The results reveal that the VO2 nanocoating is able to interrupt the mitochondrial electron transport chain and then elevate the intracellular reactive oxygen species levels, leading to the collapse of the mitochondrial membrane potential and the destruction of cell redox homeostasis. Indeed, this chain reaction can effectively trigger oxidative damage in the cholangiocarcinoma cells. Additionally, this study has provided new insights into designing a tumor‐cell‐inhibited biomaterial surface, which is modulated by the mechanism of mitochondria‐targeting tumor cell death.A biomaterials surface enabling the induction of tumor cell death is particularly desirable for implantable biomedical devices that directly contact tumor tissues. However, this specific antitumor feature is rarely found. Consequently, an antitumor‐cell nanocoating comprised of vanadium dioxide (VO Trivalent (+3), tetravalent (+4), and pentavalent (+5) vanadium are the most common forms, and their respective oxides are V2 nanocoating, and the film thickness has been optimized by tuning the deposition time. In brief, we fabricated a VO2 nanocoating on quartz glass through the reactive sputtering of vanadium metal with O2 gas. Following the characterization of the physical and chemical properties of the VO2‐modified quartz surface, the inhibition effect of this surface was tested by culturing human cholangiocarcinoma cells. Meanwhile, the cytocompatibility of this surface was also tested by culturing rat bone marrow mesenchymal stem cells. Furthermore, the detailed biological responses and underlying mechanism of tumor cell inhibition were investigated by the coincubation of glutathione antioxidant on the VO2‐modified surface.To realize the proposed concept, a customized magnetron sputtering technique has been utilized to fabricate the VO22.1Figure2 nanocoating on the substrate through reactive magnetron sputtering of the V metal target and O2 gas. Figure [qv: 1]b–d shows the surface atomic force microscopy (AFM) images of the VO2 samples on quartz glass. With the help of the AFM tapping mode, the VO‐1 sample (30 nm thickness) was seen to have a homogeneous compact film morphology consisting of tiny nanoparticles  2θ scan profiles in Figure 2 phase were detected in the XRD pattern. All of the positions of the diffraction peaks were accordant with the standard reference data (JCPDS No. 72–0514), demonstrating the exclusive generation of single‐phase VO2 polycrystalline films. Meanwhile, the increase in thickness resulted in better crystallinity for the VO2 films, as evidenced by the rising intensity of the diffraction peaks. Additionally, the full width at half maxima (FWHM) values of the dominant orientation (011) were reduced as the VO2 film thickness increased. Based on the Scherrer formula, the average grain size of the VO2 films had a positive correlation with the reciprocal of the FWHM, which thus provided more support to explain the growing nanoparticle size.gure qv: b–d showsgure qv: b–d showsgure qv: b–d showsgure qv: b–d showsFigure2 + H2O = H2SiO3). For VO2 samples, intermediate state VO2 tends to oxidize to V2O5 (4VO2 + O2 → 2V2O5), and the total charge density of the hydrated surface is zero at IEPS (V2O5 + 3H2O = 2H3VO4). Here, a negatively charged surface was generated on VO2 thin films in a wide pH range because of the pKa values for vanadate .3/2 and V2p1/2 in VO2, respectively.3/2 (517.6 eV) and V2p1/2 (524.8 eV) in V2O5 were the major peaks detected on the surface.2 nanocoating in RPMI 1640 medium are shown in Figure 2 nanocoating in a time‐dependent and thickness‐dependent manner, showing the intended difference among groups.The surface zeta potentials of the samples are given in 2.2Figure2 nanocoating with an alamarBlue reduction rate around 5%, showing no significant difference with the VO‐0 quartz control sample. However, after four days of culturing  > VO‐1 (13.9 ± 0.8%) > VO‐2 (10.9 ± 0.9%) ≈ VO‐3 (11.4 ± 0.4%). At day seven of culture . Consequently, the cell morphology observations were very accordant with the cell proliferation trend. At high magnification, the tumor cells spread well on the VO‐0 quartz glass, showing a multipolar spindle morphology  were predominant in the tumor cells cultured on the VO2 nanocoating, indicating the poor cellular viability and apoptosis of the tumor cells. Interestingly, the lamellipodia and filopodia, which are indicated by the red arrows in Figure 2,g2 showed an initial cell adhesion, spreading, and cell–material interactions, which was in agreement with the analysis of Figure 2 nanocoating at day four of culture. Moreover, increasing number of dead cells (stained in red fluorescence) were observed, further demonstrating that exposure to the VO2 nanocoating caused a severe decrease in the viability of tumor cells, especially in the VO‐2 and VO‐3 samples.Scanning electron microscopy (SEM) was performed to analyze the tumor cell morphology development and cell–material interactions after four days of culturing on the surfaces of VOy Figure e1,f1,g1.y Figure d2. Focusy Figure e2,f2,g2 2.3Figure2 nanocoating, were significantly stronger than those on VO‐0, and the levels correlated with the film thickness in a dependent manner, indicating that the intracellular ROS level was elevated in thicker films. Figure 2 nanocoating could obviously elevate the intracellular ROS levels of tumor cells, and the order of DCF/DAPI intensity ratios was as follows: VO‐3 (1.6 ± 0.1) ≈ VO‐2 (1.7 ± 0.03) > VO‐1 (0.6 ± 0.1) > VO‐0 (0.1 ± 0.01). Consequently, the ROS level obviously exceeds the threshold that cells can tolerate to maintain redox homeostasis, causing oxidative damage.The intracellular ROS levels were investigated qualitatively and quantitatively. First, ROS fluorescence staining was performed using 2′,7′‐dichlorodihydrofluorescein diacetate (DCFH‐DA) to visualize the intracellular ROS level. As shown in 2.4m) of the tumor cells cultured on various samples were detected using 5,5′,6,6′‐tetrachloro‐1,1′,3,3′‐tetraethyl benzimidazolyl carbocyanine iodide (JC‐1) dye, both qualitatively and quantitatively. A high ΔΨm facilitates the generation of JC‐1 aggregates that fluoresce red in the mitochondrial matrix, while a low ΔΨm promotes the presence of JC‐1 monomers, which fluoresce green, in cytoplasm. Figure 2‐nanocoating exposure. For unexposed tumor cells on VO‐0, well‐polarized mitochondria were stained in bright red and light green fluorescence. By contrast, the VO2‐nanocoating exposure caused significant changes in the tumor cell staining: the red fluorescence gradually decreased while the green fluorescence obviously increased. These changes in JC‐1 fluorescence following VO2‐nanocoating exposure indicated a reduction in the JC‐1 aggregates that accumulated in well‐polarized mitochondria and the diffusion of JC‐1 monomers into the cytoplasm from depolarized mitochondria, further demonstrating the collapse of the mitochondrial membrane potentials and the increase in the mitochondrial membrane permeability. Quantitatively, as seen in Figure 2‐nanocoating exposure, the gap between the red fluorescence decrease and green fluorescence increase gradually became larger in a film thickness‐dependent manner. The relative intensities (%) of red fluorescence were in the following order: VO‐0 (52.8 ± 0.9%) > VO‐1 (39.5 ± 0.6%) > VO‐2 (29.1 ± 0.6%) > VO‐3 (16.7 ± 0.2%). Accordingly, the order of the relative intensities (%) of green fluorescence was as follows: VO‐0 (47.2 ± 0.9%) < VO‐1 (60.5 ± 0.6%) < VO‐2 (70.9 ± 0.6%) < VO‐3 (83.3 ± 0.2%). This indicated the status of the ΔΨm collapse and depolarization in the mitochondria of tumor cells upon VO2‐nanocoating exposure, which was consistent with the qualitative observations. The collapse of the ΔΨm is an indicator of mitochondria depolarization, impairment, and dysfunction and is an early mark of cell apoptosis.Figure2 nanocoating samples were stained in bright red and light green fluorescence. The relative intensities (%) of red fluorescence and green fluorescence were around 60% and 40%, respectively, showing no significant differences among groups  can directly indicate the dissolution rate of metal ions of nanoparticles, where Z and r are the charge number and ionic radius (pm), respectively, of the metal cation in the nanoparticles.2 nanoparticles, the Z2/r can be estimated at ≈0.13 pm−2, which reflects the availability of vanadium ions for dissolution in aqueous solution,One crucial parameter correlating strongly with metal oxide nanoparticle toxicity is the dissolution of metal ions.TableSchemeESHE, V) and absolute potential level ,ESHE = −4.44 −E, the mitochondria‐associated redox potentials are in the range of the biological redox potential (−4.12 to −4.84 eV).2VO4− anions are the most common intracellular form in biological systems, and they can be actively transported into mammalian cells.2VO4−/VO2+ redox couple is −0.34 V at pH = 7.2VO4−) tends to be reduced to the +4 valent VO2+ (vanadyl) by the nicotinamide adenine dinucleotide phosphate/reduced nicotinamide adenine dinucleotide phosphate (NADP+/NADPH), nicotinamide adenine dinucleotide/reduced nicotinamide adenine dinucleotide (NAD+/NADH), flavin adenine dinucleotide/reduced flavin adenine dinucleotide (FAD2+/FADH2), and flavin mononucleotide/reduced flavin mononucleotide (FMN2+/FMNH2) redox couples,2VO4− + 4H+ + e− → VO2+ + 3H2O.Here, we first attempt to understand the mechanism of the electron transfer chain on the inner mitochondrial membrane from the perspective of redox chemistry. In the mitochondria, the electron transport proceeds down the redox potentials, as listed in 2O2 in cells.2+ has the potential to induce the production of hydroxyl radicals (•OH) through a Fenton‐like reaction (VO2+ + H2O2 + H+ → VO3+ + H2O +•OH).Table2+. For example, by directly reacting with O2, VO2+ could induce the production of superoxide anion radicals (•O2−), according to the following equation: VO2+ + O2 + 3H2O → H2VO4− + 4H+ +•O2−.2 nanocoating eventually caused the elevation of intracellular ROS levels, as shown in Figure Based on the above analysis, by destructive extraction of electrons from the mitochondrial membrane, the bioreduction of vanadate tends to interrupt the electron transport chain and impair mitochondrial function. Mitochondria are the major source of ROS in most mammalian cell types, and they convert 1–2% of systemically consumed oxygen to ROS under normal conditions, accounting for the production of 60–80% of H+ + 2e−) in cellular defense against xenobiotics and endogenic toxicants that cause oxidative stress.2‐nanocoating exposure, coincubation with exogenous GSH may be able to alleviate/eliminate