdifferentiation of hepatic stellate cells into a pro-contractile and pro-fibrotic phenotype have been associated with aberrant cycles of mechanosensing and mechanoresponses. The downstream consequences of disrupted mechanical homeostasis likely contribute to the progression of NAFLD and promote the development of portal hypertension, cirrhosis, and hepatocellular carcinoma. Identification of molecular targets involved in pathogenic mechanotransduction will allow for the development of novel strategies to prevent the progression of liver disease in NAFLD.

Expression of Concern: Suppression of Synaptotagmin II restrains phorbol-ester-induced downregulation of protein kinase Cα by diverting the kinase from a degradative pathway to the recycling endocytic compartment.

This Expression of Concern relates to J. Cell Sci. (2002) 115, 3083-3092 (doi:10.1242/jcs.115.15.3083).Journal of Cell Science was anonymously alerted to duplication of bands in the actin western blot shown in Fig. 1A. The authors no longer have the original western blot data for this panel and therefore cannot explain what has happened; however, they have provided western blot data from several similar experiments to support the validity of the results in Fig. 1A and state that the conclusions of the article are not affected by the error. The additional data are available from the authors upon reasonable request.The journal is publishing this note to inform readers of the issue.

Expression of Concern: Caspase-dependent initiation of apoptosis and necrosis by the Fas receptor in lymphoid cells: onset of necrosis is associated with delayed ceramide increase.

There are issues in J. Cell Sci. (2002) 115, 4671-4683 (doi:10.1242/jcs.00153).There are irregularities with the background in Fig. 3E and Fig. 9A. Also, in Fig. 4, several data points are unexpectedly similar between the two FACS plots in panel A, and the first two lanes of the blot in panel D are duplicated. The authors no longer have the original data, but state that the conclusions of the paper are not affected.A report by Instituto de Ciencias Biomédicas, Universidad de Chile, decided that there had been ‘to some extent data manipulation but not falsification’ of this and other papers authored by Claudio Hetz and concluded that the key findings of this paper are unaffected.Without the original data, it is difficult to determine how these issues arose, so the journal is publishing this note to alert readers to our concerns.

Expression of Synj2bp in mouse liver regulates the extent of wrappER-mitochondria contact to maintain hepatic lipid homeostasis

BackgroundIn mouse liver hepatocytes, nearly half of the surface area of every mitochondrion is covered by wrappER, a wrapping-type of ER that is rich in fatty acids and synthesizes lipoproteins (VLDL) (Anastasia et al. in Cell Rep 34:108873, 2021; Hurtley in Science (80- ) 372:142–143, 2021; Ilacqua et al. in J Cell Sci 135:1–11, 2021). A disruption of the ultrastructure of the wrappER-mitochondria contact results in altered fatty acid flux, leading to hepatic dyslipidemia (Anastasia et al. 2021). The molecular mechanism that regulates the extent of wrappER-mitochondria contacts is unknown.MethodsWe evaluated the expression level of the mitochondrial protein Synj2bp in the liver of normal and obese (ob/ob) mice. In addition, we silenced its expression in the liver using an AAV8 vector. We coupled quantitative EM morphometric analysis to proteomics and lipid analyses on these livers.ResultsThe expression level of Synj2bp in the liver positively correlates with the extent of wrappER-mitochondria contacts. A 50% reduction in wrappER-mitochondria contacts causes hepatic dyslipidemia, characterized by a gross accumulation of lipid droplets in the liver, an increased hepatic secretion of VLDL and triglycerides, a curtailed ApoE expression, and an increased capacity of mitochondrial fatty acid respiration.ConclusionSynj2bp regulates the extent of wrappER-mitochondria contacts in the liver, thus contributing to the control of hepatic lipid flux.

Glial regulators of ions and solutes required for specific chemosensory functions in Caenorhabditis elegans.

Glia and accessory cells regulate the microenvironment around neurons and primary sensory cells. However, the impact of specific glial regulators of ions and solutes on functionally diverse primary cells is poorly understood. Here, we systemically investigate the requirement of ion channels and transporters enriched in Caenorhabditis elegans Amsh glia for the function of chemosensory neurons. Although Amsh glia ablated worms show reduced function of ASH, AWC, AWA, and ASE neurons, we show that the loss of glial enriched ion channels and transporters impacts these neurons differently, with nociceptor ASH being the most affected. Furthermore, our analysis underscores the importance of K+, Cl-, and nucleoside homeostasis in the Amphid sensory organ and uncovers the contribution of glial genes implicated in neurological disorders. Our findings build a unique fingerprint of each glial enriched ion channel and transporter and may provide insights into the function of supporting cells of mammalian sensory organs.

