Living HepG2 Cells Research Articles

Spectral measurement of acceptor-to-donor extinction coefficient ratio in living cells

This report presents a simple method named as sp-ECR to determine the molar extinction coefficient ratio (γ(λex)) of acceptor-to-donor in living cells at excitation wavelength λex, which is closely associated with the acceptor cross-excitation, the hardest issue of FRET quantification. sp-ECR determines γ(λex) by spectrally unmixing the emission spectrum of a donor–acceptor tandem construct under λex excitation without any additional references, such that this method can be performed under optimal imaging condition. We used sp-ECR to measure the γ(458) of Venus/Cerulean in living HepG2 cells on a confocal microscope, and the measured values were consistent with those obtained by lux-FRET method. We also used sp-ECR to measure the γ(458) values of Venus/Cerulean and YFP/CFP as well as YFP/GFP, the commonly used FRET FPs pairs in other two kinds of cancer cell lines on the confocal microscope, and found that the extinction coefficients of FPs depended on cell lines. After predetermining the γ(458) of Venus to ECFP, we used sp-ECR method to monitor the staurosporine (STS)-induced dynamical caspase-3 activation in single live A549 cells expressing SCAT3 by spectrally resolving the absolute FRET efficiency of SCAT3, and found that STS-induced caspase-3 activation in single cells is a very rapid process within 20min.

Fluorescent carbon dot (C-dot) nanoclusters

Fluorescent carbon dot (C-dot) nanoclusters composed of C-dot-loaded hollow silica spheres are obtained via the dehydration of mannose, which is adsorbed onto hollow silica spheres or poly(ethylene glycol)-graft-hollow silica spheres (PEG-g-hollow silica). The structure of C-dot nanoclusters are confirmed using 1H NMR, FTIR, TEM and TGA. C-dot nanoclusters show a redshifted fluorescence emission with an increased excitation wavelength. Passivation with PEG diamines improve the quantum yields to ∼2%. Confocal laser scanning microscopy (CLSM) results reflect the fact that C-dot nanoclusters can provide good cytoplasm imaging of live Hep G2 cells and live MCF-7 cells, and the imaging obtained is brighter and more even than those from free C-dots. With their combination of good photostability and low cytotoxicity, C-dot nanoclusters are promising for the production of higher quality bioimaging.

Facile synthesis of functional gadolinium-doped CdTe quantum dots for tumor-targeted fluorescence and magnetic resonance dual-modality imaging.

Magnetic quantum dots (MQDs) are an important class of agents for fluorescence (FL)/magnetic resonance (MR) dual-modal imaging due to their excellent optical and magnetic properties. However, functional MQDs prepared by a simple room-temperature route as FL/MR dual-modal imaging probes are lacking. Herein, we report the fabrication of Gd-doped CdTe quantum dots (Gd:CdTe QDs) as an agent for FL/MR dual-modality imaging. The as-designed QDs with an ultrasmall particle size are synthesized by a facile one-pot aqueous synthesis approach at room temperature. They emit strong fluorescence at 640 nm with a quantum yield of 37% in water, and they have a high longitudinal relaxation rate (r1) value of 3.27 mM-1 s-1. With the further conjugation of folic acid, the Gd:CdTe QDs can successfully label live HepG2 cells for targeted cellular imaging and present no evidence of cellular toxicity up to the concentration of 0.5 mg mL-1. They have been employed as a suitable contrast agent successfully for tumor-targeted FL/MR dual-modal imaging in a mouse model.

Electrochemical approach for the development of a simple method for detecting cell apoptosis based on caspase-3 activity.

This paper reports a novel approach for the simple detection of cell apoptosis using an electrochemical technique. This method uses caspase-3 activity as an indicator of apoptosis. Caspase-3 activity was detected with differential plus voltammetry (DPV) as an alternative to conventional spectrometry. In this method, p-nitroaniline (pNA) released from Asp-Glu-Val-Asp-pNA by caspase-3 enzyme reaction was measured with DPV by using a glassy carbon electrode. Using this method, we successfully detected cell apoptosis occurring inside living HepG2 cells without the need for a cell lysis step. This method provides an easy assay procedure and, more importantly, allows a live cell apoptosis detection format. This novel electrochemical apoptosis assay using living cells instead of typically used cell lysates will expand the applicable range of the apoptosis assay to include cell activity assays for drug discovery and cell transplantation medicine.

