The lateral pn diode formed in this way manifests efficient self-powered detection by separating ~12% photo-generated electrons and holes. When polarized as bipolar phototransistor, the device is customized with a gain ~1000 by its transistor action, reaching the responsivity ~12 A W−1 and detectivity over 1013 Jones while keeping a fast response speed within 20 μs. A promising pathway toward high performance optoelectronics is thus opened up based on local ferroelectric polarization coupled 2D semiconductors.Ferroelectric engineered pn doping in two-dimensional (2D) semiconductors hold essential promise in realizing customized functional devices in a reconfigurable manner. Here, we report the successful pn doping in molybdenum disulfide  materials still suffer from low performance. Here, the authors tackle this issue by introducing a reconfigurable design enabled by locally tuning the doping of a 2D molybdenum disulfide film through the polarization of an underlying ferroelectric material. Using field effects coupled by high-κ dielectrics7, ionic liquids and gels9, and ferroelectric (FE) polarization12, their electric properties have been widely tuned from semiconductor to highly conductive metals and oppositely to the insulators. Such features have enriched the emerging of various gate-modulated devices13, including transistors15, logic inverters7, memories16, light-emitting diodes (LEDs)17, and photodetectors19. An ultimate pursuit to this end would be however a reconfigurable functional device that can be customized on demand, so that a universal device architecture can be deployed in various applications. FE material-coupled 2D semiconductors hold special promise in reforming these devices toward a reconfiguration, given their large, non-volatile yet rewritable feature of remnant polarization23. There is an increasing interest in integrating FE materials, such as BiFeO310, P(VDF-TrFE)12, PbZrTiO324, and LiNbO325, to 2D functional devices. However, further efforts are, to a marked degree, hampered by the ability of FE polarization in controlling pn doping of 2D materials23, which constitutes a fundamental building block for functional electronic devices27.Two-dimensional (2D) semiconductors have drawn extensive interests in functional electronic devices for their exotic optoelectronic properties by the quantum confinement in atomic thickness and feasibly changed characteristics under external modulation29. In optoelectronics, these devices based on pn junctions could manifest self-powered or fast and high gain photodetection beyond the usual photoconductors. However, early attempts to construct the kind of devices relied prominently on locally buried gates28, lateral and vertical heterojunctions33, the behavior of which are complicated to manipulate without fine-tuned pn doping. For example, the amplification gain in the resulting bipolar transistor is severely limited by the low carrier injection efficiency at the emitter–base junction, for example, based on MoS2/WSe229 or MoS2/BP heterojunctions34. Moreover, these efforts fail to satisfy reconfigurable customization on demand with the predefined gate electrodes, epitaxial sequence in lateral junction, or stacking order in van der Waals heterostructures. With the large remnant polarization, FE coupling to 2D semiconductors is favored to realize reconfigurable optoelectronics based on rewritable pn doping in the 2D components, which however has not been demonstrated yet16. For example, organic FE materials, such as P(VDF-TrFE) copolymers, have in the past manifested indispensable merits in their facile spin-coating fabrication. Sophisticate top-gate integration methods have been adopted for their integration with various 2D semiconductors, including MoS236, MoSe237, MoTe2, and WSe238. However, these efforts generally fail to achieve pn modulation even with the large remnant polarization of P(VDF-TrFE) (~8 µC cm−2) over conventional gate oxides, which draw increasing demand to improve the FE integration in 2D optoelectronic devices for reconfigurable pn doping and functionalization.Given the selective pn doping of 2D materials, a variety of fundamental devices that enable the digital technology can be developed, for example, pn diodes and amplifying bipolar transistors2 using the device configuration of bottom electrical contacts. With the rewritable FE polarization using a scanning atomic force microscope (AFM) tip, the MoS2 photoconductor is facile customized into pn diodes and bipolar phototransistors with optimal photodetection performance. This allows us to demonstrate a lateral pn diode with an ideal factor of 1.7 for self-powered photodetection that separate ~12% photogenerated electrons and holes, and a npn bipolar phototransistor with a gain ~1000 with fine-adjusted pn polarization. Such reconfigurable device characteristics may promote the evolvement of smart image sensors that respond to external light levels for balanced photoresponse gain and energy efficiency. Our study on reconfigurable optoelectronic devices using FE polarization thus unravels a pathway towards customizing novel functional optoelectronics based on 2D semiconductors.Here, we report on reconfigurable optoelectronic photodetectors with FE polarization-defined pn doping in MoS2 device, in which an AFM tip is employed to switch the local polarization. To fabricate the device, multilayer MoS2 from mechanical exfoliation is used as the semiconductor channel, while the FE copolymer P(VD-TrFE) is spin coated on the top with a thickness of ~200 nm. The copolymer film is later crystallized into its orthorhombic β phase by annealing at 135 °C for 15 min39. The coercive field of as-prepared P(VDF-TrFE) film, under which the FE polarization switches, is measured in agreement with literature to be ~5 × 107 V m−1 using capacitance–voltage measurement  in intimate contact with MoS2 is then exploited to engineer the carrier doping in MoS2 through their reversible polarization by an external poling field. The representative coupling between the FE polarization in P(VDF-TrFE) and the adjacent MoS2 is illustrated in Fig. 2, upward polarization (P↑) in P(VDF-TrFE) is required, while reversely the opposite downward polarization (P↓) is desired to enhance n-type doping. Specifically, in order to achieve widely tuned doping in MoS2, we adopt bottom electrode contacts by placing thin MoS2 flakes on top of predefined metal electrodes, as indicated in Fig. 2 transferred onto Cr/Au electrodes is shown in Fig. 2 devices studied in this work. Compared to the previous investigations with metal electrodes on the top of MoS2 or other 2D materials41, which may screen the FE polarization field near contact and result in Schottky barriers, this will ensure the intimate coupling of the FE polarization field to MoS2 at the contacts and hence uniform doping to the whole semiconductor channel.Figure 22. Despite the switched FE polarization tends to relax due to the incomplete compensation to depolarization field in P(VDF-TrFE), it enables rewritable polarization pattern on the same device, thereby allowing the direct study of the influence of device configurations without worrying about material differences. To pole the FE layer, the AFM tip is grounded while the source (S) and drain (D) electrodes of MoS2 channel are biased by a poling voltage Vp. By scanning the AFM tip over the device area, the P(VDF-TrFE) copolymer between the tip and MoS2 channel is polarized to P↑ using positive Vp and P↓ with negative Vp. The effect of resulted remnant FE polarization in P(VDF-TrFE) to the electrical conductance of MoS2 in Dev-1 is reflected by the significant hysteresis shown in Fig. Vds = 1 V after each polarization scan. With positive Vp, the current flow in MoS2 is dramatically decreased with increasing Vp due to the depletion of electrons. However, an apparent change is observed with Vp >+20 V, that is, increasing the poling voltage leads to increased conductance. Such transition is consistent with a reversal of the doping polarity in MoS2 to p-type due to the dramatically increased remnant polarization (P↑) in FE film16. This is confirmed by the field-effect modulation measurements using the Si back-gate, as will be discussed later. By wweeping the poling voltage to negative, an opposite transition from p to n happens at Vp <−10V. These turning voltages are found consistent with the previous measured coercive voltages of P(VDF-TrFE) films, thus validating the correlation between the changed pn doping states in MoS2 and the FE polarization switching. It should be noted that the successful doping polarity switching in MoS2 is in sharp contrast with the one that employed electrode-on-top configuration, which in our control experiment displays only monotonous decrease of conductance with increasing Vp  , an AFM system is employed for the facile reconfiguration of polarization pattern, as reported in the literaturesee Fig. . This co2 channel can be modulated with an on–off ratio of >107 as shown in Fig. I–V) characteristics can be found in Supplementary Fig. 2 channels by avoiding the contact issue12. To get further insight into the resulting doping characteristics in MoS2 by the coupled FE polarization, we analyze the field-effect measurements using the Si back-gate. Figure 2 back-gate transistor after poling operations at Vp = 0, −15, and +25 V. The initial MoS2 is seen n doped, that is, increasing Vg at back-gate enhanced its conductance, while after −15 V downward polarization (P↓), its threshold voltage is remarkably shifted to negative from −13 to −85 V. Considering the oxide capacitance of 300 nm SiO2, Cox = 11.5 nF cm−2, the FE-coupled effective charge carriers in MoS2 is estimated to be ~0.8 µC cm−2 by ΔQ = ΔVth∙Cox44, which accounts to only a portion (~10%) of the remnant polarization of P(VDF-TrFE). This indicates that a majority of coupled electrons were frozen in new deep energy traps formed after polarization, for example, the acceptor states by the prompt interaction between aligned F-containing groups and MoS245. However, the mobility in MoS2 is not influenced as the traps are effectively screened by the heavy concentration of electron carriers. Figure 2 with respect to different FE polarization, calculated using µ = (L/W)Vds−1Cox−1 (dIds/dVg), where L and W are, respectively, the channel length and width. The maximum electron mobility in MoS2 is ~12.3 cm2 V−1 s−1 under P↓. Notably, increasing Vg in positive leads to the decrease of electron mobility in n-doped MoS2 due to the dominant electron–electron scattering, implying that the FE polarization-enhanced n-type doping in MoS2 is reaching a heavily doped metallic behavior6. In comparison, the MoS2 after 25 V (P↑) poling manifests apparent p-type behavior, with the hole mobility decreasing with increasing Vg. Such transport behavior is attributed to the trap-dominated transport in p-type MoS2, since the inherent donor atoms in MoS2 start to act as deep energy traps to holes46. By using the carrier mobility extracted from transfer curves at each FE polarization state, the free carrier concentration tuned by FE polarization is estimated to be ~109–1012 cm−2 in MoS2 for both electrons and holes, and in WSe2 ~107–1011 cm−2  by switching the polarity of bias voltage is estimated to be ~100–180 nm  at large biases, as revealed in Supplementary Fig. 47. Nevertheless, SCLC behavior is alleviated in heavily p-doped devices by the filling of trap centers when the Fermi energy EF is close to the valance band maximum. With laterally