the oxidative stress. To verify this, a complementary study using GSH coincubation was conducted. As shown in Figure 2‐nanocoating exposure.Under normal conditions, cells can maintain redox homeostasis by self‐regulating their ROS level. For this purpose, reduced glutathione (GSH) plays a central detoxification role in cell biology by donating electrons  will cause the depolarization of mitochondria, opening of permeability transition pores, and release of cytochrome c from the mitochondria.2 nanocoating. This mitochondria‐targeting vanadate release strategy and oxidative stress‐mediated tumor cell apoptosis process are illustrated in Scheme [qv: 7]. VO2 nanocoating can release vanadate ions that purposely target mitochondria in order to disrupt electron transfer and elevate ROS production based on redox chemistry, which then causes mitochondrial depolarization and dysfunction to activate proapoptotic caspases and induce cellular apoptosis. This holds great promise for cancer cell inhibition.Therefore, VO2, Ta2O5, Nb2O5, ZrO2) have commonly been used as biocompatible coatings for medical implants.2 coatings exhibit a hydrophilic character, with a water contact angle between 20° and 70°.2 coatings are negatively charged at the physiological pH (7.4), as verified by the surface zeta potentials in Figure 2O3) than on negatively charged metal oxide surfaces ; metal oxide surfaces with negative zeta potential show little or even no protein adsorption.2O3) but higher on the surface of negatively charged metal oxides . This leads to the observation that osteoblasts stay away from Al2O3 regions, as better cell adhesion morphology and cytoskeletal arrangement are found in the other regions.2 groups and VO‐0 in initial cell adhesion within 24 h  and over longer periods of time.Protein–surface interaction is the initial process occurring at the contact of implant–biological ambiance, and the human serum proteins tend to adsorb to biomaterials. Metal oxides  also show that the VO2 nanocoating had no significant cytotoxicity on rat bone mesenchymal stem cells, especially for VO‐1 and VO‐2. However, the organic vanadium compound VO2, which generates vanadate under physiological conditions, has shown the ability to discriminate between human hepatoma cells and normal hepatocytes.Regarding the potential toxicity of vanadium, this issue was studied and found to be dosage dependent. Vanadate has a mitogenic effect on osteoblasts and their precursor cells, and a low concentration of vanadium can even promote DNA and protein (collagen and noncollagen tissue) synthesis in bone, thereby stimulating bone cell proliferation and osteogenesis in vitro.2 prior to resorption, health risks due to vanadium overload are unlikely to occur. There is currently no clinical evidence proving that vanadium compounds are toxic if administered in a sensible dosage.[[qv: 18b]] As noted by Paracelsus, “all substances are poisons; it is solely the dose that differentiates between a poison and a remedy.”[qv: 62] The current study suggests that the deposition of VO2 nanocoating on biomaterial/implant surfaces could induce the death of cancer cells. A notable example is the use of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia.Furthermore, the main source of potential vanadium toxicity is exposure to high loads of vanadium oxides in the breathing air through the respiratory or gastrointestinal systems. For example, this may occur in industrial enterprises involving vanadium processing. Most dietary vanadium can be excreted by the human body. The vanadium compounds found in the bloodstream are mainly vanadate and vanadyl bound to transferrin. Bony tissue can also store vanadate through blood circulation. Figure S2 (Supporting Information) briefly summarizes the whole process of uptake, distribution, and excretion of vanadium compounds in the human body. Since almost all of the vanadium will be excreted in the form of insoluble VO(OH)42 nanocoating was first deposited onto the substrate through customized reactive magnetron sputtering. The concept of the mitochondria‐targeting death of tumor cells induced by the controlled release of vanadium ions was proven using human cholangiocarcinoma cells. The results suggest that the released vanadium ions can disrupt the mitochondrial electron transport chain and therefore elevate the intracellular ROS level of tumor cells, thereby leading to mitochondria depolarization due to the collapse of mitochondrial membrane potential. The dysfunction of the mitochondria ultimately induces the apoptosis of the cancer cells. These findings may contribute to the exploitation of VO2 nanocoating as an antitumor surface material for implantable biomaterials/devices for cancer treatments.To improve the surface functionalization of implantable biomedical devices for human cancer treatments, a thickness‐tunable VO5Sample Fabrication: Considering float glass contains elements like Na, K, Ca, Mg, etc., which may interfere with the experimental results, fused quartz (consisting of Si and O) was chosen as deposition substrate for the VO2 thin films in this study. Homogeneous VO2 thin films of a high quality were fabricated using a reactive magnetron sputtering apparatus  with high‐purity vanadium metal target of 3 inch and O2 gas of 15 sccm at a radio frequency power of 200 W. Prior to growth process of VO2 thin films, deposition chamber was pumped down to 10−4 Pa and high pure argon gas (99.999%) was introduced at 25 sccm. During deposition process, substrates were maintained at 450 °C to improve film crystallinity and rotated along vertical axis at a speed of 10 rpm to improve film homogeneity. After the deposition of VO2 thin films, samples were cooled down spontaneously to ambient temperature. The thicknesses of VO2 thin films were determined using an F20 thin‐film analyzer (FILMSTRICS Corp.). Prepared VO2 thin films with thicknesses of 30, 80, and 120 nm were denoted as VO‐1, VO‐2, and VO‐3, respectively. Meanwhile, quartz glass was used as a control (VO‐0).Sample Characterization: AFM (Nanocute SII scanning probe microscope) was used to examine the surface morphology of the prepared VO2 thin films, operated in tapping mode under ambient conditions. An XRD (Rigaku Ultima IV), fitted with Cu Kα radiation (λ = 1.541 Å) at a voltage of 40 kV and current of 40 mA, was used to investigate the crystalline structure of the VO2 thin films. The XRD patterns were recorded in the range of 20°–80° (2θ) with a step size of 0.02° and a scanning rate of 2° min−1. For the XRD test, the glancing incidence angle was fixed at 1°. Phase identification was performed with the help of the Joint Committee on Powder Diffraction Standards (JCPDS) database. Surface chemical components and the chemical state were analyzed by XPS .Zeta Potential Measurement: A SurPASS electrokinetic analyzer  equipped with a special cell for coating/film was used to measure the surface zeta potentials (ζ) of samples VO‐0, VO‐1, VO‐2, and VO‐3 (size 20 mm × 10 mm × 1 mm). The samples were fixed on holders with double‐sided adhesive tape. An electrolyte of 0.9% NaCl aqueous solution served as measuring medium. The HCl and NaOH solutions were added to adjust the pH value. To measure streaming potential, electrolyte medium was pumped to flow along a pair of sample surfaces and the potentials resulting from ion motion in diffusion layer were recorded based on Helmholtz–Smoluchowski equationU/dP is slope of streaming potential versus pressure; η is electrolyte viscosity; ε0 is vacuum permittivity; ε is dielectric constant of electrolyte; K is conductivity. These parameters were collected automatically from SurPASS control and evaluation software and gave the ζ values.Vanadium Release Measurement: The release of the vanadium ions from the VO2 nanocoating samples immersed in Roswell Park Memorial Institute 1640  culture medium was determined by inductively coupled plasma optical emission spectrometry . The testing samples were positioned in sterile microcentrifuge tubes (15 mL) filled with 10 mL of fresh RPMI 1640 culture medium, followed by 1, 4, 7, and 14 days of sequential incubation at 37 °C in static conditions. At the end of each incubation period, all the leachates were collected for measurement and the microcentrifuge tubes were supplemented with fresh RPMI 1640 culture medium for subsequent incubation.Cancer Cell Culture: The human cholangiocarcinoma cell line was purchased from the American Type Culture Collection. The tumor cells were initially maintained in the culture medium provided by the supplier in a humidified atmosphere with 5% CO2 at 37 °C. Afterward, the cells were passaged at a ratio of 1:2–1:3 every 2–4 days according to the cellular state in RPMI 1640 culture medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin.Cell Viability Assay: The tumor cell growth and viability were measured by AlamarBlue assay (Sigma‐Aldrich).4 cells per well. Five samples were tested for each group. At the end of each culture duration of 1, 4, and 7 days, the supernatant culture medium was removed, and 500 µL of fresh culture medium with 10 v/v% of AlamarBlue reagent was added into each well. After a subsequent 4 h of incubation at 37 °C, 100 µL of culture medium was transferred into a new 96‐well plate to measure the absorbance of the reduced AlamarBlue at wavelengths of 570 and 600 nm on a microplate reader . All operating procedures followed manufacturer's instructions.Cell Morphology Observation: The tumor cell morphology and cell–material interactions were further investigated using SEM. At day four of culture, each group of samples was carefully transferred into a new 24‐well plate and gently rinsed with PBS thrice, followed by fixing with 2.5% glutaraldehyde solution overnight at 4 °C. Afterward, all of the samples were sequentially dehydrated in a series of ethanol solutions  for 10 min each. Finally, dehydration was performed in absolute ethanol twice and then completed using a series of hexamethyldisilazane ethanol solutions. Prior to SEM observation, the samples were sputter coated with platinum.Live/Dead Fluorescence Staining: The tumor cell viability was further investigated using the fluorescence‐based Live/Dead Staining Kit (Sigma‐Aldrich) following the manufacturer's instructions. In brief, the cells were seeded onto the VO‐0, VO‐1, VO‐2, and VO‐3 samples in a 24‐well plate at a density of 5 × 104 cells per well followed by a subsequent four days of culturing at 37 °C. To stain for cellular viability, calcein‐AM (green fluorescent dye) and propidium iodide  were used to stain the live and dead cells, respectively. The calcein‐AM and PI dyes were diluted to the final