Expression of Concern: Specific roles of Gi protein family members revealed by dissecting SST5 coupling in human pituitary cells.

There is an issue with J. Cell Sci. (2014) 126, 638-644 (doi:10.1242/jcs.116434).The journal was alerted anonymously to duplicated bands in the GαoB gel in Fig. S2. The authors no longer have the original data and therefore cannot explain what happened but state that they did not perform any quantification or comparison of the expression of these genes and that the conclusions of the paper are not affected.The journal is publishing this note to alert readers to the issue.

Myostatin is a negative regulator of adult neurogenesis after spinal cord injury in zebrafish.

Intrinsic and extrinsic inhibition of neuronal regeneration obstruct spinal cord (SC) repair in mammals. In contrast, adult zebrafish achieve functional recovery after complete SC transection. While studies of innate SC regeneration have focused on axon regrowth as a primary repair mechanism, how local adult neurogenesis affects functional recovery is unknown. Here, we uncover dynamic expression of zebrafish myostatin b (mstnb) in a niche of dorsal SC progenitors after injury. mstnb mutants show impaired functional recovery, normal glial and axonal bridging across the lesion, and an increase in the profiles of newborn neurons. Molecularly, neuron differentiation genes are upregulated, while the neural stem cell maintenance gene fgf1b is downregulated in mstnb mutants. Finally, we show that human fibroblast growth factor 1 (FGF1) treatment rescues the molecular and cellular phenotypes of mstnb mutants. These studies uncover unanticipated neurogenic functions for mstnb and establish the importance of local adult neurogenesis for innate SC repair.

Single-cell RNA sequencing reveals different signatures of mesenchymal stromal cell pluripotent-like and multipotent populations

Somatic stem cells are advantageous research targets for understanding the properties required to maintain stemness. Human bone marrow-mesenchymal stromal cells (BM-MSCs) were separated into pluripotent-like SSEA-3(+) Muse cells (Muse-MSCs) and multipotent SSEA-3(-) MSCs (MSCs) and were subjected to single-cell RNA sequencing analysis. Compared with MSCs, Muse-MSCs exhibited higher expression levels of the p53 repressor MDM2; signal acceptance-related genes EGF, VEGF, PDGF, WNT, TGFB, INHB, and CSF; ribosomal protein; and glycolysis and oxidative phosphorylation. Conversely, MSCs had higher expression levels of FGF and ANGPT; Rho family and caveola-related genes; amino acid and cofactor metabolism; MHC class I/II, and lysosomal enzyme genes than Muse-MSCs. Unsupervised clustering further divided Muse-MSCs into two clusters stratified by the expression of cell cycle-related genes, and MSCs into three clusters stratified by the expression of cell cycle-, cytoskeleton-, and extracellular matrix-related genes. This study evaluating the differentiation ability of BM-MSC subpopulations provides intriguing insights for understanding stemness.

Expression of Concern: Increased responsiveness of hypoxic endothelial cells to FGF2 is mediated by HIF-1α-dependent regulation of enzymes involved in synthesis of heparan sulfate FGF2-binding sites.

There are issues in Fig. 2 in J. Cell Sci. (2002) 115, 1951-1959 (doi:10.1242/jcs.115.9.1951).Bands are duplicated three times in western blots in Fig. 2, despite these blots representing different conditions. The authors no longer have the original data but state that the results have since been verified and the conclusions of the paper are not affected.Without the original data, it is difficult to determine how the issues arose, so the journal is publishing this note to alert readers to our concerns.

Optimized Decal Transfer Method for the Mitigation of Incidental Particle Deposition at the Interface of Proton Exchange Membranes and Catalyst Layers