patterned p and n doping of the channel, the formed pn diode exhibits clear rectification behavior with a large on–off ratio of ~105 at ± 1 V. The value is higher than the MoS2/GaTe heterojunction48 and larger than the lateral MoS2 homojunction enabled by chemical doping49, which are attributed to the widely tuned doping concentration in MoS2 together with the electrical contact. By fitting the junction current usingI0, Rs, and n denotes, respectively, the reverse saturation current, series resistance, and ideal factor. The formed pn junction is found to exhibit an ideal factor of 1.7 and Rs ≈10MΩ. With n ~2, the junction current shall be dominated by electron–hole recombination in the space charge region50. The recombination could however be optimized by, for example, improving the collection efficiency of metal electrodes by minimizing the size of the p region. In our work, the ideal factor is reduced to 1.4 when reducing the p-doped region in junction  of the FE polarization-defined pn junction is ~650 mV with light intensity >10 mW cm−2, which is comparable to silicon diodes and approximately half of the indirect bandgap (1.2 eV) of few-layer MoS2. The large Voc suggests efficient charge separation in devices enabled by the intense lateral built-in electric field across the pn diode. In the short-circuit condition, the photocurrent is ~177pA at an intensity of 49 mW cm−2, indicating a self-powered responsivity R ≈15 mA W−1 (R = αη(e/hν), where α and η are, respectively, the light absorption efficiency and internal quantum efficiency in device). Considering that 3.8 nm MoS2 absorbs ~30% of the incident photons at 532 nm51, η is estimated to be 12% in the diode, which could be optimized by defining the junction close to electric contact, so as to minimize the serial resistance in the channel. Given the large Voc and a fill factor of ~0.58, an overall power conversation efficiency of ~0.61% is achieved. We note that the value outperforms that of vertically stacked GaTe/MoS2 heterojunction48, MoS2 pn junctions52, and the lateral WSe2/MoS2 pn heterojunctions53, suggesting the attractive potential of FE-coupled pn junctions. As indicated in Fig. 2 device exhibits fast response within 10–20 μs  in a confocal microscope. The pn junction is defined in the middle of MoS2 channel. Figure 57. However, in all the devices we have studied, photocurrent barely appears near the contact electrodes and there is no change on the photocurrent polarity across the device area. These results thus validates the role of FE polarization-defined pn diode in bringing the high rectification in I–V characteristics and self-driven photocurrent.To elucidate the self-powered photocurrent generation, spatially resolved photocurrent distribution has been measured for a short-circuited pn diode defined in Dev-3. This is achieved by locally illuminating the device using a fine laser spot , the bipolar phototransistor could work at considerably lower operation voltages  while yielding the similar photodetection gain. A typical bipolar transistor consists of emitter (E), base (B), and collector (C) regions that are, respectively, n, p, and n doped by FE polarization, as illustrated in Fig. 28. This is elucidated by the energy band diagram in Fig. Iph as that in the photodiode. The holes separated into the base then diffuse across the forward biased E–B junction as Ih,diff, and induce a significant diffusion flux  of electrons in the opposite direction. Once the electrons diffuse across the base and reach the reverse biased B–C junction, they will be swept and collected to the collector terminal by the built-in electric field, therefore contributing to the overall collector current. The collector current IC is then amplified from the initial photocurrent Iph by the injected electrons from E. With IC = (1 + β)∙Iph, β is known as the gain factor that qualifies the performance of bipolar transistor, which is generally determined by the electron injection efficiency over the reverse hole current at the forward biased E–B junction by β ≈ Ie,diff/Ih,diff. Since the diffusion current across the forward biased junction is governed by the majority carrier density over the space charge region, β is intimately related to the doping concentration in the E and B with β∝Ne/Pb, where Ne and Pb are, respectively, the electron and hole density in E and B59. The operation principle of such bipolar phototransistor is illustrated by the equivalent circuit shown in Fig. 2 by the inherent donor atoms, hole doping by FE coupling is naturally less significant than the case of electrons. A substantial gain factor to the photocurrent could be in principle obtained by constructing a npn transistor based on FE polarization.Although pn diodes exhibit remarkably improved photoresponse speed over photoconductive devices, their responsivity is limited due to the loss of gain mechanism−2. Once configured into bipolar transistor, the photocurrent is found remarkably improved in comparison to the previous photodiode along with a fast response speed. Such improvement is consistent with the expected behavior of bipolar transistor that amplifies photocurrent with high gain. By using a fast-switching 365-nm LED source, the photoresponse speed of the bipolar transistor is estimated to be ~20 μs −1/2, where A is the device area. As a bipolar phototransistor, the device is found to exhibit 10 times higher responsivity over the initial photoconductor while preserving a fast response speed similar to the photodiode , thus to probe the amplification at collector end. The current that contributed to the overall output at VC > VB > VE is schematically illustrated in Fig. VCE under different VB input from 0 to 2 V and in the presence of the background laser of AFM feedback system. Here, the collector current IC is significantly modulated by the input VB. Note that ICE increased with increasing VCE rather than being a constant due to the modulation to the base width by the extended space charge region at reverse biased B–C junction59. Such base width modulation results in a small early voltage of ~2 V (the extrapolated intersection with VCE), which is related to the low doping concentration in base. Slight increase of VCE would dramatically modulate the effective base width and the diffusion current across the base. From the measured output characteristic, the amplification behavior of the bipolar transistor is extracted at VCE = 1 V and shown in Fig. IC scaled with increasing VBE, and is 2–3 orders of magnitude larger than IB. The gain factor observed in photodetection could be probed from the magnitude of IC variation while changing IB. As shown in the inset of Fig. VBE from 0 to 1 V, we obtain an superior gain factor β ~3000, which is on the same order of magnitude as the expected amplification ratio in bipolar phototransistor as discussed above. The gain factor β decreases gradually to 1 when VBE is increased, following closely the case in photodetection that responsivity reduces at higher light illumination intensity. Interestingly, at VB = 1.5 V, with both B–C and B–E junction forward biased, one still obtains β ~10. This clearly suggests the significant electron diffusion flux from emitter to collector in contrast to the hole flux in the same direction at the forward biased base to collector junction29. Our results therefore unambiguously confirm the high gain characteristic of FE-coupled bipolar phototransistor, which originates from the widely and fine-tuned doping states in MoS2.In order to confirm the high performance of the bipolar transistor, we fabricate a three-terminal device by using MoS2 optoelectronic devices using the rigid pn doping engineering enabled by switchable FE polarization. By polarizing the MoS2 device into homojunctions of pn diode and npn bipolar transistors, the device is configured into self-powered or high gain photodetectors of optimal performances without the assistance of external gate bias. The gate-free yet reconfigurable methodology introduced the great potential of exploiting locally coupled FE polarization in customizing high-performance optoelectronic devices based on the thriving 2D semiconductors and in the future their van der Waals heterojunctions, which in principle could offer even larger speed and gain product than present homojunctions. Further maturation of such strategy towards array-structured functional optoelectronic devices with high stability shall be viable based on predefined top-gate patterns or alternatively flexible electrical imprint methods.In summary, we have demonstrated the facile reconfigurable customization of MoS2 with thickness ranging from 2 to 5 nm are exfoliated from MoS2 single crystals  using scotch tape and transferred to SiO2/Si substrate by polydimethylsiloxane stamp. The electrode contacts are made by Cr/Au electrode. In the case of bottom electrode contacts, Cr/Au (5/5 nm) electrodes are first defined on the substrate using lithography processes with direct laser writing. The exfoliated MoS2 thin flakes are later transferred. The as-prepared devices are then annealed at 150 ℃ with Ar protection for 1 h to release stresses and improve electrical contact. The FE thin film of P(VDF-TrFE) copolymer is then spin coated on the device using a solution of 2–5  wt% P(VDF-TrFE) (Piezotech FC 25) dispersed in anhydrous N,N-dimethylformamide . The copolymer film is then annealed at 135 °C for 15 min to improve the crystallinity.Few-layer MoS2 source and drain electrode biased by a poling voltage Vp. A potential bias of 0.1 V is kept between source and drain for the in situ observation of the MoS2 conductance during the polarization scan. To obtain local polarization coupling to MoS2, Vp is programmed during the AFM scan.An AFM system  is used to realize the FE poling using scanning conductive AFM tip with the MoSThe device after polarization is placed in a Lakeshore probe station  equipped with a semiconductor device analyzer  for electrical measurement. After AFM poling, the FE polarization in device tends to degrade within 1–2 h because of the lack of screening to depolarization field without top metal contacts. The device performance is therefore studied within this period; out of the period the device is repolarized at the same conditions for measurements. A 532-nm laser with tansistor-trasistor logic triggering is used as the light source for photodetection measurements, in which a power meter  is used to calibrate the light intensity. To probe the photoresponse speed, a 365 nm fast-switching LED  and fast measurement unit  is used.Supplementary informationPeer Review File