Native Chemical Ligation Combined with Spirocyclization of Benzopyrylium Dyes for the Ratiometric and Selective Fluorescence Detection of Cysteine and Homocysteine

Spirocyclization of xanthene dyes has become a powerful technique for developing fluorescent probes. Herein, we extend this unique fluorescence switching mechanism to a near-infrared (NIR) dye, 2-(7-diethylamino-2-oxo-2H-1-benzopyran-3-yl)-4-(2-carboxyphenyl)-7-diethylamino-1-benzopyrylium (CB), and construct a ratiometric fluorescent probe 1 for cysteine (Cys)/homocysteine (Hcy). The ratiometric sensing of probe 1 toward Cys/Hcy is realized by utilizing a tandem native chemical ligation/spirocyclization reaction to interrupt the large π-conjugated system of CB fluorophore, thereby affording remarkable blue shifts in the spectra of sensing system (from 669 to 423 nm in absorption spectra and from 694 to 474 nm in emission spectra). Probe 1 shows a high sensitivity for Cys/Hcy, and the detection limits (3 δ) for Cys and Hcy are 1.6 × 10(-7) and 1.8 × 10(-7) M, respectively. Moreover, since both the sulfhydril and the adjacent amino groups are involved in the sensing process, probe 1 is selective toward Cys/Hcy over other thiols such as glutathione. All these unique features make it particularly favorable for ratiometric Cys/Hcy sensing and bioimaging applications. It has been preliminarily used for Cys detection in rabbit serum samples and the ratiometric fluorescent imaging of Cys in living HepG2 cells.

A Self‐Assembled Nanohybrid Composed of Fluorophore–Phenylamine Nanorods and Ag Nanocrystals: Energy Transfer, Wavelength Shift of Fluorescence and TPEF Applications for Live‐Cell Imaging

A fluorophore-phenylamine derivative (L) has been coupled with silver nanocrystals (NCs) to construct an L-Ag nanohybrid. Owing to synergic effects of the L and Ag components, the exciton-plasmon interactions between L and Ag increase the strength of the donor-acceptor interaction within the nanohybrid, a fact that results in an energy-transfer process and further brings about a dramatic redshift of single-photon absorption and fluorescence, and a decreased fluorescence FL lifetime. The coupling effect also leads to enhancement of a series of nonlinear optical properties, including two-photon-excited fluorescence (TPEF), two-photon-absorption (TPA) cross section (δ), two-photon-absorption coefficient (β), nonlinear refractive index (γ), and third order nonlinear optical susceptibility (χ((3))). The enhanced two-photon fluorescence of the nanohybrid is proven to be potentially useful for two-photon microscopy of live cells, such as HepG2. Moreover, cytotoxicity tests show that the low-micromolar concentrations of the nanohybrid do not cause significant reduction in cell viability over a period of at least 24 h and should be safe for further biological studies.

A New Near-Infrared Neutral pH Fluorescent Probe for Monitoring Minor pH Changes and its Application in Imaging of HepG2 Cells

A new near-neutral pH near-infrared (NIR) fluorescent probe utilizing a fluorophore-receptor molecular framework that can modulate the fluorescence emission intensity through a fast photoinduced electron transfer process was developed. Our strategy was to choose tricarbocyanine (Cy), a NIR fluorescent dye with high extinction coefficients, as a fluorophore, and N-methylpiperazine (MP) as a receptor. The pH titration indicated that MP-Cy can monitor the minor physiological pH fluctuations with a pKa of ∼7.10 near physiological pH, which is valuable for intracellular pH researches. The probe responds linearly and rapidly to minor pH fluctuations within the range of 3.05-7.10 and exhibits strong dependence on pH changes. As expected, the real-time imaging of cellular pH and the detection of pH in situ was achieved successfully in living HepG2 cells by this probe. It is shown that the probe effectively avoids the influence of autofluorescence and native cellular species in biological systems and meanwhile exhibits high sensitivity, good photostability, and excellent cell membrane permeability.