concentrations of 2 and 5 × 10−6m in PBS, respectively. After carefully transferring the samples into a new 24‐well plate and washing them gently in PBS thrice, 100 µL of the prepared dye solutions were added into each well of a sample followed by 15 min of incubation at 37 °C. Afterward, both the live and dead cells were visualized by confocal laser scanning microscopy .Mitochondrial Membrane Potential Detection: As an early marker of cell apoptosis, the mitochondrial membrane potentials of the tumor cells cultured on the VO‐0, VO‐1, VO‐2, and VO‐3 samples were detected by the cationic fluorescent probe JC‐1. JC‐1 aggregates in the mitochondria, indicated by red fluorescence, at a high ΔΨm (negative inside), while at low ΔΨm, the JC‐1 monomer exists in the cytoplasm and fluoresces green.m JC‐1 Assay Kit was purchased from Sigma‐Aldrich. All of the operation procedures strictly followed the manufacturer's instructions. To visualize mitochondrial damage, the cells were seeded on the VO‐0, VO‐1, VO‐2, and VO‐3 samples in a 24‐well plate at a density of 5 × 104 cells per well and then were cultured in a 5% CO2 incubator at 37 °C for four days. Prior to use, the JC‐1 staining solution (1 µg mL−1 of working solution) was freshly prepared by diluting a 200× stock solution of JC‐1 in warm (37 °C) RPMI 1640 culture medium supplemented with 10% FBS. Then the JC‐1 staining solution was gently added into each well followed by 20 min of incubation at 37 °C. The cells were gently washed twice with the JC‐1 stain buffered solution at 4 °C (ice bath) and then observed immediately using CLSM. The JC‐1 fluorochrome was excited at 488 nm, and the light emission was collected at 570–600 nm for red fluorescence (JC‐1 aggregate) and at 515–545 nm for green fluorescence (JC‐1 monomer). All image acquisition and analyses were carried out using the Leica LAS AF software. The laser beam intensity and photodetector sensitivity were kept constant in order to compare the relative fluorescence intensities between the various groups. Further quantitative analysis was performed by trypsinizing the cells stained with JC‐1 followed by reading the fluorescence intensities on a microplate reader (DTX 800 Series Multimode Detector). The results were expressed as a relative intensity (%) of green and red fluorescence.Intracellular Reactive Oxygen Species Determination: The oxidative stress levels in the tumor cells were investigated using the Intracellular ROS Assay Kit (Sigma‐Aldrich) that contains DCFH‐DA as a fluorescent probe. The DCFH‐DA is able to penetrate the cell membrane where it is then deacetylated into the nonfluorescent DCFH by intracellular esterases. The DCFH remains in cells and can be oxidized into the highly fluorescent DCF by intracellular ROS.4 cells per well followed by four days of culturing at 37 °C in a 5% CO2 incubator. Afterward, the cells were gently washed thrice with PBS. Then the DCFH‐DA was diluted to a final concentration of 10 × 10−6m of staining solution using FBS‐free RPMI 1640 culture medium. Subsequently, the above DCFH‐DA staining solution was added into each well followed by 20 min of incubation at 37 °C and then observed immediately using CLSM. The DCF fluorochrome was excited at 488 nm and light emission was collected at 535 nm. All image acquisition and analyses were carried out using the Leica LAS AF software. The laser beam intensity and photodetector sensitivity were kept constant in order to compare the relative fluorescence intensities between various groups. Further quantitative analysis was conducted by trypsinizing the cells stained with DCFH‐DA and DAPI (cell nuclei staining) followed by reading the fluorescence intensities on a microplate reader (DTX 800 Series Multimode Detectors). The intracellular ROS levels were obtained from the fluorescence reading of DCF and were normalized to the number of tumor cells.Glutathione Antioxidant Investigation: To explore the potential role of oxidative stress in the VO2‐induced cell apoptosis, the tumor cells were coincubated with 10 × 10−3m of GSH, the major endogenous antioxidant, to attenuate/eliminate the contribution of oxidative stress in the cellular response to VO2‐nanocoating exposure. This strategy has previously demonstrated the effective alleviation of the oxidative stress induced by cellular pro‐oxidants.−3m stock solution, sterile filtered, and then diluted in RPMI 1640 culture medium to prepare a 10 × 10−3m working solution. Afterward, the cells were immediately seeded onto the VO‐0, VO‐1, VO‐2, and VO‐3 samples in a 24‐well plate using the freshly prepared cell culture medium containing 10 × 10−3m GSH followed by four days of culturing at 37 °C in a 5% CO2 incubator. After that, the following assays were performed according to the manufacturer's instructions. The tumor cell viability after four days of coincubation with 10 × 10−3m of GSH antioxidant on various VO2 samples was further investigated using the fluorescence‐based Live/Dead Cell Staining Kit. The mitochondrial membrane potentials of the cultured tumor cells after four days of coincubation with 10 × 10−3m of GSH antioxidant on various VO2 samples were further investigated using the fluorescence‐based ΔΨm JC‐1 Assay Kit. The intracellular ROS levels in the cells after four days of coincubation with 10 × 10−3m of GSH antioxidant on various VO2 samples were further investigated using the fluorescence‐based DCFH‐DA Assay Kit.Statistical Analysis: Statistically significant differences  between various groups were measured using one‐way analysis of variance and Tukey's multiple comparison tests using a GraphPad Prism 5 statistical software package. All of the data were expressed as a mean ± standard deviation. A value of P < 0.05 was considered to be statistically significant and denoted as “*”, P < 0.01 was “**”, and P < 0.001 was “***”.The authors declare no conflict of interest.SupplementaryClick here for additional data file.


Gata4 using Cre‐recombinase under the control of the Col1a1 2.3 kb promoter showed significantly reduced values for trabecular bone properties by microCT analysis of femur and tibia of 14‐week‐old male and female mice, suggesting GATA4 is necessary for maintaining normal adult bone phenotype. Quantitative PCR analysis revealed higher expression of Gata4 in trabecular bone compared with cortical bone, suggesting a role for GATA4 in maintaining normal trabecular bone mass. In vivo and in vitro, reduction of Gata4 correlates with reduced Runx2 gene expression, along with reduced osteoblast mineralization. To determine if Runx2 is a direct target of GATA4, chromatin immunoprecipitation (ChIP) was performed, and it demonstrated that GATA4 is recruited to the two Runx2 promoters and an enhancer region. Furthermore, when Gata4 is knocked down, the chromatin at the Runx2 region is not open, as detected by DNase assays and ChIP with antibodies to the open chromatin marks H3K4me2 (histone 3 lysine 4 dimethylation) and H3K27ac (histone 3 lysine 27 acetylation) and the closed chromatin mark H3K27me2 (histone 3 lysine 27 trimethylation). Together, the data suggest that GATA4 binds near the Runx2 promoter and enhancer and helps maintain open chromatin to regulate Runx2 expression leading to bone mineralization. © 2017 The Authors. JBMR Plus is published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.GATA4 is a zinc‐finger transcription factor that is a pioneer factor in various tissues and regulates tissue‐specific gene regulation. In vivo deletion of  Runx2 is expressed in undifferentiated mesenchymal cells, hypertrophic chondrocytes, and early in osteoblast differentiation, where it turns on other differentiation and mineralization genes, including alkaline phosphatase and osteocalcin (Bglap).Runx2 promoter is controlled epigenetically, as increased levels of histone 3 lysine 4 methylation (H3K4me) and histone 3 lysine 27 acetylation (H3K27ac) correlate with osteoblast‐specific expression.Runx2 promoter epigenetics and subsequent mRNA expression early in osteoblastogenesis are not well understood.Osteoblast differentiation is controlled by directly inducing the expression of, or regulating the activities of, the “master” osteoblast transcription factor RUNX2. Gata4 knockout mice die early in embryogenesis due to the heart and intestinal defects.Gata4 knockout mice (cKO) with Cre expression driven by the 2.3‐kb promoter of Col1a1 showed reduced trabecular bone mass and exhibited both appendicular and cranial defects in newborn mice. GATA4 has also been shown to act as a regulator of osteoblasts by controlling estrogen receptor recruitment to the chromatin in a tissue‐specific manner.GATA4 is a zinc‐finger transcription factor that binds to the consensus region “GATA.” It is important for the development of the heart and intestines early in embryogenesis, as it mediates tissue specificity. Runx2 in vitro and in vivo. We present evidence that GATA4 maintains open chromatin at both the Runx2 promoters and an enhancer to upregulate Runx2, the master regulator of osteoblast differentiation, and to control proper adult bone formation in vivo. We suggest that GATA4 is critical for the epigenetic mechanisms of tissue‐specific differentiation of osteoblasts.Given the early role of GATA4 in regulating bone mass in newborn cKO mice and the co‐localization of GATA4 and RUNX2 in trabecular bone and in calvaria,ad libitum. GATA4fl/fl mice were purchased from The Jackson Laboratory , where they were generated by homologous recombination in which the region containing the GATA4 coding sequence upstream of exon 3 and downstream of exon 5 was replaced with a loxP‐flanked thymidine kinase/neomycin phosphotransferase expression (tk/neo).fl/fl mice was backcrossed for 10 generations to FVB background.