This approach greatly simplifies the synthesis of currently known materials, and provides a general framework for synthesising both predicted and unexpected new 2D compounds.Only a few of the vast range of potential two-dimensional materials (2D) have been isolated or synthesised to date. Typically, 2D materials are discovered by mechanically exfoliating naturally occurring bulk crystals to produce atomically thin layers, after which a material-specific vapour synthesis method must be developed to grow interesting candidates in a scalable manner. Here we show a general approach for synthesising thin layers of two-dimensional binary compounds. We apply the method to obtain high quality, epitaxial MoS The scalable synthesis of 2D materials critically relies on finding appropriate vapour-phase metal precursors and careful fine-tuning of growth parameters. Here, the authors instead use solid elemental precursors and a single recipe to demonstrate a general approach for synthesising thin epitaxial layers of 20 different 2D binary compounds, including transition metal sulphides, selenides, tellurides, and nitrides. MXn, where M is typically a transition metal and X a chalcogen or non-metal from groups IV, V, or VI5. The molybdenum and tungsten disulphides and diselenides remain the most commonly studied 2D binary compounds—other than hexagonal boron nitride (hBN)—due to the ready availability of naturally occurring bulk crystals amenable to exfoliation. Chemical vapour deposition (CVD) techniques for the scalable synthesis of these materials are available6; however, controlling the stoichiometry and hence the defect density can be challenging. Such techniques typically employ solid metal oxide10 or metal-organic11 precursors which are chalcogenated at elevated temperatures. Finding appropriate metal precursors can be a limiting challenge for extending these methods to other 2D transition metal compounds, and as a result requires single-process dedicated equipment that is highly optimised for growing one specific material. A more general method using simpler precursors would thus be beneficial. Published CVD growth models for binary compounds on metal catalysts stipulate that both M and X elements be insoluble in the catalyst to ensure surface-limited growth14, by analogy with CVD graphene growth on copper15. In fact, the synthesis of monolayer hBN films on copper—despite the high solubility of boron in copper16—suggests that only one component need be insoluble.Many of the possible 2D materials are binary compounds of the form M films with gold, which has limited solubility of the X elements . In brief, a thin layer (~20 nm) of metal M is sputtered onto a c-plane sapphire substrate followed by a thick layer (~ 500 nm) of gold (see Methods). The M-Au layer is then heated to 850 °C to form an alloy with an Au {111} surface. The relative thicknesses of the M and Au layers determines the concentration of M in the final alloy, which here is deliberately limited to ≤5 at%24 in order to maintain single-phase alloying conditions. The Au-M alloy is subsequently exposed to a vapour-phase precursor of element X. The limited solubility of X in the gold restricts the formation of MXn compounds to the surface of the alloy—at the solid–gas interface—resulting in few-atom thick layers of binary compounds that are epitaxially aligned to the underlying Au substrate.Here we present a general method for synthesising two-dimensional compounds on a metal catalyst from solid elemental precursors Fig. . We arraThe process enables the epitaxial synthesis of both known and new 2D materials using a single recipe and simple elemental precursors, demonstrated here by the synthesis of 20 compounds including sulphides, selenides, tellurides, and nitrides.2 layers   Fig.  and anglED) Fig.  of as-grely Fig. . While t2 layers  and selected area electron diffraction (SAED) images of crystals transferred to holey carbon support grids confirms the crystal structure of the MoSers Fig.  and that2 domains transferred onto 90 nm SiO2 on Si substrates (solid line), compared to a monolayer exfoliated MoS2 crystal control sample (dashed line). Identical acquisition parameters and substrates were used in both cases. The intensity of the PL response is comparable in both cases, while the PL peak for transferred crystals is blue-shifted with respect to exfoliated samples. Electric field-effect measurements were performed on 100 µm × 100 µm unencapsulated devices of continuous MoS2 films transferred onto 300 nm SiO2 on Si substrates with pre-patterned contacts (see Methods). Four-point sheet conductivity σS was calculated at varying gate bias (VG) as described in ref. 29, with the results for a representative device shown in Fig. 4 over the gate bias range, and small maximum hysteresis of about 16 V. The low level of hysteresis suggests that the overall number of charge traps is low, including intrinsic charge traps within MoS2 itself30. The field-effect mobility µ was calculated using the formula31Cox is the capacitance per unit area of the back gate. Our measured devices showed a range of µ between 5 and 30 cm2 V−1 s−1  under identical growth conditions results in the structures visible in the SEM images in Fig. Varying 2 layers whose properties—namely Raman spectroscopic response, nanoscale crystalline structure, intensity of PL response, and electric field-effect properties—are comparable to mechanically exfoliated monolayers from bulk crystals or high-quality films from more complex CVD processes. This process is selective towards monolayer synthesis despite the fact that the solubility of molybdenum in gold is appreciable (~1 at%) at 850 °C17, demonstrating that the strict insolubility of all component elements is not required when extending surface-limited growth models to multi-elemental 2D compounds.We have shown the synthesis of epitaxially aligned MoSM and X elemental precursors, and additional Raman, atomic force microscopy (AFM), PL, and TEM characterisation for the materials which are stable during transfer to oxidised silicon or on the catalyst layer. Without further optimisation, a large selection of materials which display morphologies, stoichiometries, and chemical bonding expected of binary 2D transition metal dichalcogenides can be grown. While exhaustive characterisation of all of these materials is beyond the scope of this manuscript—in particular for candidate materials which have not previously been synthesised or isolated from bulk crystallites before—the simplicity of this growth strategy enables very rapid experimentation in terms of the range of precursor combinations that can be tested and materials which can be grown. In practice since multiple M-Au alloy samples can be simultaneously exposed to the same X precursor, the time needed to fully characterise such materials individually far exceeds the time needed to produce them in parallel.Furthermore, we show XPS and SEM data for a wide range of other materials produced simply by selecting different pairs of M, but shows limited solubility for X elements at our growth temperatures. Moreover, gold is unique in that it does not react with X precursors at these temperatures. Gold is catalytically active, which aids in the formation of crystalline atomically thin materials, and the {111} surface formed on c-plane sapphire also templates the epitaxial alignment of the grown materials. The method presented here is distinct from vapour–liquid–solid (VLS) and vapour–solid–solid (VSS) growth models32 of III–V nanowires since we actively avoid supersaturation of component metals in gold—a key hallmark and necessary prerequisite for VLS/VSS growth mechanisms—by limiting the amount of metal available to <5 at%. This ensures that the resulting alloy behaves as a single-phase, simple solid solution at the growth temperature. The role of the gold here is to facilitate phase separation of M and X precursors such that they react only at the vapour–solid interface. Once this interface has been passivated by the 2D MXn layer, the growth terminates, similar to the case of graphene on copper.The surface-limited growth of a wide variety of binary 2D materials presented in this work relies on the use of gold as a catalyst layer. Gold readily alloys with most transition metals 35; however, a key advantage of the present scheme is that solid elemental metal films are used as precursors rather than metal-organic vapours or volatile metal compounds, which makes our process significantly simpler to implement for both known and new TMDs. We also note that similar methods for growing atomically thin carbides and sulphides have been previously reported37, but only for systems where both M and X components have limited solubility (<0.1 at%) in the catalyst. These represent a subset of the general approach presented here, i.e. one where the solubility of M in the catalyst approaches zero. In such cases, growth is dominated by surface-mediated diffusion of both M and X. Here instead the growth is dominated by precipitation of component M from the catalyst bulk upon reacting with X at the solid–gas interface at the surface of the catalyst. The crucial point is that surface-limited growth does not require the metal precursor to be insoluble in the catalyst layer. This fact allows our approach to be compatible with all precursor metals, in contrast to previous reports.The use of gold as a catalyst for synthesising 2D TMD layers has been previously reported in literature2 monolayer coverage—achieved here by increasing the growth temperature—can result in adlayers. In practice, adlayer growth can be reduced by tuning the growth temperature and time, reducing the flux of X, or limiting the alloy M content of the gold catalyst layer. Controlling the M content can be particularly important in determining the layer coverage, maintaining single-phase alloying conditions, and in preventing precipitation of excess metal upon cooling. Such precipitation can interfere with 2D growth and lead to 3D structures on the surface, as seen for Co21 or Fe20. In general we expect that optimisation of additional deposition and growth parameters for individual M–X combinations will be necessary, as has been the case for growth strategies for other 2D materials.In the presented work, growth targeted at complete MoS38 or metal-organic chemical vapour deposition (MOCVD) of 2D materials11. Notably, solid elemental precursors  or simple compounds (NH3) are the only feedstocks required to grow the presented materials, which are in general more readily available and easier and safer to handle than metal-organic precursors and lead to less contaminants (such as carbon) being incorporated into the films. Critically, this approach provides a universal method for epitaxial synthesis of 2D materials without the need for salt additives38, which may have a detrimental impact on material performance through loss of epitaxy and alkali metal doping39. While we have employed a tube furnace operating under low pressure, the growth scheme presented does not rely on a flow but simply the presence of X precursor and as such can be performed in a sealed chamber. Such a scheme might be particularly beneficial to reduce the amount of oxygen and water in the growth system—the alloys of certain transition metals  are highly sensitive to oxidising impurities in the growth chamber41, which can interfere with growth by passivating the catalyst surface with metal oxides. This issue can be addressed by operating in a highly reducing environment or under high vacuum.The present approach holds a number of advantages over state-of-the-art salt-assisted44 suggests that similar strategies might be successfully employed here.Some of the materials grown are unstable under ambient conditions—no special steps were taken to limit the exposure of samples to ambient atmosphere before characterisation. As such, transfer of many of these materials to, e.g., oxidised silicon substrates or TEM grids is challenging, as they rapidly degrade on contact with aqueous or oxygen-containing solutions, which has hampered extended characterisation in many cases. Recent progress in the solution-phase exfoliation of air and water-sensitive 2D crystalsMXenes, based on the insight that only one component of a binary compound need be insoluble to achieve surface-limited growth. The strategy employs only a volatile X precursor, while all the presented materials are grown under identical