The membrane electrolyte assembly (MEA) is the key component of a proton exchange membrane fuel cell (PEMFC). The MEA usually consists of gas diffusion layers as outer layers to the inner catalyst coated membrane (CCM). There are various methods to prepare CCMs and decal transfer is currently a common method which was first introduced by Wilson and Gottesfeld [1] and further developed by other researchers [2]–[4]. The initial step of this method is that the catalyst ink is coated onto an intermediate substrate material creating a catalyst coated film (CCF). This step is followed by a transfer of the active layer onto the membrane by hot pressing the membrane and CCF between heated press platens maintained at a specific high temperature for an optimized time and pressure to ensure bonding.Extensive studies on the procedure of decal transfer, substrate material and its preparation, catalyst solvents, etc. have increased the transfer yield of decal transfer to more than 95% [5]. Additionally, in the last decade, industrial development has reduced its cost; however, the total cost for fuel cell manufacturing is still relatively high. One reason for this high production cost is the contamination sensitivity of the CCMs by external particles that can potentially be detrimental to fuel cell operation [6]. This requirement necessitates the use of cleanrooms and quality control equipment to prevent contaminants entering the manufactured components, thus increasing the overall costs. The purpose of the present research is to improve the understanding of the interactions between foreign contaminants and the manufacturing process in the context of MEA production quality. The specific objectives are to i) understand the impact of external particles on the catalyst layer decal transfer process and ii) improve the robustness of the decal transfer method in the presence of external particles.This research investigates the impact of incidental external particles that may be found on the membrane surface or the CCF prior to hot pressing. 60µm Silica microspheres (Si-M) were selected as a representative of solid particles and CCMs with purposely introduced Si-Ms were fabricated and several decal transfer methods and support materials were tested and imaged using the X-ray computed tomography (XCT) technique to analyze the impact on the fuel cell integrity and operation at the presence of the Si-M. While regular decal transfer protocols result in excessive membrane thinning in the presence of these particles, it was observed that by changing the rate of applied pressure and using alternative support materials when transferring the cathode catalyst onto a half-coated membrane, it was possible to reduce membrane thinning under the particles by more than 20% while maintaining transfer quality and cell performance (Fig. 1). In addition, it was observed that although anisotropic mechanical properties of the membrane can adversely affect the CCM topography after the decal transfer, a tuned protocol is able to prevent unwanted deformations and potential stress concentrations. Overall, it is envisioned that the outcomes of this work may enable relaxed quality control measures and manufacturing site cleanroom standards by reducing the potential effects of external particles on MEA production quality. Acknowledgements This research was supported by the Natural Sciences and Engineering Research Council of Canada, Canada Foundation for Innovation, British Columbia Knowledge Development Fund, Western Economic Diversification Canada, Ballard Power Systems, and W.L. Gore & Associates. This research was undertaken, in part, thanks to funding from the Canada Research Chairs program. Keywords: fuel cell; membrane durability; X-ray computed tomography; decal transfer; manufacturing References Wilson, M. S. & Gottesfeld, S. Thin-film catalyst layers for polymer electrolyte fuel cell electrodes. J. Appl. Electrochem. 22, 1–7 (1992).Shahgaldi, S., Alaefour, I. & Li, X. Impact of manufacturing processes on proton exchange membrane fuel cell performance. Appl. Energy 225, 1022–1032 (2018).Cho, H. J. et al. Development of a novel decal transfer process for fabrication of high-performance and reliable membrane electrode assemblies for PEMFCs. Int. J. Hydrogen Energy 36, 12465–12473 (2011).Thanasilp, S. & Hunsom, M. Effect of MEA fabrication techniques on the cell performance of Pt-Pd/C electrocatalyst for oxygen reduction in PEM fuel cell. Fuel 89, 3847–3852 (2010).Liang, X., Pan, G., Xu, L. & Wang, J. A modified decal method for preparing the membrane electrode assembly of proton exchange membrane fuel cells. Fuel 139, 393–400 (2015).James, B. D., Moton, J. M. & Colella, W. G. Mass Production Cost Estimation of Direct H2 PEM Fuel Cell Systems for Transportation Applications: 2018 Update. ASME 2014 12th Int. Conf. Fuel Cell Sci. Eng. Technol. collocated with ASME 2014 8th Int. Conf. Energy Sustain. V001T07A002–V001T07A002 (2018). Figure 1

Study of Mechanical Properties of Membrane-Electrode Assemblies for Proton Exchange Membrane Fuel Cells By the Small-Punch Technique