(5) Mice that were heterozygous for the deletion were crossed to collagen A1(Col1A1) (2.3 kb)‐Cre mice (FVB‐Tg(Col1a1‐cre)1Kry/Mmucd)  to produce homozygous GATA4 cKO and wild‐type littermate control mice. Fourteen‐week‐old adult (12 wild type [WT] and 12 knockouts [cKO]) were euthanized and the right hindlimbs cleaned and stored in phosphate‐buffered saline (PBS) at −20°C for micro‐computed tomography (μCT) until measurements were made. The left femur was transferred to formalin and the left tibia was transferred to ethanol and stored at 4°C.Animal experiments were approved by the Institutional Animal Care and Use Committee at the University of Tennessee Health Science Center. Animals were maintained in a specific pathogen‐free environment at 20°C to 26°C with a relative humidity of 30% to 70% and a 12‐hour light/dark cycle. Commercial rodent chow  and drinking water were available 5 vertebrae were isolated using the μCT X‐ray scout view and scanned with an 8‐µm voxel size. A section of the vertebral body measuring 0.5 mm immediately cranial to the caudal end plate was selected for analysis.Tibias, femora, and vertebrae from 14‐week‐old mice were stored in PBS and then scanned using a Scanco μCT 40  set at 55 kVp/109 µA. The entire femur and tibia were scanned in the sample holder with 12.3‐mm diameter at medium resolution. These tubes were filled with PBS and the top of the tube was covered with Parafilm  to prevent dehydration. A scout view of each bone was taken and the sample height was adjusted to ensure the bone was within the field of view. The tibias and femora images were obtained at 6‐μm resolution. The integration time and Gaussian filter used for these samples was 300 ms and 1, respectively. Solid three‐dimensional models were reconstructed from these images automatically after completion of each cone‐beam image stack with the built‐in software. The trabecular parameters were calculated on 200 slices of trabecular bone from a region just below the growth plate as described in Khalid and colleaguesBone histology was performed at the Histomorphometry and Molecular Analyses Core (HMAC) at the University of Alabama at Birmingham School of Medicine.Formalin‐fixed paraffin‐embedded samples were processed through standard deparaffinization protocols. Antigen retrieval was performed in 90°C citrate buffer until the temperature reached 55°C. The tissue was then incubated in blocking buffer  for 30 minutes. Anti‐RUNX2  antibodies were incubated overnight at 4°C in a humidified chamber followed by the DAKO Envision Visualization system and counterstaining with hematoxylin.5 cells/well and allowed to adhere overnight. To determine the effect of knocking out GATA4 on mineralization, the silenced shC and shGATA4 mouse calvarial cells (lentivirus silencing as described below) were differentiated in mineralization medium supplemented with 50 μg/mL L‐ascorbic acid 2‐phosphate and 5 mM β glycerophosphate. Cells were maintained under these conditions for 14 days with media replaced every 3 days. Experiments were performed in α‐MEM media with 10% fetal bovine serum . Differentiation was confirmed by gene expression and fixing the cells and staining with Alizarin Red.Mouse calvarial osteoblasts were isolated from 2‐day‐old CD1 mice by sequential collagenase digestion, as previously described.6 cells/well and left undisturbed for 5 days in MesenCult MSC Basal Medium . To determine the effect of knocking out Gata4 on mineralization, the bone marrow cells were treated for 14 days with α‐MEM media supplemented with 50 μg/mL L‐ascorbic acid 2‐phosphate and 5 mM β glycerophosphate with media changed every 3 days. After 14 days, the difference between WT and cKO mineralization was confirmed by fixing the cells and staining with Alizarin Red.Mouse bone marrow cells were isolated from long bones of 3‐month‐old WT and GATA4 KO mice. Each hindlimb was cleaned off by scraping the diaphysis and pulling the tissue toward the end of the bones. The ends of the femur and tibia just below the end of marrow cavity were chopped with sharp rongeur. A 27‐gauge needle attached to a 10‐mL syringe containing basal media was then used to flush out the bone marrow. The bone marrow cells were then seeded in a 6‐well plate at a seeding density of 2 × 10Wild‐type femurs were prepared by removing muscle and flushing the bone marrow. The epiphysis was cut from the diaphysis for the trabecular and cortical bone, respectively.2.U2OS cells were purchased from American Type Culture Collection  and validated yearly by Genetica DNA Laboratories. The cells were grown in DMEM‐F12 with 10% fetal bovine serum. The cells were maintained at 37°C with 5% COGata4 (TRCN0000095215: CCGGCCCAATCTCGATATGTTTGATCTCGAGATCAAACATATCGAGATTGGGTTTTTG and TRCN0000095217: CCGGCATCTCCTGTCACTCAGACATCTCGAGATGTCTGAGTGACAGGAGATGTTTTTG). The calvarial cells were plated in six‐well plates at a seeding density of 2 × 105 cells per well. After 24 hours, the cells were infected with lentivirus in α‐MEM with 8 µg/mL polybrene per well. The plates were centrifuged at 1400g at 30°C for 45 minutes and left undisturbed for 24 hours after which the cells were washed with PBS and mineralization media was added. The mineralization assay was continued for 14 days with media replacement every 3 days. The knockdown of Gata4 was confirmed by qPCR.Lentivirus shC  and shGATA4 were purchased from Sigma‐Aldrich . Two different shRNA from The RNAi Consortium (TRC) in pLKO vector were used to knock down mouse Knockdown of GATA4 in U2OS cells was performed as described.Total RNA was isolated from mouse cells differentiated in osteogenic media for 14 days using TRIzol . cDNA was prepared using Superscript III First Strand Synthesis Kit according to the manufacturer's guideline and then quantified using TaqMan Universal Master Mix II  or SYBR Green . The qPCR cycling conditions for TaqMan were initial denaturation at 50°C for 2 minutes; 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. The SYBR Green conditions used were 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. The oligonucleotide‐specific primers and probes used are listed in Supplemental Table S2. For analysis of the data, the values were normalized to β‐actin values.Immunoblotting was performed on the osteoblasts from shC and shGATA4 samples. The cells were lysed on ice in EBC lysis buffer  supplemented with a protease inhibitor cocktail . Protein concentrations were quantified using the Bradford method. Proteins samples were boiled for 10 minutes at 95°C in sodium dodecyl sulfate (SDS) loading buffer and separated using SDS‐PAGE and then transferred on a polyvinylidene difluoride membrane  via transfer apparatus according to the manufacturer's protocols . The membrane was then probed with Runx2 (1:100) (Santa Cruz Biotechnology) and β‐actin (1:5000)  after incubating with 5% milk in TBST overnight at 4°C. The proteins were detected by using 1:10,000 dilution of horseradish peroxidase‐conjugated secondary antibodies (anti‐rabbit or anti‐mouse) along with peroxidase substrate for chemiluminescent detection (ThermoFisher Scientific). Membranes were exposed to X‐ray film and developed with ECL system (ThermoFisher Scientific). Each immunoblot was performed in triplicate and the protein levels were normalized to β‐actin for individual samples.Osteoblasts were differentiated for 14 days and then fixed in 50% ethanol for 15 minutes at 4°C, followed by a 30‐minute incubation with 1% Alizarin Red S (wt/vol with 0.1% ammonium hydroxide). The stain was then washed with distilled water, dried, and photographed. The amount of mineral content was measured by eluting the Alizarin Red stain with 10% cetylpyridinium chloride and the optical density was measured at OD 570 nm.5 cells and left undisturbed for 24 hours before silencing using lentivirus directed toward shC or shGATA4 as mentioned above. After 24 hours of silencing, the cells were washed with PBS and complete α‐MEM media was added to each well and left for and additional 3 days after which ChIP was performed using truChIP Ultra Low Cell Chromatin Shearing Kit  . qPCR using SYBR Green Mastermix  was used to amplify the immunoprecipitated DNA.For chromatin immunoprecipitation, the calvarial cells were plated at a seeding density of 2 × 10‐biotin mice (Gt(ROSA)26Sortm1(birA)Mejr Gata4tm3.1Wtp/J)‐biotin pups were isolated as described above for wild‐type calvaria and grown under appropriate cell culture conditions in α‐MEM media with 10% fetal bovine serum in 15‐cm dishes. Once confluent, the cells were fixed with 37% formaldehyde for 10 minutes, and the excess formaldehyde was quenched with glycine for an additional 5 minutes as per the Magna ChIP A/G chromatin immunoprecipitation kit protocol . Wild‐type CD1 mice were used as a control under identical conditions. The chromatin was lysed according to Millipore protocol and sonicated using a Biorupter sonicator  and incubated for 1 hour at 4 °C with protein A beads to preclear the sheared chromatin followed by overnight incubation at 4 °C with streptavidin beads (ThermoFisher Scientific). After the incubation, the samples were sequentially washed with cold SDS wash buffer (2% SDS), high‐salt buffer  sodium deoxycholate, 1% (v/v) Triton X‐100), LiCl buffer  Nonidet P‐40 (NP‐40), 0.5% (w/v) sodium deoxycholate), TE buffer , and Low Stringency IP Wash Buffer (EMD Millipore).GATA4‐FlagChromatin with an equivalent of 2 μg of DNA was treated with variable amounts of DNase I  in 200 μL total in 1× RSB  for 3 minutes at 37°C. DNA was then purified using DNeasy Tissue kit  and qPCR was performed with primers in Supplemental Table S2.U2OS osteosarcoma cells were plated in 24‐well tissue culture dishes and transfected with the rat 0.6 kb Runx2‐promoter‐luciferase (a kind gift from Dr Gary S Stein),The Runx2‐promoter luciferase was mutated by PCR mutagenesis,Each transfection was performed in triplicate and the entire assay was repeated at least 3 times. Twenty‐four hours after transfection, cells were assayed according to the manufacturer's protocol . To obtain RLU (relative luciferase units), all values were normalized as follows: The raw firefly luciferase value was divided by the internal Renilla Luciferase value (pRL‐SV40) and then divided by the vector control.Gata4 and Runx2 mRNA were detected in situ by RNAscope technology  in paraffin‐embedded wild‐type and cKO femurs according to the manufacturer's recommendations. Probes were designed by Advanced Cell Diagnostics.t test and a p value less than 0.05 was considered statistically significant. All experiments were performed with both biological and technical triplicates, except for mouse studies where n = 12.The data shown are the mean ± standard deviation. Statistical analyses were made using Sigma Plot . Data were analyzed using a two‐tailed Student's fl/flGata4/Cre+) mice were not born at the expected Mendelian ratio, and μCT analysis of the newborn femur and vertebra showed reduced trabecular bone properties and skeletal defects.fl/flGata4 female mice with Gata4fl/+/Cre+ male mice). A statistically significant cohort of 14‐week‐old Gata4 cKO mice was obtained to analyze the adult bone phenotype. To ensure that the surviving mice are still knockouts, RNA in situ using RNAscope technology was used to monitor Gata4 gene expression in the trabecular bone of femurs from wild‐type and cKO mice. Indeed, there is less Gata4 expression in the osteoblasts (Runx2‐positive cells) of cKO mice, as would be expected ; however, the relative expression of GATA4 in different tissues and cells may vary.Previously, we found that newborn