conditions. Notably, this method enables the growth of epitaxially oriented MXn layers on Au. New 2D materials can be made through the free choice of M and X, and different compounds can be obtained on the same growth substrate simply by switching the precursor X gas. As such, this scalable growth technique simplifies the production of existing binary 2D materials with a quality comparable to exfoliated crystals, and at the same time greatly increases the range of such materials available. We do not doubt that growth conditions for materials can be individually optimised, and that with further research, growth of in-plane and out-of-plane heterostructures could also be accessible.In summary, we have demonstrated a simple and universal strategy for approaching the growth of few-atom thick binary compounds, including transition metal mono- and dichalcogenides and nitride M followed by a thick layer  of gold  on a <001> sapphire substrate. Oxidation of the metal M is avoided by immediate encapsulation with gold before exposure to ambient conditions.Substrates were prepared by physical vapour deposition of a thin layer (~20 nm) of metal X vapours. The vapours were generated by heating solid chalcogen precursors situated upstream from the samples: ~110 °C for sulphur , ~220 °C for selenium , and ~420 °C for tellurium . After growth, the samples were naturally cooled to room temperature under 100 sccm Ar flow. Synthesis of continuous MoS2 films for electrical device measurements was achieved by increasing the growth temperature to 950 °C.Transition metal chalcogenides were synthesised in a hot-wall quartz tube reactor under low-pressure conditions. The chamber was flushed three times with argon (Ar), and the samples were subsequently heated to 850 °C under 100 sccm Ar. The samples were annealed at this temperature for 30–60 min, and growth was subsequently carried out for 10–15 min by exposing the samples to volatised chalcogen 2 flow instead of argon in order to mitigate surface oxidation of the alloys. The growth of nitrides was performed by introducing 5 sccm NH3 into the chamber for 5 min The samples were then naturally cooled down to room temperature under 100 sccm H2.Identical processing conditions were used to synthesise transition metal nitrides in a cold-wall reactor (AIXTRON Black Magic), except that the entire process was done under 100 sccm H2 gold etchant solution . After the gold was completely etched, the films were washed in DI water and transferred onto oxidised silicon substrates, where they were baked at 160 °C for 10 min. PMMA was subsequently removed in acetone. Transfer of 2D materials onto TEM grids (Quantifoil GmbH) was done by wedging transfer46 from transferred films on oxidised silicon.Samples were transferred from the gold substrate by etching transfer. A solution of 10% wt. PMMA 950K in anisole (Sigma Aldrich) was spincoated onto the samples at 1500 rpm for 1 min, after which the samples were baked at 160 °C for 15 min. The polymer film was then manually removed at the edges of the sample. The samples were then put in a KI/I2 was done in an FEI Tecnai T20 G2 operated at 200 kV. ARPES and LEED measurements were conducted at the SGM-3 beamline endstation at ASTRID2 in Aarhus, Denmark. ARPES measurements for MoS2 were acquired at T = 30 K and hv = 49 eV, using an energy resolution <25 meV and angular resolution <0.2°47. LEED images were acquired at T = 30 K and EK = 113 eV.SEM images were taken in a Zeiss Supra 40VP operated in in-lens detection mode at 5 keV. TEM characterisation of transferred MoSRaman spectroscopy was conducted in a Thermo Fisher DXR microscope equipped with a 455 nm laser. Measurements were made using an incident power of 5 mW and a 50× objective, and 5 acquisitions with 10 s exposure time were collected for each Raman spectrum. Photoluminescence spectra were obtained using a custom spectroscopy setup built from a Nikon Eclipse Ti-U inverted microscope. The excitation source was a 407 nm diode laser from Integrated Optics. The light was focused to a diffraction limited spot on the sample with a TU Plan Fluor objective from Nikon  resulting in an incident power of 30 µW. The emitted fluorescent light was collected with the same objective, and the spectra were recorded using a Shamrock 303i Spectrometer equipped with a 450 nm longpass filter (FELH0450 from Thorlabs) and an electronically cooled Newton 970 EMCCD. A total of five acquisitions with 1 s exposure time each were collected for each PL spectrum.2 films were transferred from the catalyst surface using the above procedure onto 300 nm SiO2/Si substrates with predefined electrical contacts48. Typical channel length was 100×100 µm2. Devices were electrically characterised in a Linkam LTS600P probe station after desorbing water from the surface49 via baking at 225 °C for 30 min in dry nitrogen. Subsequent measurements were performed under dry nitrogen at room temperature.Continuous MoSSupplementary InformationPeer Review File


When cells with a mesenchymal type of motility come into contact with an adhesive substrate they adhere and start spreading by the formation of lamellipodia. Using a label-free approach and virtual synchronization approach we analyzed spreading in fibroblasts and cancer cells. In all cell lines spreading is a non-linear process undergoing isotropic or anisotropic modes with first fast (5–20 min) and then slow (30–120 min) phases. In the first 10 min cell area increases 2–4 times, while the absolute rate of initial spreading decreases 2–8 times. Fast spreading depends on actin polymerization and dynamic microtubules. Inhibition of microtubule growth was sufficient for a slowdown of initial spreading. Inhibition of myosin II in the presence of stable microtubules restored fast spreading. Inhibition of actin polymerization or complete depolymerization of microtubules slowed down fast spreading. However, in these cases inhibition of myosin II only partially restored spreading kinetics. We conclude that rapid growth of microtubules towards cell margins at the first stage of cell spreading temporarily inhibits phosphorylation of myosin II and is essential for the fast isotropic spreading. Comparison of the fibroblasts with cancer cells shows that fast spreading in different cell types shares similar kinetics and mechanisms, and strongly depends on dynamic microtubules. Summary: Cell spreading is a non-linear process. The fast spreading phase depends on dynamic microtubules (MTs). Rapid growth of MTs towards the cell membrane promotes the temporal relaxation of acto-myosin contractility. Cell spreading is a complex process  driven bUnderstanding of mechanisms underlying cell adhesion and spreading is of great importance since these events preface invasion of cancer cells and are essential for metastasis.Spreading of an individual cell on the adhesive substrate consists of three major stages: initial attachment , radial spreading (fast or P1) and polarization (contractile or P2) . FormatiEarly works demonstrated large heterogeneity of the cell population according to spreading kinetics . HoweverMicrotubules (MTs) are involved in cell polarization and play an essential role in fibroblast and cancer cells migration . MTs regMTs regulate cell cortex behavior by binding GEF-H1 and inducing phosphorylation of myosin II via RhoA activation when depolymerized . HoweverWhile the necessity of MTs for cell migration has been demonstrated in numerous assays, the role of MTs in adhesion and spreading processes that preface invasion of cancer cells is poorly understood. Particularly, the role of MTs in the P0 phase of cell spreading is thought to be negligible . InvolveOur previous observations showed that stabilization or complete depolymerization of MTs significantly decelerated spreading of Vero cells . We propWe demonstrated that non-linear spreading kinetics is a universal feature of normal and cancer cells with a mesenchymal type of motility, and initial fast spreading, either isotropic or anisotropic, always depended on the dynamic MTs. Our model allowed us to advance the understanding of cell polarization and spreading and provided general insight into how MT dynamics and myosin II-mediated actin contraction coordinate cell spreading. We show that MTs' overall impact on initial stages of cell spreading is realized through temporal inhibition of phosphorylation of myosin II light chain and subsequent promotion of lamellipodia advancement at the cell edge.Cell spreading as a function of time at the population level was described by a sigmoid curve . HoweverOur preliminary observations showed that substantial parts of cells always undergo anisotropic spreading and different treatments of cells resulted in the absence of an isotropically spread subpopulation (see below). So far the subpopulation of cells with anisotropic spreading have to be central to all types of inhibitory analysis. To analyze cell behavior life histories were built; time point of attachment was determined as a frame after which the cell became immotile, the beginning of spreading was determined as a frame after which cell area on the substrate continued to increase for at least 2 min.Table S1).Vero cells on the glass surface started to form lamella in 7.2±3.5 min after attachment. Initial spreading rate was maximal in the first 10 min; in the next 10 min it decreased fivefold and continued to decrease over time . Cells with isotropic spreading extended smooth round lamella, cells with anisotropic spreading extended by simultaneous formation of several short lamellae separated by concave edges. The rate of initial spreading was higher in the isotropic group (Table S2). In next 10 min, the difference between groups was still obvious. After 20 min of spreading all cells acquired an irregular shape and morphological differences between the two groups disappeared.Spreading cells underwent two scenarios: isotropic (40%) and anisotropic (60%) spreading with nearly equal probabilities  Fig. 1BBFig. 