Capturing intercellular sugar-mediated ligand-receptor recognitions via a simple yet highly biospecific interfacial system

Intercellular ligand-receptor recognitions are crucial natural interactions that initiate a number of biological and pathological events. We present here the simple construction of a unique class of biomimetic interfaces based on a graphene-mediated self-assembly of glycosyl anthraquinones to a screen-printed electrode for the detection of transmembrane glycoprotein receptors expressed on a hepatoma cell line. We show that an electroactive interface confined with densely clustered galactosyl ligands is able to ingeniously recognize the asialoglycoprotein receptors on live Hep-G2 cells employing simple electrochemical techniques. The only facility used is a personal laptop in connection with a cheap and portable electrochemical workstation.

Pathway-focused bioassays and transcriptome analysis contribute to a better activity monitoring of complex herbal remedies

BackgroundTranscriptome analysis in combination with pathway-focused bioassays is suggested to be a helpful approach for gaining deeper insights into the complex mechanisms of action of herbal multicomponent preparations in living cells. The polyherbalism based concept of Tibetan and Ayurvedic medicine considers therapeutic efficacy through multi-target effects. A polyherbal Indo-Tibetan preparation, Padma 28, approved by the Swiss drug authorities (Swissmedic Nr. 58436), was applied to a more detailed dissection of mechanism of action in human hepatoma HepG2 cells. Cell-free and cell-based assays were employed to evaluate the antioxidant capacity. Genome-wide expression profiling was done by applying Human Genome U133 Plus 2.0 Affymetrix arrays. Pathway- and network-oriented analysis elucidated the affected biological processes. The results were validated using reporter gene assays and quantitative real-time PCR.ResultsTo reveal the direct radical scavenging effects of the ethanolic extract of the Indo-Tibetan polyherbal remedy Padma 28, an in vitro oxygen radical absorbance capacity assay (ORAC) was employed, which resulted in a peroxyl-radical scavenging activity of 2006 ± 235 μmol TE/g. Furthermore, the antioxidant capacity of Padma 28 was analysed in living HepG2 cells, by measuring its scavenging potential against radical induced ROS. This formulation showed a considerable antioxidant capacity by significantly reducing ROS levels in a dose-dependent manner.Integrated transcriptome analysis revealed a major influence on phase I and phase II detoxification and the oxidative stress response. Selected target genes, such as heme oxygenase 1, were validated in qPCR experiments. Network analysis showed 18 interrelated networks involved in important biological functions such as drug and bio-molecule metabolism, molecular transport and cellular communication. Some molecules are part of signaling cascades that are active during development and morphogenesis or are involved in pathological conditions and inflammatory response.ConclusionsThe identified molecular targets and pathways suggest several mechanisms that underlie the biological activity of the preparation. Although extrapolation of these findings to the in vivo situation is not possible, the results obtained might be the basis for further investigations and new hypotheses to be tested. This study demonstrates the potential of the combination of focused and unbiased research strategies in the mode of action analysis of multicomponent herbal mixtures.

Two-photon fluorescent probe for cadmium imaging in cells

A novel two-photon excited fluorescent probe for cadmium (named as TPCd) was designed and synthesized utilizing a prodan (6-acetyl-2-methoxynaphthalene) derivative as the two-photon fluorophore and an o-phenylenediamine derivative as the Cd(2+) chelator, which possessed favorable photophysical properties and good water-solubility. The probe was designed with a photoinduced electron transfer (PET) mechanism and thus was weakly fluorescent itself. After binding with Cd(2+) which blocked the PET process, the fluorescence intensity of the probe was enhanced by up to 15-fold under one-photon excitation (OPE) and 27-fold under two-photon excitation (TPE), respectively. The two-photon action cross-section (Φδ) of the TPCd-Cd complex at 740 nm reached 109 GM compared to 3.6 GM for free TPCd, indicating the promising prospect of the probe in two-photon application. TPCd chelated Cd(2+) with 1 : 1 stoichiometry, and the apparent dissociation constant (K(d)) was 6.1 × 10(-5) M for the one-photon mode and 7.2 × 10(-5) M for the two-photon mode. The probe responded to Cd(2+) over a wide linear range from 0.1 to 30 μM with a detection limit of 0.04 μM. High selectivity of the probe towards Cd(2+) was acquired in Tris-HCl/sodium phosphate buffer. The probe was pH-independent in the biologically relevant pH range and non-toxic to living cells at reasonable concentration levels, warranting its in vivo applications. Through two-photon microscopy imaging, the probe was successfully applied to detect Cd(2+) uptake in living HepG2 cells.