cKO (A–F). No significant changes in cortical bone thickness nor porosity were detected between Gata4 cKO adult mice and their littermate controls . The expression of Gata4 mRNA was compared in trabecular and cortical bone, and Gata4 is sixfold higher in trabecular bone , suggesting that GATA4 has a specific role in trabecular bone. Similar μCT results were obtained in male mice  and in the femurs of both male and female mice . The vertebrae of cKO also showed reduced BV/TV, decreased trabecular number, and increased trabecular separation .The percentage bone volume (bone volume [BV]/tissue volume [TV]) was significantly reduced in tibia of adult female cKO mice compared with WT littermates, together with a significant decrease in the trabecular number and a significant increase in trabecular separation, whereas no change was found in trabecular thickness Fig. A–F. No Gata4 cKO were also confirmed by histological analyses of femurs from 14‐week‐old mice. Hematoxylin and eosin (H&E) staining of bone sections from Gata4 cKO mice showed a reduced percentage bone volume and number of trabeculae, whereas the trabecular spacing was increased compared with wild‐type mice . H&E staining also showed abnormalities in the cKO chondrocytes; the cKO proliferative zone was increased  compared with the WT bone. In addition, there are more binucleate chondrocytes in the cKO bones.Trabecular changes in the (16) Significantly less RUNX2 staining was observed in the cKO growth plate and trabecular bone . Furthermore, there is less Runx2 mRNA in the femurs of cKO mice . These results suggest that Runx2 is a target of GATA4 gene regulation at the growth plate, affecting both normal osteoblast and chondrocyte differentiations.Because the observed differences in the chondrocytes and trabecular bone at the growth plate mimic the expression pattern of RUNX2,6 SignifiGata4 (or control) knockdown with lentiviral shRNA, were differentiated for 14 days, and the cDNA expression level of Gata4 and Alizarin Red mineralization were quantified. Gata4 mRNA was reduced twofold in shGata4 samples, confirming that Gata4 was successfully knocked down in shGata4 samples compared with control silencing (shC) . The primary calvarial cells transduced with shC showed efficient mineralization as visualized by Alizarin Red staining, but when Gata4 was silenced, there was a significant reduction in Alizarin Red staining . The mineral content, measured by eluting Alizarin Red, was significantly reduced in shGata4 silenced cells . To further confirm that GATA4 affects mineralization, bone marrow stromal cells were isolated from WT and cKO femur and tibia and differentiated ex vivo for 14 days, and the Gata4 expression level and mineralization were quantified. The Gata4 expression was significantly reduced in cKO mice compared with WT mice , along with reduced Alizarin Red staining and a significant reduction in mineral content . This demonstrates that GATA4 affects mineralization both in primary calvarial osteoblasts, as well as in bone marrow‐derived osteoblasts from adult mice.To confirm that GATA4 plays a crucial role in osteoblast differentiation and/or mineralization, primary calvarial osteoblasts, following hC) Fig. A. The ping Fig. B. The mlls Fig. C. To fuice Fig. D, alongent Fig. E, F. ThGata4 and Runx2 are co‐expressed early in osteoblastogenesis.Runx2 in vitro, primary calvarial osteoblasts were silenced for Gata4, as described above. In a parallel experiment, bone marrow stromal cells from Gata4 cKO and WT mice were differentiated to osteoblasts . Knockdown of Gata4, as well as Gata4 cKO mice, showed significant reductions in Runx2 mRNA expression. We confirmed that absence of GATA4 has a functional consequence as knockdown of Gata4 in the primary calvarial osteoblasts exhibits reduced RUNX2 protein levels . Together, these data demonstrate a misregulation of Runx2 in Gata4‐ablated cells.We had previously demonstrated that sts Fig. A, B. Knels Fig. C. TogetRunx2 is regulated by two different promoter regions: one specific for osteoblasts (P1) and the other promoter region specifically for both osteoblast and/or other mesenchymal tissues (P2).Runx2 by binding to the Runx2 promoter, a luciferase assay was performed, using the osteoblast‐specific P1 promoter.Runx2‐promoter luciferase reporter plasmid, along with pcDNA3‐GATA4 or control pcDNA3 plasmid. GATA4 increased the luciferase expression by more than threefold. When the canonical GATA4 binding motif “GATA” was mutated to “GCCA,” GATA4 failed to induce luciferase activity . These results suggest that Runx2 transcriptional activity is activated by GATA4 both in vivo and in vitro.Runx2 gene expression in normal osteoblast differentiation. To determine the precise binding loci in cells requires chromatin immunoprecipitation (ChIP) by GATA4 antibodies; unfortunately, commercially available antibodies do not perform well in ChIP. Therefore, a mouse with GATA4 tagged with the Flag epitope and a biotinylation site at the endogenous locus was created and has been successful for GATA4 ChIP.A). We had identified an enhancer region for Runx2 associated with open chromatin, marked by histone 3 lysine 4 dimethylation (H3K4me2) in human osteoblasts that we translated to the mouse genome . The isolated DNA was assayed for regions near the Runx2 gene and to a negative control genomic region . GATA4 was found to be occupied in all three regions of the Runx2 loci, and the highest occupancy was found at promoter 1, which is specific for osteoblast differentiation, and less occupancy at promoter 2 and the enhancer region that is necessary for both osteoblasts and/or other mesenchymal tissues.Based on the above, we hypothesized that GATA4 directly regulates RUNX2 by directly binding to chromatin at promoter regions that are essential for regulating Runx2 expression to be “open” and/or epigenetically marked for transcription.Runx2 proximal promoters and/or the enhancer region, we analyzed the histone marks at those sites in primary calvarial osteoblasts by ChIP in the presence and/or absence of GATA4 . The two promoter sites and one enhancer site are positive for the open marks H3K4me2  and H3K27ac , and negative for the closed mark H3K27me3  in normal calvarial osteoblasts. More importantly, when Gata4 is silenced, both the H3K4me2 and H3K27ac open marks are significantly reduced; concurrently, the H3K27me3 mark is increased over baseline at promoter 1 and the enhancer region but not on the promoter 2 site.Mechanistically it is likely that GATA4 helps maintain the chromatin regions regulating Runx2 promoters and enhancer region, as in primary calvarial osteoblasts, and silencing of Gata4 reduces Runx2 expression . Silencing of Gata4 in U2OS cells also reduces the H3K4me2 and H3K27ac at the Runx2 loci .U2OS osteosarcoma cells are a good model for osteoblasts,cis regions have “open” or “closed” chromatin, DNase assays were performed in primary calvarial osteoblasts . At day 14 of differentiation, the cells are mineralizing the matrix, as evidenced by Alizarin Red staining . In undifferentiated cells, H3K4me2 at the osteoblast‐specific promoter 1 is 13‐fold higher than at day 14 . Gata4 knockdown no longer has a significant effect on H3K4me2 at day 14 at any of the three Runx2 regulatory regions. Similarly, H3K27ac at promoter 1 is decreased by Gata4 silencing at day 0 but not at day 14 . H3K27me3 is increased by Gata4 silencing at promoter 1 and the enhancer at day 0 and at promoter 1 and the enhancer at day 14 . Thus, GATA4 functions to directly control Runx2 expression in a temporal and spatial manner.sis Fig. A, B. Atcer Fig. C. In un 14 Fig. D. Gata4 14 Fig. E. H3K27 14 Fig. F. Thus,Gata4 is expressed in mesenchymal stem cells and in osteoblastogenesis.Runx2 expression, by opening the chromatin region and having a direct effect on bone properties in vivo.Gata4 knockout in mice is embryonic lethal.Gata4 knockout in mice (cKO) with Cre expression driven by the 2.3‐kb promoter of Col1a1 is partially embryonic lethal,Gata4 cKO mice did not survive to adulthood, possibly because of cranial or spinal defects. The neonatal and the adult Gata4 cKO mice had lower BV/TV, decreased trabecular number, and increased trabecular spacing. We further describe that GATA4 regulates Runx2 expression via binding directly to three loci near Runx2.Runx2 expression in cKO mice with the Col1A1 2.3‐kb promoter‐Cre expression, causing a conundrum, as it is believed that Col1A1 expression begins at E14.5 and Runx2 expression peaks at E12.5. However, Runx2 is expressed throughout osteoblastogenesis, as is Gata4. It is possible that GATA4 is necessary to maintain open chromatin near Runx2, instead of initializing chromatin opening. The Col1A1 2.3‐kb‐cre;ROSA26 (R26R LacZ) mouse demonstrates a high level of bone‐specific expression of Cre recombinase;Our results determined that GATA4 regulates Gata4 expression, allowed, for the first time, a precise mapping of the chromatin epigenetic remodeling regulating Runx2 mRNA expression. These data provide very strong evidence, mechanistically, how and when Runx2 can either influence both osteoblast differentiation alone and/or concurrently regulate other mesenchymal tissue  in vivo. At very specific osteoblast differentiation timing, in adult mice, we identified differential occupancy of Gata4 on both the promoters and an enhancer. In support of this notion, we provide in vivo analysis of RUNX2 protein alteration in the trabecular bone at the growth plate and/or near the hypertrophic chondrocytes, which happens only during endochondral ossification. The direct role of GATA4 and/or RUNX2 on chondrocyte differentiation will require further in vivo and in vitro experiments. Here, we propose that GATA4 helps drive osteoblast‐specific gene expression by regulating open chromatin. Whether H3K27ac is dependent on p300 at the RUNX2 loci will also need to be experimentally confirmed in future work.GATA4 has been shown to direct tissue‐specific gene expression, particularly in the heart and liver.Gnai3 and bone sialoprotein.GATA4's regulation of osteoblast differentiation is multifactorial. With estrogen receptor alpha, GATA4 regulates FasL, FoxC1, and alkaline phosphatase.Runx2 expression.Runx2 promoter to drive Runx2 expression. We have previously demonstrated that GATA4 regulates BMP and TGFβ signaling pathway components in osteoblasts.Runx2 expression directly at the Runx2 gene and indirectly via regulating the BMP and TGFβ pathways.RUNX2 activity is regulated by transcription, protein‐protein interactions,All authors state that they have no conflicts of interest.Supporting Data S1.Click here for additional data file.