1B.Table S1). Up until that point all cells had formed stable edges and some started crawling on the substrate (data not shown). Later on most cells continued expanding their area slowly .Further spreading (P2 phase) continued as a slow process of the formation of anisotropic extensions and at the end of the first hour total cell area increased 6.64±0.35 times compared to initial values .Cell spreading is accompanied by changes in the cell form. Upon attachment, cells were nearly round shaped with EF (elongation factor) 1.15±0.02 and had smooth contours (circularity 0.87±0.02). With the beginning of morphologically visible polarization (1 h after plating), cells continued elongation (EF increased up to 1.41±0.07) and acquired an incurved shape (circularity 0.68±0.04). In 3 h when all cells had started crawling, circularity remained the same, while elongation factor increased slightly (Table S1). In 3T3 cells, relatively fast spreading continued for the first 10 min and then also slowed down  was observed in the first few minutes after attachment and then the spreading rate gradually decreased as described elsewhere , 2008. ITable S5), relative area increase for cells spread on poly-L-lysine was 4.78±0.46, within 3 h cells on poly-L-lysine enlarged 7.21±0.82 times, the main difference between cells on regular glass and poly-L-lysine was that after 3 h cells spread on poly-L-lysine were less elongated and the transition to migration was less frequent.We assessed whether the same type of kinetic curve is retained when cells are spread on non-specific and ligand-specific binding surfaces, as the ECM composition had been reported to define the adhesion type and dynamics . We obseTable S5), though they started to form initial lamella 2.2±1.5 min after initial attachment (three times faster than cells on a regular coverslip did). When cells spread in the presence of both fibronectin and serum vitronectin, the transition between slow and fast spreading modules was less evident and cells continued spreading without transition to free migration stage after 3 h.To assess whether cell spreading depends on integrin-mediated adhesion, we used the fibronectin-covered surface. Cells plated on fibronectin in the absence of serum demonstrated lower rates of initial spreading  and inhibition of dynamic instability of MTs with nocodazole and paclitaxel applied at nanomolar concentrations .After initial attachment, cells with depolymerized MTs retained a nearly round shape for a long time (80±113 min) undergoing intensive blebbing without formation of lamellipodia. Some cells were unable to start spreading during the entire time-lapse series (6 h). The spreading process started with the formation of small pseudopodia that flattened upon attachment and then formed small lamellae. Blebbing did not stop after lamellae formation and continued for 1–3 h during cell spreading A,C.Table S6) and more homogenous spreading kinetics than in control (Table S7).The spreading process after depolymerization of MTs was always anisotropic and in some cases reversible; a cell that already began spreading returned to rounded form and then began to spread again. Overall, cells without MTs demonstrated a slower spreading rate  a tenfold prolongation of the blebbing phase and an overlapping with the spreading phase, (ii) a slowdown of initial spreading and loss of isotropic spreading and (iii) prevention of cells forming wide lamellae and (iv) slow and sometimes reversible spreading within 3 h.Since many effects attributed to the lack of MTs could come from the stabilization of MTs  we furthN=65) to 2.29±0.78 µm after treatment (N=61) (N=98) to 7.31±2.27 µm/min (N=99). After treatment, the overall number of tracks decreased in the perinuclear area, and spatial distribution of tracks became more random. Moreover, in cells with stabilized MTs most of the tracks were shorter than 3 μm  decreased from 5.33±0.67 µm before treatment (t (N=61) A–B,E andhan 3 μm E.Fig. 3Second, we analyzed the spatial distribution of MTs in Vero cells fixed 20 min after plating and stained with antibodies against actin and tubulin. In control cells, numerous MTs were visualized as a radial array, and the distance from MTs' ends to the cell edge was 1.99±0.09 µm. In cells with stabilized MTs radial array was less evident, and the distance between MTs' plus ends and the cell margin was doubled – 3.86±0.18 µm.Movies 1 and 2). In normal cells during radial spreading MTs grew directly towards the cell edge, while after treatment MT growth was randomized and significant parts of nascent lamella had no MTs. Thus, nocodazole and paclitaxel applied in nanomolar concentrations prevents MTs' plus ends from growing into nascent lamella.Finally, MT growth was analyzed directly during spreading of Vero cells . During spreading these cells remained smooth edged and became elongated .Initial spreading of cells with stabilized MTs was mainly anisotropic and blebbing continued on the unstretched cell surface. Spreading kinetics was linear and significantly slower compared to control cells .The same kinetics of spreading was observed for MEF (mouse embryonic fibroblasts) fibroblasts treated with MT inhibitors: during the first 20 min the spreading of cells with depolymerized MTs was almost linear and relative area increase was only 2.08±0.09 times, in 3 h relative area increased 3.84±0.34 times. Cells with stabilized MTs spread slightly faster, but in 20 min their relative area increased only in 2.64±0.23 times and in 3 h relative area increased up to 4.29±0.36 times . MTs in EB3-depleted cells were almost quiescent RNA expressing vector was cloned into pGPV expression vector , shRNA sequence  prolongation of the blebbing phase (P0) and its overlapping with the spreading phase, (ii) slowdown of initial spreading (P1) and (iii) slowdown of spreading in the hourly scale (P2). The difference between cells with stabilized MTs and cells without MTs is in the morphology of lamellae and the ability of cells to elongate.It is generally assumed that cell spreading is limited by the contraction since spread fibroblasts generate significant contraction force applied to the substrate . To testFig. S4).Cells treated with inhibitors of myosin II phosphorylation pathway demonstrated nearly normal kinetics of spreading during the first 20–30 min after plating. Upon attachment, cells had no blebbing and started to expand their area within 10.5±4.2 min (Y-27632 treatment) and 7.5±4.5 min (blebbistatin treatment). The major difference from the untreated cells was a lack of isotropically spreading cells. From the very beginning, lamellae in all cells started to expand simultaneously with long protrusions in several directions, so the cell boundary became incurved and dissected and cells gradually acquired irregular stellate form with numerous protrusions that continued to elongate . Spreading kinetics for P0, P1 and P2 modules was nearly the same for blebbistatin- and Y-27632-treated MEF fibroblasts and was similar to control cells at all time-points .The rate of spreading after blebbistatin treatment in the first 10 min was slightly less than for control cells. Further spreading was similar to that of the control cells .During early phases of spreading, circularity and elongation factor values for Vero cells were similar to those for untreated cells, but later cell circuits became more tortuous , while elongation factor was similar to the untreated cells . After 3 h cells treated with nocodazole and blebbistatin remained almost completely round, but gained complex dissected boundaries, while cells treated with nocodazole and Y-27632 become elongated and exhibited relatively smooth boundaries (Table S4). In both cases blebbing was completely inhibited while overall cell morphology was similar to the cells treated with nocodazole only; spreading began with protrusion of small pseudopodia which immediately formed several flat narrow lamellae .Addition of blebbistatin had no effect on cell morphology compared to the treatment with nocodazole only, while addition of Y-27632 recovered nearly all changes in elongation factor and circularity values in hourly scale (Table S6).Treatment of cells with depolymerized MTs with blebbistatin partially recovered the fast initial spreading . After stabilization of MTs inhibitors and blebbistatin treatment cell area increased similarly to control cells (Table S6).Addition of blebbistatin to cells with stabilized MTs recovered kinetics of the fast spreading stage in the same manner as for cells with depolymerized MTs . Notably, as for Vero cells, treatment with myosin inhibitors facilitated spreading of MEF cells with stabilized MTs and the recovering effect of blebbistatin as a direct inhibitor of myosin II was more apparent than that of Y-27632.The same effects were observed for MEF fibroblasts  and became common 1 h after plating .In the cells undergoing isotropic spreading, no actin bundle was determined for the first 10 min. After 10–20 min some cells already had thin actin bundles along the cell margin, and myosin was distributed uniformly through the cell lamellae. Between 20–60 min, actin arches were gradually substituted with straight thick actin fibers. Stress fibers with regular myosin patches were found in a few cells 20 min after plating  and between 60–180 min actin bundles remained thin and no stress fiber formed .Treatment with blebbistatin and Y-27632 prevented formation of thick actin bundles and reorganization of myosin II during cell flattening; in 20 min a few of very thin actin bundles could be determined . This data confirms the hypothesis about the temporal relaxation of myosin II by dynamic MT network and provides a mechanistic insight into the role of dynamic MTs in fast spreading.Based on the results obtained, we hypothesized that dynamic MTs affect the phosphorylation state of myosin II light chain (MLCII). Addressing this question cells with normal, stabilized and disrupted MT network were stained with antibodies against phosphorylated MLCII at 5 and 20 min after initial attachment. At 5 min, in cells with stabilized or depolymerized MTs, we observed prominent actin bundles colocalized with ring-shaped clusters of phosphorylated MLCII near the cell margin , while iFig. S8; Tables S10–S11). The slower initial spreading of PC-3 cells compared to other cell lines may be attributed to the lower percentage of cells with isotropic spreading in this culture (data not shown). Nevertheless, the switch between fast and slow spreading was also obvious for the PC-3 cell line. In all cases alteration of MT dynamics  led to the significant deceleration of initial spreading and decrease of the overall area within 3 h after cell attachment. Thus, the phenomenon of fast spreading stimulated by dynamic MTs is nearly the same for normal and cancer cells.Basic mechanisms that underlie cell spreading and migration are central to the process of metastatic growth. The role of MTs in cancer cell spreading was tested using three cell lines – HT1080, A549 and PC-3. For all these cells , we observed the same type of kinetic curve with the spreading rate being maximal in the first few minutes and rapidly decreasing over time  inhibition of myosin II has no effect on the fast initial spreading, while it changes cell behavior afterwards, (ii) complete depolymerization of MTs has a dramatic effect on cell spreading and polarization, (iii) partial stabilization of MTs is sufficient to slow down cell spreading with minor changes in cell morphology and (iv) myosin II pathway inhibitors make cell early spreading insensitive to stabilization and even depolymerization of MTs.Cell spreading as a function of time was originally described by a sigmoid curve ; howeverEarly spreading was a subject of a detailed investigation for several studies . In thesTable S9). Thus it can be concluded that the initial stages of cell spreading onto a substrate exhibit a physiologically universal behavior being cell-type and substrate independent.Anisotropic spreading is slower than isotropic, yet also non-linear . This fiOur observations confirm that the spreading process occurs asynchronously at the population level and for a given cell is largely non-linear both in a minute and hourly scales. However, when cells are virtually synchronized at the time when fast spreading starts , the detRadial spreading occurs through isotropic and anisotropic pathways and in both cases is quickly decelerated. In accordance with previous observations , the aveAs initial steps of cell spreading might depend on the formation of integrin-mediated adhesions, we evaluated the spreading rate for Vero cells plated on fibronectin and obtained the same type of kinetic with obvious transition from P1 to P2, although the values of total area increase in the presence of fibronectin only were 1.5 times less than for cells that spread on unspecific substrate (in the presence of serum). In presence of both integrin specific and unspecific ligands (fibronectin and vitronectin) the spreading curve became more linear and cells