Isolation and identification of native membrane glycoproteins from living cell by concanavalin A–magnetic particle conjugates

The discovery, isolation, and subsequent identification of cell membrane glycoproteins involved in the structure and function of the cell surface are becoming more and more important. Here, concanavalin A–magnetic particle conjugates were employed to isolate the special membrane glycoproteins from living HepG-2 cells. The isolated glycoproteins were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and liquid chromatography–tandem mass spectrometry as well as annotated. A total of 37 membrane glycoproteins were identified, and 25 of them were ascertained to locate in the extracellular region.

A New Ratiometric Fluorescent Probe for Detection of Fe2+ with High Sensitivity and Its Intracellular Imaging Applications

Good sense: A highly sensitive and selective ratiometric fluorescent probe for Fe2+ was developed by linking an Fe2+-insensitive fluorophore to an Fe2+-sensitive reporter–fluorophore fragment. The fluorescence intensity ratio increased linearly with the concentration of Fe2+ (see scheme). The probe was applied to the ratiometric imaging of intracellular and adscititious fluctuations of Fe2+ in live HL-7702 and HepG2 cells.

Chemical Modification and Organelle‐Specific Localization of Orlistat‐Like Natural‐Product‐Based Probes

Orlistat, also known as tetrahydrolipstatin (THL), is an FDA-approved anti-obesity drug with potential anti-cancer activity. Previously, we developed a chemical proteomic approach, based on the Orlistat-like probe (1a) for large-scale identification of unknown cellular targets of Orlistat in human hepatocytes. In this article, we report the chemical synthesis and biological evaluation of an expanded set of Orlistat-like compounds, with the intention to further dissect and manipulate potential cellular targets of Orlistat. In doing so, we carried out proteome-wide activity-based profiling and large-scale pull-down/LCMS analysis of these compounds in live HepG2 cells, and successfully identified many putative cellular targets for Orlistat and its structural analogues. By qualitatively assessing the spectra counts of potential protein hits against each of the seventeen Orlistat analogues, we obtained both common and unique targets of these probes. Our results revealed that subtle structural modifications of Orlistat led to noticeable changes in both the cellular potency and target profiles of the drug. In order to further improve the cellular activity of Orlistat, we successfully applied the well-established AGT/SNAP-tag technology to our cell-permeable, benzylguanine (BG)-containing Orlistat variant (4). We showed that the drug could be delivered and effectively retained in different sub-cellular organelles of living cells. This strategy may provide a general and highly effective chemical tool for the potential sub-cellular targeting of small molecule drugs.

A ratiometric fluorescent pH probe based on aggregation-induced emission enhancement and its application in live-cell imaging

A ratiometric fluorescent pH probe, 4-carboxylaniline-5-chlorosalicylaldehyde Schiff base (1) was synthesized via a facile reaction and used for pH sensing in live cells. While exhibiting weak fluorescence when dispersed in solution, 1 displayed aggregation-induced emission enhancement (AIEE) characteristics in its aggregate/solid state, as a result of the restriction of free intramolecular rotation of a C–N bond and the non-planar configuration in the aggregate/solid state. The integration of hydroxyl and carboxyl groups provided 1 with a ratiometric fluorescent response to pH based on AIEE and the pH-dependent spectral characteristics of compound 1, with proton dissociation constants pKa1 and pKa2 of 4.8 ± 0.1 and 7.4 ± 0.1, respectively. The probe exhibited a significant fluorescence color change from orange to green with an intensity ratio (I516 nm/ I559 nm) enhanced when the pH increased from 5.0 to 7.0 in aqueous solution. Confocal fluorescence imaging of intracellular pH through ratiometric response was successfully achieved in live HepG2 cells. The results demonstrate that probe 1 is a good candidate for monitoring pH fluctuations in live cells with good selectivity, stability, and excellent membrane permeability.