The hydrogels formed by different mixtures of the two peptides are made up mainly of antiparallel β‐sheet nanofibers entangling in an intricate network. The effect of the addition of SA21 on the morphology of the hydrogels was investigated by atomic force microscopy and transmission electron microscopy and correlated to the mechanical properties of the hydrogel. Finally, the biocompatibility of the hydrogels using 2D cell cultures was tested. © 2018 The Authors. journal Of Biomedical Materials Research Part A Published By Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 535–544, 2019.Self‐assembling peptide hydrogels (SAPHs) represent emerging cell cultures systems in several biomedical applications. The advantages of SAPHs are mainly ascribed to the absence of toxic chemical cross‐linkers, the presence of ECM‐like fibrillar structures and the possibility to produce hydrogels with a large range of different mechanical properties. We will present a two‐component peptide system with tuneable mechanical properties, consisting of a small pentapeptide (SFFSF‐NH The proposed way to control mechanical properties of the hydrogels is by introducing physical crosslinks in the nanofibers structure by an elastin‐inspired three‐block peptide that contains at both ends sequences identical to the self‐assembling peptide.We propose a two‐component peptide system designed to produce a wide range of stiffness values for the hydrogel by the appropriate combination of two designed self‐assembling peptides. The peptides are SFFSF‐NH22.1N‐[(1H‐benzotriazol‐1‐yloxy)(dimethylamino) methylene]‐N‐methylmethaniminium hexafluorophosphate (HBTU) was acquired from Matrix Innovation . Reagent used for cleavage from resin was an aqueous solution of 95% 2,2,2‐trifluoroacetic acid, . The peptides were purified by semi‐preparative reversed‐phase HPLC, using binary gradient H2O (0.1% TFA) and CH3CN (0.1 %TFA) as solvents. The purity of the peptides was assessed by 1H‐NMR spectroscopy and MS (ESI) mass spectrometry .The peptides, SA5N and SA21 were synthesized by solid phase peptide synthesis (SPPS) on a Tribute automatic peptide synthesizer (Protein Technologies Inc.) with a standard 9‐fluorenylmethoxycarbonyl (Fmoc) protection peptide synthesis protocol. Fmoc‐α‐amino acids were purchased from Inbios , and coupling reagent 2.2Hydrogels at a final concentration of 0.7%, 1% and 2% (w/v) were prepared by dissolving the weighed SA5N peptide powder in 10 μL of dimethyl sulfoxide (DMSO) and 200 μL of milliQ water. The solution was mildly agitated (5 min) at 100°C and then left to gel for 1 h. Hydrogels composed of a blend of SA5N and SA21 were prepared by adding SA21 dissolved in pure DMSO to the weighed amount of SA5N powder, followed by MilliQ water to a final concentration of 5% of DMSO. The mixture was mildly agitated (5 min) at 100°C and then left to gel for 1 h.2.3−1. All spectra are absorbance spectra obtained after background subtraction. Smoothing of spectra was carried out with a step of 11 or 13 data points, using the Savitzky–Golay function. Second derivatives of the spectra were obtained using a step of 13 datapoints to identify discrete absorption bands that make up the complex amide profiles. Quantitative analysis of the individual component bands of amide I was achieved by using the peak fitting module implemented in the Origins® Software .About 1–2% of lyophilized hydrogel was mixed with potassium bromide (KBr), ground to a fine powder and pressed to form a pellet. IR spectra were recorded on a Jasco FTIR‐460 spectrometer. Each spectrum is the result of signal‐averaging of 256 scans at a resolution of 2 cm2.4γ = 0.04–10%) were performed at constant angular frequency (ω = 1 rad s−1) to identify the linear viscoelastic region (LVR). Subsequently, frequency sweeps (ω = 0.06–94.2 rad s−1) at constant strain amplitude (γ = 0.2%) were performed to determine the elastic (G′) and viscous (G″) moduli within the LVR. All measurements were performed at 25°C, and all measurements were repeated at least three times to ensure reproducibility.Rheological studies were performed on TA instrument discovery HR‐2 rheometer equipped with a Peltier device to control the temperature. Parallel plate geometry with 20 mm diameter with a 0.250 mm gap was used. To minimize the evaporation a solvent trap was applied. Initially, strain amplitude sweeps  at room temperature. Data acquisition was carried out in intermittent contact mode at scan rates between 0.2 Hz and 1.5 Hz, using rectangular Si cantilevers  having a radius of curvature less than 10 nm and with the nominal resonance frequency and force constant of 300 kHz and 40 N m2.6Samples were prepared after 24 h of gelling by diluting the hydrogel tenfold. The solutions were vortexed until they were fully homogeneous. A total of 10 μL of sample was deposited on a carbon coated copper grid  for 1 min. The loaded grids were negatively stained with freshly prepared and filtered 2% (w/v) uranyl acetate for 2 min and then washed with double‐distilled water twice, blotting at each stage using Whatman filter paper. After air‐drying, grids were observed by transmission electron microscopy (Fei Tecnai G2 20 Twin) operating at 100 kV. Fiber width and morphology analysis was performed by ImageJ.2.72. All cell culture reagents were purchased from Invitrogen, U.K. Cell morphology was observed with a Leica light microscope.Mouse NIH 3T3 fibroblasts were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% foetal bovine serum (FBS) and 1% penicillin and streptomycin. Cells were maintained in a humidified incubator at 37°C with 5% CO2.8−1). Once cooled (5 min) the media (DMEM) was added on above (0.5 mL) and below (1.5 mL). Further media changes (3×) within 1 h were carried out at 37°C to obtain pH 7. Subsequently, 500 μL of cell suspension (3 × 104 cells) was transferred to the surface of each gel. The hydrogels with cells were incubated at 37°C and confocal microscope images were collected at 5 h, 24 h, and 48 h.While the gels were cooling they were pipetted into 12‐well cell culture inserts (ThinCert™ Greiner Bio‐One) (0.3 mL gel insert2.9−6M ethidium homodimer‐1 (EthD‐1) and 2 × 10−6M calcein AM was prepared. The live/dead assay solution was pipetted on top of samples, which were incubated for 30 min at 37°C. Thereafter, the staining solution was removed. Imaging of samples was obtained by Leica TCS SP5 confocal microscope at 2 h, 24 h, and 48 h.Cell viability was qualitatively tested using a live/dead assay (Invitrogen) following instruction of the manufacturer. PBS containing 6.4 × 102.10Quantitative cell proliferation was evaluated using Alamar blue (AB) assay, which measures the metabolic activity of cells through a redox reaction. The fluorescence derived from the reduction of the oxidized Alamar Blue is an indicator of aerobic respiration.Alamar blue solution (10%) was added on top of samples 3 h before the timepoint, incubated for 3 h at 37°C and then removed into a 96 well plate for analysis at the following timepoints; . Samples were analyzed at 545 nm/590 nm (excitation/emission) using the BMG LABTECH plate reader. The number of viable cells is proportional to the level of fluorescence. Duplicates were used.33.12 peptide (SA5N) positively net charged at physiological conditions, avoiding precipitation. The introduction of the amide group shifts the isoelectric point from pH 6 to pH 14, which promotes the gelation at pH 7.The aim of the present work is to design a two‐component peptide system that forms hydrogels with tuneable mechanical properties. The system is composed of a basic self‐assembling peptide (gelator) and a bridging peptide  that combined in different ratios can modulate the stiffness of the hydrogels by introducing physical cross‐links between nanofibers Fig. . The gel3.2The SA5N peptide‐based hydrogel formation at different concentrations was visually monitored by inversion test. It is considered successful if the hydrogels are self‐supporting and do not flow upon inversion of the vial, as shown in Figure 3.3−1, assigned to β‐sheet structures. The concomitant presence of a high wavenumber band at 1695 and 1696 cm−1 suggests that the β‐sheet structure could be antiparallel. In all the samples, a low wavelength band at 1617 cm−1 is also present, assigned to a strongly H‐bonded amide group, usually observed in intermolecular aggregates and amyloid cross‐β structures.−1 range assigned to unordered and loop structures. The results are compatible with the presence of a central elastin sequence that previous studies on elastin peptides have shown to adopt type II β‐turn together with unordered conformations.−1 and 1522 cm−1, assigned to β‐structures. The spectrum of H5 hydrogel shows an additional band at 1542 cm−1, assigned to unordered conformations, which might be due to the higher amount of SA21 peptide present in the hydrogel.Insights into the molecular structure of the peptides forming the hydrogel network were gained by Fourier transform infrared (FT‐IR) spectroscopy. This technique is useful for determining the secondary structure of the peptide chain. In particular, Amide I and Amide II bands are very sensitive to the conformational state of the peptides. The decomposed amide I and II FTIR spectra of H2, H4, and H5 hydrogels are shown in Figure 3.4In order to investigate the viscoelastic properties of the SA5N peptide based hydrogels, the elastic (G′) and loss (G″) moduli were measured by shear oscillatory rheology. Hydrogels H1‐H3, formed by SA5N peptide at different concentrations were analyzed. The data as a function of radial frequency are shown in Figure The rheological behavior of the SA5N hydrogels is modified by the addition of three‐block SA21 peptide  was used to gain deeper insight into the nanoscale morphology of the hydrogels assemblies. Figure 3.7The suitability of SA5N/SA21 hydrogels to support the culture of mammalian cells was investigated through the evaluation of murine fibroblasts (3T3 cells) viability when cultured on SA5N/SA21 gels. These cells are able to proliferate on scaffolds with a wide range of stiffness values, although they prefer more stiff matrices, where they undergo less apoptosis and increased proliferation.To confirm the suitability of SA5N/SA21 gels to support cellular growth, the metabolic activity of fibroblasts on hydrogels was evaluated using Alamar Blue assay (AB). Cells cultured on cell culture plates (TCP) were used as a reference Fig. . Cells cAfter 3 h incubation metabolic activity was observed to be similar in all samples, which shows that there were no immediate cytotoxic effects of the gels on the cells. Metabolic activity measured at 24 h post‐seeding was reduced compared to 3 h incubation and the TCP control for all samples. This observation is commonly seen when cells are seeded on hydrogels.4de novo designed and are based on a ionic complementary design with alternating hydrophilic/hydrophobic sequences, which promote aggregation and gelation under appropriate conditions.Several peptides are able to form hydrogels and were explored as scaffolds in tissue engineering. Some of them were inspired by the yeast protein zuotin,de novo design of a two‐peptide system, that allows to formulate hydrogels with tailored mechanical properties. This was achieved by designing a short peptide SA5N that self‐assembles and forms beta‐sheet fibers that above a critical gelation concentration forms a percolated networks and an hydrogel. The mechanical properties were controlled by the concentration of the peptide and the introduction of a second longer peptide designed with a central elastin‐inspired flexible sequence end‐capped at both ends with the self‐assembling peptide (SA21 peptide). The aim was to introduce physical cross‐links between beta‐sheet fibers to control the hydrogel mechanical properties. The occurrence in SA21 peptide of the elastin sequence (VPGVG)2 containing proline residues hinders the adoption of α‐helices as well as β‐sheet structures conferring high disorder to the backbone chain and introducing an elastic bridge between the SA5N fibers.In the present work, we were interested in β‐sheet forming peptides, inspired by amyloid‐like sequences.