continued to spread radially even after 20 min from initial attachment. These data are consistent with other studies, as it was shown that serum promotes spreading but not initial attachment of 3T3, chicken embryonal fibroblasts and human skin fibroblasts , and serObtaining a detailed time scale of spreading events, we further addressed the question on the contribution of cytoskeletal components in switching between phases. Blebbistatin and Y-27632 completely inhibited blebbing at the first stage, i.e. decelerated tension force that is supposed to keep a cell rounded , but neiThe lack of myosin regulation of the initial spreading could be explained by taking into account the relatively slow development of the contractile system in the spreading cell. Myosin-dependent retraction in spreading fibroblasts requires formation of actin bundles attached to the cell surface , which tTherefore we suggest that myosin II-dependent contractility is absent during phase P1 and addressed the question whether temporal relaxation of myosin II in spreading cells depends on MTs.The first clues about the role of MTs in cell spreading came from the comparative study of fibroblasts spread on 3D and 2D substrates . FibroblUsing inhibitory analysis we uncovered the dual role of MTs in cell spreading on the 2D substrate. Depolymerization of MTs in fibroblasts or fibroblast-like cells is well known to inhibit cell motility . More reTo stabilize MTs we used a treatment with simultaneously added nocodazole and paclitaxel as described earlier . StabiliComplete depolymerization of MTs prolonged P0 phase and made this phase indefinitely long for some cells. After depolymerization of MTs, no lamellipodia formation was observed and no fast spreading occurred – instead overall increase of cell area was slow and linear in time.In cells with stabilized MTs, we observed tug-of-war in the formation of lamellae via small lamellipodia from the beginning of spreading, but the overall spreading was irreversible. Thus the major difference between cells with stabilized MTs and depolymerized MTs during the spreading process was the absence of real lamellipodia formation. Our data suggest that the cell morphology and spreading kinetics depend on the ability of the cell to switch between amoeboid stage with blebbing (P0) and formation of lamellipodia (P1 and later on) that can be described as a mesenchymal type of behavior. Complete depolymerization of MTs keeps fibroblasts with the amoeboid-like behavior for a rather long time, while fibroblasts with stabilized MTs maintain the ability to go through amoeboid-to-mesenchymal transition, yet slower than cells with dynamic MTs. Thus MTs are responsible for the mesenchymal type of fibroblast behavior.To test the hypothesis that deceleration of cell spreading induced by perturbations with MT system depends on myosin contraction, we treated cells plated on the glass surface simultaneously with MT and myosin II pathway inhibitors.In the presence of myosin inhibitors stabilization and even complete depolymerization of MTs have a minor effect on the initial attachment and spreading stages (P0–P1) – duration of P0 and spreading rate during fast spreading (P1) are both similar to the cells treated with myosin inhibitors only. By direct analysis of MT dynamics and distribution in the presence of myosin II pathway inhibitors, we confirmed that there is no feedback between myosin II activity and MTs – MT dynamics were not reversed when myosin II had been inhibited. Thus, return to the normal dynamics of spreading under the simultaneous action of two inhibitors could be explained solely by the lack of myosin II induced contraction.In normal cells, switching between blebbing and lamellipodia formation depends on the balance between actin polymerization and actomyosin contraction . This shBased on the results obtained the following model of cell spreading is proposed : after iFormation of the stress fibers and focal adhesions during cell spreading largely depends on the cooperative work of FAK kinase and RhoA signaling pathway . DynamicTaking into account the well-known role of MTs in the interaction with focal adhesions at the cell edge , we suggTaken together, our data show that in cells with mesenchymal type of motility both in physiological and pathological conditions MTs have a strong impact on the early stages of cell spreading. MTs’ overall impact on initial spreading stages occurs through the inhibition of myosin II light chain cascade and promotion of actin polymerization at the cell edge in the form of lamellipodia. Since stabilization of MTs results in the profound effect on all stages of cell spreading it could be speculated that several molecular pathways are involved in this process. Uncovered players are MT-plus TIPs , and sma2. Cells were grown to 70% confluence, trypsinized quickly, washed with soybean trypsin inhibitor, centrifuged, and plated in 35 mm Petri dishes. The cell concentration was adjusted so that spreading cells did not make contact with each other. Cover glasses were washed for 1 h in concentrated % sulfuric acid, washed for 30 min with tap water and then with ddH2O and then coated the cover glass with 500 μl of a 20 μg/ml pure fibronectin (Sigma-Aldrich) solution for 1 h at 37°C, aspirated the excess of solution and let the coverslips dry at room temperature.Vero (ATCC#CCL-81), 3T3 (ATCC #CRL-1658), HT1080 (ATCC# CCL-121), MEF (ATCC # SCRC-1040), PC-3 (ATCC#CRL-1435) and A549 (ATCC# CCL185) cells were maintained in DMEM medium supplemented with 25 mM HEPES , 10% fetal calf serum , and 0.8 mg/ml gentamycin at 37°C, at 5% CO−6 М. To stabilize microtubules 100 nM nocodazole and 50 nM paclitaxel (Sigma-Aldrich) were simultaneously added to the culture medium. For inhibition of non-muscle myosin II, blebbistatin (Sigma-Aldrich) (45 µM) and Rho-kinase inhibitor Y-27632 (Sigma-Aldrich) (10 µM) were used. For inhibition of actin polymerization, 30 nM of cytochalasin D (Sigma-Aldrich) was added to the cell suspension prior to plating.To depolymerize, MT cells were treated with nocodazole (Sigma-Aldrich) in the concentration of 13.2×102. Cells were grown to 70% confluence, trypsinized quickly, washed with soybean trypsin inhibitor, centrifuged and plated in 35 mm Petri dishes. The cell concentration was adjusted so that spreading cells did not make contact with each other. Cover glasses were washed for 1 h in concentrated % sulfuric acid, washed for 30 min with tap water and then with ddH2O and then coated the cover glass with 500 μl of a 20 μg/ml pure fibronectin (Sigma-Aldrich) solution for 1 h at 37°C, aspirated the excess of solution and let the coverslips dry at room temperature.In all cases the dilution of antibodies was 1:100 for primary antibodies and 1:150 for secondary antibodies according to the manufacturer's protocol. Cytoskeleton structures were visualized by staining with phalloidin [conjugated with Alexa 568  or Atto390 ], antibodies to non-muscle myosin II , antibodies to a phosphorylated light chain of nonmuscle myosin II  and antibodies to α-tubulin . Cells were fixed with 2.5% glutaraldehyde in PBS, pH 7.2, for 15 min at 4°C, washed three times, permeabilized with 0.1% Triton X100 in PBS for 1 h at room temperature, washed three times with 1% sodium borohydride, and stained with antibodies. To visualize MT plus-ends, cells were stained with monoclonal antibodies to EB3  and then stained with second goat anti-rat antibodies conjugated with AlexaFluor 488 (Abcam). For triple staining, cells were incubated first with monoclonal antibodies to α-tubulin  and myosin II , then with second anti-mouse antibodies conjugated with Alexa488 (Sigma-Aldrich) and second anti-rabbit antibodies conjugated with AlexaFluor 555 (Invitrogen) correspondingly, actin structures were visualized by staining with phalloidin conjugated with Atto390. Specimens were mounted in Mowiol 488 (Sigma-Aldrich). Fixed cells were imaged on Nikon TiE microscope under PlanApo 60×/1.4 objective (phase contrast) with CoolSnapHQ digital camera using filter sets for FITC and Texas red. Vero, 3T3, HT1080, MEF, PC-3 and A549 cells were maintained in DMEM medium supplemented with 25 mM HEPES , 10% fetal calf serum , and 0.8 mg/ml gentamycin at 37°C at 5% COVero or 3T3 fibroblasts were transfected with X-tremeGENE HP DNA transfection reagent according to the manufacturer's protocol. Briefly, 1 μl of EB3-RFP plasmid  and 2 μl of the agent were added to 100 μl of PBS, incubated for 20 min and then transferred into a Petri dish with cells in 2 ml of DMEM medium. After 24 h of incubation we observed an acceptable plasmid expression level, according to flow cytometry data, about 80% of cells were successfully transfected. EB3 shRNA probe  was cloRNA was extracted from thawed suspensions of cells using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. The RNA concentration was measured using a NanoPhotometer , and its purity was assessed according to the А260/А280 and А260/А230 ratios. cDNA was transcribed using the ImProm-II AMV-Reverse Transcription Kit (Promega) according to the manufacturer's instructions.EB3 were 5′- CAA GAA ACT CAT TGG CAC AGC A -3′ (sense) and 5′- TCG TTC TTT CTC AAG CCC GT -3′ (antisense).Real-time qPCR was further performed on CFX96  cycler with Taq-polymerase in SYBR Green I buffer . The reaction protocol included denaturation , followed by 40 amplification cycles . All samples were processed in triplicate. All primers were synthesized and HPLC-purified by Syntol (Russia). Primer sequences for YWHAZ, UBC and HPRT1.The data were normalized according to the method proposed by 2-independent media (Gibco) with 10% of fetal calf serum . CoolSnap HQ2  or Hamamatsu ORCA-Flash4.0 V2  digital cameras were used for image recording, with 1 min time intervals between frames.Live imaging was carried out on inverted Nikon TiE fluorescent microscope operating under MicroManager software with ×20/0.45 objective (phase contrast) at 36.5–37°C in a COMT dynamics was analyzed by fluorescent microscopy of transfected cells on the same microscope. Time-lapse was recorded using PlanApo 60/1.4 oil immersion objective with a time interval of 2 s between frames and exposure of 300 ms. For visualization of GFP, standard FITC filter cube was used (emission 510–540 nm), for RFP–Cy-3 filter cube (emission 575–640 nm).2)/(4πS), where P is the length of cell outline (perimeter), S is the cell area, and elongation factor (EF) is the ratio of the major and minor axes of the equimomental ellipse of cell projection. The spreading rate was evaluated as the rate of cell area enlargement per time unit for each cell and then normalized using initial area of a given cell as the denominator.Microscopic data were analyzed in ImageJ program (NIH). Cell area was measured on phase contrast images, to obtain more precise data, cell boundaries were contoured manually. The last image before the first lamellipodia protrusion was considered the zero time point for each cell. Spreading speed on each time interval was estimated as the average difference between cell area on first and last frames of the interval. For quantitative description of cell morphology we used the parameters of form factor and elongation factor, where the first allows estimating the complexity of cell edge and the second indicates the extent of cell polarization: form factor was calculated as , and data are presented as mean values with a standard error of mean. Fluorescent images were processed using ImageJ and finalized with Adobe Photoshop  software.