Mechanical properties of membrane-electrode assemblies (MEAs) are most relevant for the durability of proton-exchange membrane fuel cells (PEMFCs) [1]. During operation, the MEA must endure eventual changes in the static pressure of anodes and cathodes caused by changes in power demand, gases flow rate, and/or water generation, accompanied by hygrothermal cycles, that cause mechanical stresses in the membrane, leading to cracks and possible fatal failure [2]. In parallel, the mechanical properties of MEAs change irreversibly during power production as a consequence of membrane and electrodes corrosion [3]. Therefore, it is of interest to characterize the mechanical properties of MEAs before assembly, and after operation.The small-punch is a new technique for testing mechanical properties of solids. It consists of measuring the deformation of a disc of the testing material with a ‘small puncher’, while recording the load (force) exerted by the puncher and the deflection produced on the specimen (Fig. 1a) [4]. The plot load vs deflection allows estimating mechanical strength, fracture toughness, impact, and tensile properties. Most interesting is that the disk specimen can be as small as a few millimeters, which makes the small-punch technique very appropriate when the amount of sample is limited, and for localized studies of mechanical properties.In this communication, results of the small-punch technique are shown for the study of MEAs operated in single PEMFCs (Fig. 1b). The analysis is carried out of the local mechanical properties on new and degraded MEAs. First results show that local mechanical properties of the aged MEA correlate well with gas feeding conditions and reflect laterally inhomogeneous degradation (Fig. 1b).Acknowledgement: The work is partially financed by the ELHYPORT project (PID2019−110896RB-I00), Spanish Ministry of Science and Innovation.[1] X. Huang, R, Solasi, Y. Zou, M. Feshler, K. Reifsnider, D. Condit, S. Burtlatsky, T. Madden, J. Polymer Science: Part B: Polymer Physics, Vol. 44, 2346–2357 (2006).[2] Z. Lu, M. Lugo, M.H. Santare, A.M. Karlsson, F.C. Busby, P. Walsh, J. Power Sources 214 (2012) 130-136.[3] M. Pestrak, Y. Li, S.W. Case, D.A. Dillard, M.W. Ellis, Y-H. Lai, C. S. Gittleman, J. Fuel Cell Sci. Technol. (2010), 7(4): 041009.[4] EN 10371:2021 Metallic materials - Small punch test method. CEN, Brussels (2021) Figure 1

High-content phenotyping of Parkinson's disease patient stem cell-derived midbrain dopaminergic neurons using machine learning classification.

SummaryCombining multiple Parkinson's disease (PD) relevant cellular phenotypes might increase the accuracy of midbrain dopaminergic neuron (mDAN) in vitro models. We differentiated patient-derived induced pluripotent stem cells (iPSCs) with a LRRK2 G2019S mutation, isogenic control, and genetically unrelated iPSCs into mDANs. Using automated fluorescence microscopy in 384-well-plate format, we identified elevated levels of α-synuclein (αSyn) and serine 129 phosphorylation, reduced dendritic complexity, and mitochondrial dysfunction. Next, we measured additional image-based phenotypes and used machine learning (ML) to accurately classify mDANs according to their genotype. Additionally, we show that chemical compound treatments, targeting LRRK2 kinase activity or αSyn levels, are detectable when using ML classification based on multiple image-based phenotypes. We validated our approach using a second isogenic patient-derived SNCA gene triplication mDAN model which overexpresses αSyn. This phenotyping and classification strategy improves the practical exploitability of mDANs for disease modeling and the identification of novel LRRK2-associated drug targets.

Keratin 75 Is a Component of the LINC Complex and Has an Essential Role in Mediating the SOX2 Rapid Healing Response during Wound Repair

Keratin 75 Is a Component of the LINC Complex and Has an Essential Role in Mediating the SOX2 Rapid Healing Response during Wound Repair

Expression of Concern: CENP-C binds the alpha-satellite DNA in vivo at specific centromere domains.

This Expression of Concern relates to J. Cell Sci. (2002) 115, 2317-2327 (doi:10.1242/jcs.115.11.2317).A reader contacted Journal of Cell Science with concerns about a potential image duplication in Fig. 2A of this article, which was also highlighted on the PubPeer website. Possible duplication was highlighted for the α-sat (1, 5, 19) and α-sat (X) total DNA dilution series dot blots in Fig. 2A.We contacted the authors of the article to request the original data for this experiment. We were informed by Professor Giovanni Perini, co-first author of the article, that the corresponding author, Professor Giuliano Della Valle, retired in 2010 and passed away in 2016. Consequently, the data and laboratory records relating to Fig. 2A are no longer available.Professor Perini informed us that he cannot exclude the possibility that the α-sat (1, 5, 19) and α-sat (X) total DNA dilution series dot blots in Fig. 2A are duplicated images. However, Professor Perini also highlighted that the legend for Fig. 2A states that the filters were sequentially hybridised with the alpha-satellite probes, raising the possibility that the similarities between these two dot blots, which are shown for semi-quantitative assessment of the corresponding ChIP dot blots, could be the result of rehybridisation of the same filter with the different probes. The authors state that the major conclusions of the article are not affected by this issue.Because the original data and records for this experiment are not available, we are unable to determine whether Fig. 2A shows duplicated data for the α-sat (1, 5, 19) and α-sat (X) total DNA dilution series. The journal is therefore publishing this Expression of Concern to make readers aware of this issue.