π stacking of the aromatic fluorenyl moiety.Rheological studies showed that G’ values observed for the SA5N hydrogels are dependent of peptide concentration and are an order of magnitude higher compared to those reported in the literature for other small peptides.From TEM images we observe that the insertion of SA21 in the nanofiber promotes branching, probably responsible for changes the rheological behavior of the hydrogels. In particular, doping of the SA5N peptide hydrogel with small amounts of SA21 peptide (H4 hydrogel) promotes the formation of a small number of “Y” shaped branching points that, however, significantly increase the storage modulus. On increasing the amount of SA21 peptide, the presence of a higher number of branching points could determine the formation of shorter nanofibers. In this way, the strengthening effect due to branching is counterbalanced by the shortening and kinking of the nanofibers. Branching of nanofibers was observed in several self‐assembling peptide, like branched peptides,5The potential of the two‐component SAPH in tissue engineering is mainly due to the versatility of the system, as an appropriate sequence design can tune several properties, including stiffness and biological effects. In comparison to other self‐assembling peptides used in regenerative medicine, these peptides are extremely short, spontaneously self‐assemble in aqueous conditions, and form very strong scaffolds without chemical cross‐linkers.The aggregation of the fibrils within the two‐component peptide system gives rise to a dense 3D network, which entraps high quantities of water and provides high and steady mechanical properties, favoring cellular growth. These kinds of scaffolds have shown a high biocompatibility and low cellular toxicity, supporting the attachment and proliferation of NIH 3T3 fibroblast cells. The inherent amino acid composition of SA5N/SA21 gels is advantageous in terms of biocompatibility, since the elastin sequence has allowed the growth of fibroblast cells. In order to use SA5N/SA21 gels for tissue engineering and regenerative medicine applications, the suitability of such gels to support different 3D culture cells has to be tested and is part of a forthcoming work.Figure S1: RP‐HPLC chromatograms of SA5N peptide after purification.Figure S2:1H‐NMR of SA5N peptide recorded in DMSO‐d6Figure S3: ESI‐MS spectrum of SA5N peptide Figure S4: RP‐HPLC chromatograms of SA21 peptide after purification.Figure S5:1H‐NMR of SA5N peptide recorded in DMSO‐d6Figure S6: ESI‐MS spectrum of SA21 peptide Figure S7: AFM topography image of H2 hydrogel ; height profile along the fiber 1; cross‐section of fiber 5 as indicated in the topography image.Figure S8: AFM topography image of SA21 peptide deposited on Silicon wafer after dilution in 5% DMSO in water. (top); 3D rendering of a magnified region (bottom).Click here for additional data file.


Here, monolayer germanene is successfully fabricated on semiconducting germanium film with the support of a Ag(111) substrate. Its linear‐like energy–momentum dispersion and large Fermi velocity are derived from the pronounced quasiparticle interference patterns in a √3 × √3 superstructure. In addition to Dirac fermion characteristics, the theoretical simulations reveal that the energy gap opens at the Brillouin zone center of the √3 × √3 restructured germanene, which is evoked by the symmetry‐breaking perturbation potential. These results demonstrate that the germanium nanosheets with √3 × √3 germanene can be an ideal platform for fundamental research and for the realization of high‐speed and low‐energy‐consumption field‐effect transistors. Recently, 2D germanene with a low‐buckled honeycomb atomic arrangement has been stabilized by the support of metallic substrates, including Au(111),z orbitals of Ge atoms, which give the π electrons near the Dirac points with the almost linear energy dispersion, are strongly hybridized with the metallic substrate states. As a result, wave functions derived from the pz orbitals are delocalized into the metallic substrate. This may lead to the absence of Dirac fermion characteristics.The Dirac equation dictates that the propagation of a 2D electron gas (2DEG) in a honeycomb periodic potential results in electronic Dirac fermion systems, in which electrons behave as massless quasiparticles with high mobility due to their linear energy–momentum dispersion. For example, graphene, silicene, and germanene are typical 2D electronic Dirac materials because the dynamics of their low‐energy electrons, modulated by two equivalent atomic sublattices, fulfills such requirements.luding Au1,11 Al(1R30° supercell on a semiconducting Ge(111) surface with the support of a Ag(111) substrate, which is verified by both scanning tunneling microscopy (STM) and scanning transmission electron microscopy (STEM). A linear‐like energy–momentum band dispersion relation has been deduced from the quasiparticle interference (QPI) patterns, regardless of the thickness of the underlying Ge(111) film. Furthermore, the supercell provides the reciprocal lattice vector connecting the two inequivalent Dirac cones in germanene, which breaks the chiral symmetry through the interaction between different valley states as well as splits the Dirac cone in the Brillion zone center by different on‐site potentials. Our work provides a feasible way to fabricate a Dirac electronic material with an energy gap, which paves the way to the development of high‐performance electronics.In this work, we successfully fabricated monolayer germanene with a (√3 × √3)2Figure2Ge surface alloy , which is consistent with the previous reports.2Ge structure could be driven into the disordered honeycomb arrangement by additional Ge deposition atoms with surface adatoms assembling themselves in the forms of dots, dimers, trimers, tetramers, and hexamers . Thus, the additional Ge atoms “pull out” the Ge atoms in Ag2Ge alloy to form the disordered structure due to the stronger strength of Ge—Ge covalent than that of Ge—Ag interfacial bonds. Further deposition leads to the formation of germanium nanosheets, as shown in Figure The growth dynamics and surface reconstructions of germanium nanosheets were studied in detail by STM, as shown in FigureZ (contrast ≈Z3/2) and shows unambiguous contrast between the Ag(111) substrate, deposited germanium nanosheets, and amorphous Pt capping layer. The germanium nanosheets exist in the form of islands, consistent with the STM results  and indicating a Volmer–Weber growth mode. The germanium nanosheets grow over the substrate step edges, suggesting a structure that is distinct from the Ag(111) substrate. The high‐resolution image , combined with the focused ion beam technique, to investigate the cross‐sectional structure. The image acquired with the high‐angle annular dark field (HAADF) detector Figure2a is sFigureThe interactions between the Ge atoms on the top layer and the interactions between the top‐layer Ge atoms and beneath Ge atoms in bulk Ge(111) are investigated by the calculation of the electron localization function (ELF), as it gives information about the nature of the interactions between atoms via electron localization. dI/dV curves and maps). A typical dI/dV curve taken at 77 K is shown in FigureE(κ) curve, where κ is the radius of the CEC at the Γ point with 2κ = |q|, where q is the intravalley scattering wave vector determined from radius of the fast Fourier transform (FFT) patterns of the STS maps. The linear‐like energy–momentum dispersion was explored , but it becomes trigonal at positions far away from the DP due to the interactions with the three nearest conical bands.k space  are √3 times that of 1 × 1 FS germanene  with a 30° rotation  couple the K and K′ points of the Brillouin zone (BZ) of 1 × 1 phase, causing the Dirac cones located at the K and K′ points of the BZ of 1 × 1 phase to be folded into the center Γ point of the BZ of the √3 × √3 superstructure . In fact, the on‐site energies are different in the superstructure from our density functional theory (DFT) calculations. The hopping parameter t2 is set to 1.30 eV, which is also determined by the DFT calculations. The t1 is tuned to see how the band structure changes with the coupling strength. After considering the variation of on‐site energies of different Ge atoms, the four bands degenerate and the energy gap opens at Γ point of BZ . The two bands close to zero energy become more and more flat with the decrease in the value of t1 . The band structure around the Γ point is similar to the DFT results  model with different order parameters were obtained. The Hamiltonian of the superstructure could be written ass Figure f with tWe have noticed the debate on the existence of √3 × √3 multilayer silicene on Ag(111).Figured core‐level XPS spectra of √3 × √3 germanene formed on Ge(111) with the support of Ag(111). The experimental data points are displayed with black dots, while the overall fitted curves are red line. The fitting results make it clear that there are two groups of bonding components, labeled as Ge1 and Ge2, respectively. The energy gap of the two peaks in each group is a constant value, indicating that the two fitting peaks in one group are related to two Ge 3d3/2 and 3d5/2 peaks, respectively. The Ge 3d5/2 spectrum appears as two peaks, 29.34 and 28.96 eV, both of which are close to that measured for germanene on Au(111)Figure med on Ge11 with t3In summary, we have demonstrated the formation of √3 × √3 germanene on Ge(111) nanosheets with the support of a Ag(111) substrate. A linear‐like energy–momentum dispersion is deduced from the QPI patterns under different energy levels, indicating the existence of Dirac fermions in the √3 × √3 restructured germanene. The DFT simulations exhibit a gap that has opened at the Dirac point due to the periodically altered hopping amplitudes and on‐site energies evoked by the Kekulé distortion. The high charge carrier mobility, a sizable energy gap corresponding to room temperature, and semiconducting Ge(111) nanosheets with tunable thickness acting as a dielectric layer present a possible avenue to realize nanotechnology applications of this material.4Synthesis of Germanium Nanosheets: All samples used in this work were fabricated in an ultrahigh vacuum  preparation chamber equipped on STM. Clean Ag(111) substrates were prepared by argon ion sputtering and annealed at 750 K for several cycles. The germanium nanosheets were then deposited on the Ag(111) surfaces by evaporation of germanium from a heated germanium wafer. The deposition flux of Ge was 0.1 monolayers per minute (ML min−1). The Ag(111) substrate temperature was maintained at 450 K during the deposition process.Characterization of Structural and Electronic Properties: The STM and Raman spectroscopy measurements were carried out by using a low‐temperature UHV STM/scanning near‐field optical microscopy system  in UHV (<8 × 10−11Torr) at 77 K. The Raman spectra were acquired using a laser excitation of 532 nm (2.33 eV), delivered through a single‐mode optical fiber at 77 K in UHV. The spot size of the incident laser in the in situ Raman spectroscopy was about 3 µm in diameter. The differential conductance, dI/dV, spectra were acquired by using a standard lock‐in technique with a 10 mV modulation at 967 Hz. HAADF images and STEM spectra were obtained using a probe corrected JEOLARM‐200F operating at 200 kV with a Centurio energy dispersive spectroscopy solid‐state X‐ray detector. In situ XPS characterizations were performed at the Photoelectron Spectroscopy Station (Beamline 4B9B) in the Beijing Synchrotron Radiation Facility (BSRF) using a SCIENTA R4000 analyze, and variable photon energies were referenced to a fresh Au polycrystalline film.DFT Calculations: All calculations were performed under the framework of DFT as implemented in the Vienna ab initio simulation package.Γ‐center 13 × 13 × 1 K‐mesh sampling for the FS model and a Γ‐center 7 × 7 × 1 K‐mesh sampling for the √3 × √3 model with a 400 eV cut‐off energy on plane wave basis sets were used in the calculations. All models were full relaxed. The convergence criteria were 10−5eV for total energy and 0.02 eV Å−1 for the force on each atom.The authors declare no conflict of interest.SupplementaryClick here for additional data file.