We aimed to investigate the relation between regenerative deficit in elderly and increase in O2•− production along with mitochondrial alterations. Myoblasts and myotubes from skeletal muscle of young and elderly healthy subjects (27.8 ± 6 and 72.4 ± 6.5 years old) were measured: (1) superoxide dismutase activity and protein content, (2) mitochondrial O2•− production levels, (3) O2•− production variability, and (4) mitochondrial bioenergetic profile. Compared to young myoblasts, elderly myoblasts displayed decreased SOD2 protein expression, elevated mitochondrial O2•− baseline levels, and decreased oxidative phosphorylation and glycolysis. Additionally, elderly versus young myotubes showed elevated mitochondrial O2•− levels when stressed with N-acetyl cysteine or high glucose and higher glycolysis despite showing comparable oxidative phosphorylation levels. Altogether, the elderly may have less metabolic plasticity due to the impaired mitochondrial function caused by O2•−. However, the increased energy demand related to the differentiation process appears to activate compensatory mechanisms for the partial mitochondrial dysfunction.Sarcopenia is the age-related loss of skeletal muscle mass, strength, and function. It is associated with regenerative difficulties by satellite cells, adult muscle stem cells, and alteration of oxidative management, mainly the increase in superoxide anions (O The isoforms of SOD are different in their cellular location: SOD1 is located in cytosol and mitochondrial intermembrane space, SOD2 in mitochondrial matrix, and SOD3 in extracellular space. Mitochondria are also the “powerhouse” of the cells producing energy in the form of adenosine triphosphate (ATP) [2 and H2O via the mitochondrial tricarboxylic acid cycle (TCA) and associated OXPHOS. OXPHOS takes places through the respiratory chain, namely, complexes I–V, that transfers electrons obtained from TCA NADH or FADH2 across the mitochondrial inner membrane and the terminal electron acceptor, oxygen (O2). More in detail, the electron transport chain passes a high energy electron at the inner mitochondrial membrane pumping hydrogen out of the matrix space, creating a proton motive force (Δp) across the inner membrane with both electrical (ΔΨm) and chemical (ΔpH) components [2 produced can be converted to bicarbonate and protons as catalyzed by carbonic anhydrase [Sarcopenia is defined as the age-related loss of muscle mass, strength, and function . In part2•− . The maje (H2O2) . Subsequte (ATP) , 16. In te (ATP) , and thete (ATP) , 19. Themponents . The granhydrase , another2•− towards the mitochondrial matrix, while complex III leaks O2•− towards both the intermembrane space and the mitochondrial matrix [Although the major part of oxygen is used for ATP synthesis (coupled respiration), the mitochondrial membrane is leaky and protons may leak back through the membrane, increasing oxygen consumption (uncoupled respiration). There are three leak events: complex I leaks Ol matrix , 22.2•− production and mitochondrial alterations. To verify this, primary myoblasts were isolated from human skeletal muscle of young and aged healthy subjects and measured as undifferentiated and differentiated cells: (1) SOD activity and protein content, (2) mitochondrial O2•− production, (3) variability in O2•− production, and (4) mitochondrial bioenergetics profile. The data from this study suggest that in elderly primary myoblasts, the metabolic machinery may not work properly. The analysis of the bioenergetic profile in elderly myoblasts seems to suggest a defect in mitochondrial function and a different metabolic plasticity with respect to young ones. Although elderly and young myotubes showed a similar oxidative metabolism, surprisingly in elderly myotubes, we found an increased level of mitochondrial superoxide anion relying more on the glycolysis processes. Taken together, these findings would suggest a glycolytic compensation in response to mitochondrial oxidative damage.We hypothesized that the deficit in regeneration in elderly could be related to the increased On = 8)Young subjects, 27.8 ± 6 years old (n = 5)Elderly subjects, 72.4 ± 6.5 years old  at FACSAria III (BD) as described in di Filippo et al. . The pri2. The loading procedure is previously described by di Filippo et al. [Videomicroscopy experiments are performed on cells plated on the bottom of special 96-well plates at a confluence of 10,000 cells/cmo et al. . After ro et al. . The livAntioxidant enzyme assays and Western blotting were performed using myoblasts and myotubes lysed in RIPA buffer , supplemented with 1 : 100 protease and phosphatase inhibitor cocktail , incubated 1 hour at 4°C, and centrifuged at 10000 rpm for 10 minutes at 4°C. Cytosol protein concentrations were measured on the resulting supernatant according to Bradford's method .2CO3, pH 10, 10 mM cytochrome c , and 1 mM xanthine  and xanthine oxidase . As the xanthine oxidase activity varies, the amount used for the assay was sufficient to stimulate cytochrome c reduction at 550 nm at a rate of 0.025 per minute without SOD addition. SOD units were calculated on the basis of the definition that one unit represents the activity that inhibits cytochrome c reduction by 50%.Superoxide dismutase (SOD) activity was determined using the modified method of L'Abbé and Fischer , 31. Theμg lysates from young and old myoblasts and myotubes, using SOD1 (71G8) mouse mAb  at 1 : 1000, SOD2 (D9V9C) rabbit mAb  at 1 : 1000, and β-actin (8H10D10) mouse mAb  at 1 : 1000, as primary antibody, and secondary HRP-conjugated antibodies  at 1 : 5000. Bands were detected and pictured at Bio-Rad GelDoc by LiteAblot PLUS enhanced chemiluminiscent substrate (EuroClone); densitometry analyses were performed with ImageJ software.Western blotting (WB) analysis was performed on 40 μM N-acetyl cysteine (NAC) for 24 hours or with NES containing 10 mM glucose plus 5 mM sodium pyruvate (BURST) solution, as acute stimulus and for 15 minutes and 2 hours. The cells were loaded with 5 μM MitoSOX and incubated for 10 min at 37°C in culture media and washed twice with normal external solution  NaOH, pH 7.3). During measurements, samples that received NAC or BURST pretreatment were in NES plus 200 μM NAC or BURST solution. Images were acquired at 512 × 512 pixels using a LSM 510 META system  equipped with an Axiovert 200 inverted microscope and a Plan Neofluar oil immersion objective (40x/1.3 NA), and they were pseudocolored in green. In a typical time-series recording, 14 frames were collected at frame rate of 0.1 Hz. MitoSOX fluorescence was acquired by exciting at 543 nm and collecting the emission using an LP filter set to 560 nm. Image analysis was performed by LSM 3.0 software (Zeiss). For each sample, fluorescence images were acquired from five randomly selected fields and off-line analysis of intensity was performed in ROI-representing cell areas, while in time lapse experiment ROIs were selected inside obvious mitochondrial areas. All experiments were performed at room temperature (22–26°C).Superoxide production was measured using the indicator MitoSOX Red  according to the manufacturer's instructions. Satellite cells were cultured as both myoblasts and myotubes. Before experiments, some of the cultures receive a treatment with 200 4/well density in 100 μl of growth medium and incubated for 24 hours at 37°C, 5% CO2. Before the performance of the Cell Mito Stress Test, some of the cultures received a 200 μM NAC treatment for 24 h. Briefly, myoblasts and myotubes were incubated for 45 min at 37°C in ambient CO2 in free-buffered DMEM  (pH = 7.4) containing 1.0 mM sodium pyruvate , 10 mM glucose, and 2 mM glutamine . Baseline oxygen consumption  rate and parameters calculated after the addition of each drug were measured 3 times for 3 min each separated by a 3 min mix . Following the baseline, 1.6 μM oligomycin , the ATP synthase inhibitor, was injected into each well, followed by 3 cycles of 3 min mix and 3 min measurement. Then, 0.5 μM of carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone  was injected to uncouple the mitochondrial inner membrane and measure maximal respiration . Lastly, 1 μM antimycin A  is injected to inhibit electron flux through complex III and so measurement of nonmitochondrial O2 consumption. At the end of each experiment, the cells were lysed to determinate the total protein content (BCA assay). The mitochondrial OCR is referred to μg of total cellular proteins and reported as pmol·min−1·μg −1.The XF Cell Mito Stress Test was used to measure the oxygen consumption rate (OCR) of myoblasts and myotubes using the Seahorse XFp Extracellular Flux Analyzer . This technique uses specific mitochondrial inhibitors which enable the determination of six parameters for describing the key aspects of mitochondrial function: basal OCR, ATP-linked OCR, spare respiratory capacity (SRC), maximal respiratory capacity, proton leak, and nonmitochondrial respiration. Extracellular flux technology enables rapid, real-time detection of metabolic changes in cellular respiration and glycolysis rate. Cells were seeded in an XFp 8-well cell culture miniplate (Seahorse Bioscience) in three replicates at 25 × 10μM oligomycin was injected to inhibit OXPHOS and further shift the energy production to glycolysis; the consequent increase in ECAR would reveal the cellular maximum glycolytic capacity. Finally, 50 mM 2-deoxy-glucose  was added; such compound is a glucose analogue that inhibits glycolysis through competitive binding to glucose hexokinase. The resulting decrease in ECAR confirms that the ECAR produced in the experiment is due to glycolysis. The ECAR is referred to μg of total cellular proteins and reported as mpH·min−1·μg−1.In order to quantify changes in the glycolytic function, the glycolysis stress test was performed. It measures the extracellular acidification rate , which reflects the rate of glycolysis. It was carried out under similar conditions as the mitochondrial stress test with some differences. The assay media contained no glucose or pyruvate but supplemented with 2 mM glutamine. After the measurement of the basal ECAR, the cells enter into glycolysis with the injection of 10 mM glucose. Then, 1 t test . Analysis of mitochondrial fragmentation has been performed using Bonferroni's multiple comparison test. The significance has been codified as ∗p ≤ 0.05 and ∗∗p ≤ 0.005.The Seahorse XF instrument was controlled by Seahorse software for the generation of data files. For data processing, Seahorse Wave software (version 2.3.0) was used. The analysis of OCR and ECAR data was carried out also using the XF Stress Test Report Generator (version 3.0.6). Figure and statistical analyses were generated with the help of GraphPad Prism version 5.0 . Results are presented as means ± standard errors (SEM) or as representative traces. The statistical analysis was carried out using the ve+ cells); ability to fuse, calculated as fusion index (% FI), % of unfused desmin/ve+ cells; and population doubling level at day 25 of culture. In ve+ cell %) does not significantly change with the donor's age, the fusion index was significantly lower in primary human elderly myoblasts differentiated for 7 days . Moreover, as we previously reported [ve+ cells calculated after 7 days of differentiation increased with age (from 32.6% in young versus 66.8% in elderly), while the myoblasts' proliferative rate (PDL) at 25 days of culture was significantly decreased.Human myoblasts from muscle biopsies were isolated, expanded in culture, and characterized by myogenic purity, calculated as the number of cells expressing desmin  and CD56/ve+/5.1H11/ve+ cells 70.1 ± 5.6 and 68.4 ± 4.3, resp.) was not significantly different, confirming a good myogenicity of our cultures and the validity of the choice of characterization markers.Moreover, for our experiments, we have used myogenic precursors sorted for two specific surface markers: CD56 and 5.1H11. CD56 is a surface marker expressed by human satellite cells either in situ or upon dissociation . 5.1H11 ve+ cell %) is shown for young and elderly cultures and analyzed by the expression of desmin. To validate whether the expression of desmin was enough and sufficient as myogenic purity index, we further sorted by FACS the primary myoblast for two surface myogenic markers (CD56 and 5.1H11). The fusion index was determined by counting the number of nuclei in differentiated myotubes (positive for MyHC) after 7 days of culture in differentiation medium and reported as a percentage of the total number of nuclei; further, the unfused desmin-positive cells were counted after 7 days of differentiation. Population doubling level calculation at day 25 of culture was used to assess cell proliferative capability. Immunostaining and FI data were derived from at least 1000 cells counted in at least 10 different randomly chosen optical fields of each culture. All data  are presented as mean ± SEM, ∗p < 0.05.The table summarizes the characterization parameters obtained from young and elderly samples. The percentage of myogenic cells . In 2+ transients in undifferentiated cells and 6- and 9-day-old differentiated cells. KCl 40 mM caused a transient rise in cytosolic Ca2+ levels in myotubes but not in myoblasts as expected .We measured the activity of the cytosolic isoform of the antioxidant enzyme SOD1 in cell lysates obtained from myoblast and myotubes both from young and elderly donors. No difference was found comparing young versus old myoblasts or young versus old myotubes. A significant increase in SOD1 activity was observed comparing myoblasts versus myotubes on both young and elderly conditions  of the images at basal condition in untreated control condition at time zero of the time series recordings .We investigated the production of Ocordings , and we 2•− production when treated for 24 hours with 200 μM of the general antioxidant NAC or for 2 hours with high glucose and sodium pyruvate solution to BURST the increase in mitochondrial superoxide.In addition, myoblasts  or myotun = 357, elderly n = 111, ∗∗∗p ≤ 0.0005). The pretreatment with 200 μM NAC  and BURST  statistically increased the O2•− production in the elderly with respect to the young ones (∗p ≤ 0.05 in NAC condition and ∗∗∗p ≤ 0.0005 in BURST condition). In myotubes . The MFI of myoblasts (panel c) and myotubes (panel d) are compared to the control and NAC sample conditions. Young myoblasts and myotubes in NAC condition showed a statistically significant decrease in MFI compared to the control condition (∗∗∗p ≤ 0.0005). The NAC exposure of elderly myotubes resulted in a statistically significant increase in MFI compared to the control condition (∗∗p ≤ 0.005).Elderly myoblasts in the control condition showed a statistically significant decrease in the MFI compared to young samples . On the contrary, elderly myotubes showed a statistically significant reduction of this parameter with respect to the young .While performing our MitoSOX analysis, we also acquired short videos recording 14 frames every 3.5 seconds (about 50 seconds) of the MitoSOX signal. We graphed the MFI of young and elderly primary myoblasts versus time  observin∗∗∗p ≤ 0.0005). Concerning the ECAR profile, the glycolysis parameter is a measure of extracellular acidification reached by the cells following the addition of saturating amounts of glucose. We found that glycolysis was significantly lower in the elderly compared to the young (∗p ≤ 0.05).Young and elderly samples (either myoblasts or myotubes) were assayed in untreated control condition and 24 hours after NAC treatment Figures –9. In ∗p ≤ 0.05), suggesting that NAC is more effective in scavenging ROS in the young than in the elderly. The glycolysis parameter, obtained by the ECAR profile, was found significantly decreased in elderly myoblasts compared to young ones (∗∗p ≤ 0.005).Following NAC exposure , the difOther glycolytic parameters  were not statistically different comparing young versus elderly myoblasts.∗p ≤ 0.05).After 7 days of differentiation , we did ∗p ≤ 0.05 and ∗∗∗p ≤ 0.0005, resp.) in elderly versus young ones, suggesting that the scavenging activity of the antioxidant may be more considerable on the young than on the elderly. After NAC exposure, glycolysis value did not change comparing young and elderly myotubes.After NAC treatment , the spaSimilarly to the values obtained for myoblasts, also in myotubes other glycolytic parameters  were not statistically different comparing young versus elderly.2•−, and scavenger activity of antioxidant enzymes [ve+ cells). The difficulty in functional differentiation, which we already previously demonstrated [Aging is known to be characterized by an increase in oxidative stress related to an imbalance between ROS production, in particular O enzymes , 36. Thi enzymes , 29. Thenstrated , is conf2•− radical, which we had already shown to be higher in the elderly's whole myoblasts [2•−, readily reduced by scavenger enzymes, seems to be positively correlated with a sustained metabolism, as occurs during the normal mitotic process or during the early phase of regeneration [The Oyoblasts , could tneration , whereasneration . On the 2•− in the mitochondria of elderly myoblasts . These results suggest that first, an increase in O2•− production might be not associated with a proportional increase in SOD, and second, solely in samples from young donors might NAC be able to promote cell fitness and viability, since elderly cells even showed significantly higher O2•− levels in the presence of NAC compared to the control condition (∗∗p ≤ 0.005).Elderly myoblasts and myotubes Figures  did not 2•− production Consistently, treatment with an antioxidant agent like NAC, which acts as glutathione precursor [2•− production expressed as coefficient of variation (CV) in young and elderly samples under control conditions. The CV was found higher in elderly myoblasts with respect to young myoblasts, while an opposite result was observed in myotubes. We can argue that the increased variability in O2•− production in elderly myoblasts could be due to a large dispersion in the frequency distribution of O2•− flickering or, in other words, we can imagine that the elderly mitochondria produce O2•− in a slow and continuous regimen with respect to young mitochondria. In elderly myotubes, the O2•− flickering has a narrow frequency distribution, significantly reduced with respect to the young ones, probably because those rely more on glycolysis than mitochondrial OXPHOS.In aged muscles  as well recursor , 42, migrecursor . FurtherκB transcription factor-activating IκB kinase (IKK) that phosphorylates the IκB inhibitor in turn upregulating NF-κB [2•− found in our elderly myotubes with respect to young ones (∗∗p ≤ 0.005) could result in the activation of NF-κB that in turn could boost cell catabolism. Consistent with this, we and other groups previously demonstrated that elderly samples' upregulation of FOXO1A, myostatin genes, and NF-κB protein expression, together with downregulation of AKT1/PIK3CA/MTOR genes and phospho-Akt protein expression, could lead to the activation of the atrophic/catabolic pathway at the expense of the anabolic ones and finally inhibit muscle differentiation [in vitro and to regenerate muscle in vivo [ROS not only have a detrimental effect on biological molecules, such as DNA, proteins, and lipids , due to ng NF-κB . In the ng NF-κB . Consequntiation , 9, 32.  in vivo  could beFurthermore, to deeply investigate mitochondria's functions, we also analyzed mitochondrial bioenergetics in our samples via the Seahorse Flux analyzer. This instrument monitors OXPHOS by measuring in real time OCR and glycolysis by measuring in real time ECAR. Revising literature on aged muscle bioenergetics, many studies demonstrated a significant decrease in the activity of several respiratory chain complexes and a substantial increase in ROS production, thus confirming the mitochondrial theory of aging , 45. MeaIn myoblasts, we observed a decreasing trend for many OCR parameters when elderly and young samples were compared, especially considering the nonmitochondrial respiration Figures . Notably2•− produced by young myoblasts  is associated with downregulation of OXPHOS, and aging in vivo (young versus elderly) caused an altered metabolic profile by favoring the glycolytic pathway. Our results are in agreement also with another recent study demonstrating that senescent myoblasts show a metabolic shift leading to glycolytic enzymes a pronounced downregulation in glycolysis [This hypothesis would be in agreement with the recent study conducted by Pääsuke et al.  on primaycolysis .In myotubes, we found a similar trend for the OCR parameters of elderly versus young donors. The myogenic differentiation process is usually accompanied by an increase in oxidative metabolism with a shift from glycolysis to OXPHOS, as the major energy demand occurs , 48. Thi2•− levels, neither contrasted by an adequate amount of SOD2 nor scavenged by a NAC or BURST stressor. The proliferative state shows a slower metabolism characterized by less OXPHOS and glycolysis, probably to the detriment of the aerobic glycolytic component.The aging process produces increased mitochondrial OThe metabolic burst due to the onset of differentiation leads to a pronounced increase in glycolysis which does not occur in young myotubes. This result suggests that OXPHOS in elderly myotubes may not be as effective at producing ATP as it is in young myotubes. The increased glycolytic rate observed in elderly myotubes could be therefore considered as part of a compensatory mechanism mounted by the aging cells in response to the sudden increase in energy demand and in the absence of an extra OXPHOS support.The data collected in the present study lead us to conclude that the elderly may have less metabolic plasticity due to the impaired mitochondrial function caused by oxidative stress. Further studies on the expression and activity of mitochondrial complexes in myotubes with respect to myoblasts could lay the foundations for future investigations about the detailed causes of impaired muscle regeneration during aging.