Histone lysine demethylases (KDMs) are involved in the dynamic regulation of gene expression and they play a critical role in several biological processes. Achieving selectivity over the different KDMs has been a major challenge for KDM inhibitor development. Here we report potent and selective KDM5 covalent inhibitors designed to target cysteine residues only present in the KDM5 sub‐family. The covalent binding to the targeted proteins was confirmed by MS and time‐dependent inhibition. Additional competition assays show that compounds were non 2‐OG competitive. Target engagement and ChIP‐seq analysis showed that the compounds inhibited the KDM5 members in cells at nano‐ to micromolar levels and induce a global increase of the H3K4me3 mark at transcriptional start sites. II and 2‐oxoglutarate (2‐OG) to oxidise methylated lysine substrates to form an unstable hemiaminal which fragments to the demethylated lysine and formaldehyde.Histone proteins can be covalently modified by a plethora of post‐translational marks including methylation. These modifications modulate chromatin structure which impacts gene expression, chromosome packaging and DNA damage repair. Methylation is a reversible process regulated by histone methyltransferases (HMTs) and lysine demethylases (KDMs) that control the methylation state  of lysine residues in histone tails.JmjC‐KDMs are a family of 20 human enzymes which are further categorized into subfamilies KDM2‐7.II in the 2‐OG binding site and form a salt bridge with a conserved lysine or hydrogen bond to a conserved tyrosine residue in the KDM5 subfamily. Although nanomolar enzyme IC50 values are reported, cellular activities are commonly much weaker which has been attributed to competition of the inhibitors with higher levels of competing substrate in cells (2‐OG≈1 mm)m range), a factor known to reduce kinase inhibitor efficacy.CPI‐455 is a potent, selective KDM5 inhibitor with nanomolar enzyme IC50, but micromolar cellular IC50.tert‐butyl pyrazole 12, incorporating a 8‐pyrazolopyridopyrimidinone core were published, however this series suffered from poor selectivity over KDM4 and poor cellular potency despite cellular permeability.Several small‐molecule inhibitors of KDM5s have been reported.lls 2‐OG≈ mmm rangCPI‐455 and pyridopyrimidinone 2 with improved selectivity that target key cysteines exclusive to the KDM5s. Irreversible binding of the protein to the inhibitor resulted in reduced competition with the 2‐OG cosubstrate and maintained their cellular activity despite low cellular permeability. Covalent inhibitors are potentially more selective and potent, with better cellular efficiency and can require less frequent and lower doses, although this can be offset by poor selectivity with the cellular cysteinome. Covalent inhibitors can also be used as proteomic tools for both covalent and non‐covalent drugs.Here we report two series of KDM5 selective covalent inhibitors based on cyanopyrazole Based on alignments of the JmjC‐KDMs, two cysteine residues were identified as potential nucleophiles for covalent modifications in the KDM5 family. C497 in KDM5B was identified as a non‐catalytic residue close to the binding site present only in the KDM5 members. In addition, C480 was identified as a unique residue in KDM5B, so improved selectivity could be achieved over KDM4s as well as KDM5A/C/D Figure  A. The i2 and was suitable for the design of cysteine‐selective electrophiles based on this scaffold. 8‐Pyridopyrimidinone‐based covalent inhibitors 3–6 (PP series)  Figure  D were s7–11 (PZ series)   although no covalent bond was seen in the crystal structure. The acrylamide of 10 was positioned further from C497 however compound flexibility could position it within covalent bonding distance. Both compounds maintained the key coordination of the nitrile to the metal centre and H‐bonding interactions of the carbonyl oxygen.A second series of KDM5 covalent inhibitors kinact/Ki, rather than a simple IC50. These parameters were calculated by using an established method to derive Ki and kinact directly from time‐dependant IC50 values (Table S1). IC50 values were measured using an AlphaScreen assaykinact and Ki values determined as previously described.Since covalent inhibitors possess time‐dependent inhibition due to the kinetics of covalent binding to the protein, their activity was better assessed through determination of the kinetic parameter 5 was the most potent with the highest kinact/Ki (40×103 m−1 s−1) and although acrylamide 4 was less active than 5 with (kinact/Ki 7.4×103 m−1 s−1) it showed a drop in IC50 of 12‐fold after an hour incubation .In the PP series, chloroacetamide 7 showed the greatest kinact/Ki (25×103 m−1 s−1) with an IC50 drop of 27‐fold upon pre‐incubation  and covalent inhibitors (4 and 7) were therefore determined at different concentrations of 2‐OG up to 1000 μm  and showed the best selectivity profile (Table S3). In the PP series the covalent compounds tested showed high activity with IC50 in the low nanomolar range against most KDM5 family members.A panel of JmjC KDMs was tested by AlphaScreen to determine the selectivity of the compounds Table S2). IC. IC50 va4–6 showed greater than 20‐fold selectivity over the KDM4 family, in particular 5 was more than 60‐fold less active on KDM4B. We attribute this selectivity for KDM5B with the covalent PP inhibitors to binding to C480. In the PZ series, all compounds were more than 50‐fold selective over all the KDM4 family members, with compounds 7 and 10 having >500‐fold selectivity over KDM4A/B.Both PP and PZ series showed the greatest selectivity over KDM2A and 3A with selectivity from 200‐ to 1500‐fold. The covalent inhibitors also demonstrated good selectivity over the most closely related KDM4 subfamily members. In the PP series, methylbenzylamine covalent inhibitors 6 was the most selective with greater than 7‐fold selectivity for KDM5B over KDM5A/C/D which could be explained by specific binding to C480. In the PZ series, the most selective compound was chloroacetamide 11 showing the highest selectivity for KDM5B with more than 100‐fold over KDM5C.When comparing the activity of the compounds in the KDM5 family members, both series were equipotent against KDM5A/B/D, but 30–100‐fold selective over KDM5C. In the PP series, compound 3 was incubated with KDM5B and the resulting MS spectra showed a minor set of peaks relating to unmodified KDM5B (m/z 55 153) and a major peak relating to protein–inhibitor adduct with the correct mass shift (Δm/z 379)  and MS showed only unmodified protein peaks as expected for these proteins that do not contain a cysteine at C480 as in KDM5B. The specific covalent binding to KDM5B explains the improved selectivity over KDM5C and KDM4B compared to non‐covalent inhibitor 2.Covalent binding of the inhibitors with KDM5B was confirmed through MS‐labelling experiments Figure . PP comp3 was binding specifically to the desired C480 and not to other cysteines in KDM5B  .With confirmation of potent and selective binding to recombinant KDM5 targets, we wanted to show cellular target engagement. We developed a PZ‐based cell‐permeable fluorescent tracer assay to quantify the engagement of compounds with KDM targets that are fused to NanoLuciferase.1, showing that the tracer produced significant BRET signal in a dose‐dependent manner with all the KDM5 family members (KDM5A‐D), but no BRET signal for any of the KDM4s or KDM6B, demonstrating the selectivity of the tracer . Cellular activity of the most relevant PP and PZ compounds was then evaluated . However, compounds 7, 10 and 11 showed micromolar IC50 values, which are more than a 100‐fold weaker than their in vitro activity, probably due to their low logP and poor cell permeability.The tracer was tested in transfected NanoLuc‐KDM4s, KDM5s and KDM6B in the presence of compound 4 was the only example of the PP series that showed some activity in cells, with an IC50 of 10.6 μm, in the same range as the non‐covalent compound 2 (IC50 8.8 μm). For both the PP and PZ compounds, the similar cellular potency compared to their non‐covalent analogues can be explained due to their high polarity as measured clogP and poor cell permeability as measured by Caco‐2  but despite being much less cell permeable (Caco‐2 AB<1.5×10−6 cm s−1), the covalent compounds are as potent in cells as their non‐covalent analogues, presumably due to their better 2‐OG competition.Compound 5, compounds were not cytotoxic in HEK cells . Compounds 4, 7 and 9 were selected for H3K4me3 ChIP‐seq experiments and quantification.Immunofluorescence‐based assays have been previously used to analyse global changes in H3K4me3 as a measure of KDM5 activity, but the global change can be affected by other factors such as cytotoxicity.After sequencing and normalisation, read coverage was evaluated and coverage plots were plotted across all genes revealing significant increases of H3K4me3 levels around TSS  should be addressed to the authors.SupplementaryClick here for additional data file.