Effect of cytoskeleton inhibitors on deadhesion kinetics of HepG2 cells on biomimetic surface

Cytochalasin-D (Cyto-D) and latrunculin-A (Lat-A) are known inhibitors of actin microfilaments and adversely affect the physiological functions of anchorage-dependent cells. Alternatively, doxorubicin (Dox), a chemotherapeutic drug is known to induce apoptosis and cell detachment of tumor cells. However, the intricate interplay between drug administration, cytoskeletal rearrangement and biophysical responses of live cells on immobilized layer of extracellular matrix (ECM) protein remains unknown. In this study, the deadhesion kinetics and actin remodeling of live HepG2 cells following the addition of the three drugs are probed with confocal reflectance interference contrast microscopy (C-RICM) and fluorescence confocal microscopy. First, it is shown that the reduction in two-dimensional spread area of HepG2 cells is 10.5%, 15.4% and 21.9% under the influence of 5 μM of Lat-A, Cyto-D and Dox, respectively. Secondly, C-RICM demonstrates the recession of strong adhesion contact against time of cell seeding upon the addition of the three drugs. Thirdly, the initial cell detachment rate and extent of reduction in the degree of cell deformation ( a/ R) are dependent on both the drug types and concentration. Lastly, oscillation-like responses of a/ R and adhesion energy are uniquely found in Lat-A induced cell detachment. Overall, our biophysical approaches have been proven as a highly quantitative platform for elucidating the interfacial properties of adherent cells on biomimetic surfaces under cytoskeleton disruption.

Enhanced Cellular Uptake and Cytotoxicity Studies of Organometallic Bioconjugates of the NLS Peptide in Hep G2 Cells

SPACE INVADERS: Organometallic fragments such as the ferrocenyl group (shown in red in the picture) help to enhance cellular entry of NLS peptides. Eventually, these nontoxic conjugates find their way to the cellular nucleus as shown by fluorescence microscopy studies in this work. Intracellular delivery to biomolecular targets is still a major challenge in molecular and cell biology. We recently found that attaching an organometallic group, namely the cobaltocenium cation, to the SV 40 large T antigen nuclear localisation signal (NLS) greatly enhances cellular uptake of the conjugate (Noor et al., Angew. Chem. Int. Ed. 2005, 45, 2429). In addition, nuclear localisation of the conjugate was observed. In this work, we present a thorough investigation of this novel cellular delivery system with respect to the nature of the metal complex and the peptide sequence. A number of ferrocene ((Fe(II)), neutral metal complex) and cobaltocenium ((Co(III)), cationic metal complex) bioconjugates with both the NLS wild-type sequence PKKKRKV and a scrambled sequence (NLS(scr), KKVKPKR) were prepared by solid-phase peptide synthesis (SPPS). Cellular and nuclear uptake of these bioconjugates was studied by fluorescence microscopy on living Hep G2 cells. In addition, cytotoxicity screening on the conjugates was carried out, as the toxic effects of several simple metallocenes have been noted previously. Rapid cellular uptake as well as nuclear localisation was observed for the metal-NLS conjugates, but not for any dipeptide controls, the metal-NLS(scr) conjugates or any metal-free conjugates. It thus appears that the presence of a metallocene, but not its charge, and the correct NLS sequence is essential for cellular uptake. Fluorescence microscopy co-localisation studies did not reveal a significant endosomal entrapment of the conjugates. The metallocene not only provides a hydrophobic handle for membrane translocation but also facilitates the localisation and distribution of the conjugate in the cytoplasm. The NLS peptide on the other hand is responsible for the nuclear localisation of the bioconjugate. Finally, none of the conjugates were found to be toxic up to the highest concentrations that was tested (1 mM) against the Hep G2 cells that were used in this study. In conclusion, this work supports metallocene-NLS bioconjugates, in particular with the very robust cobaltocenium group, as a simple but potent, nontoxic system for cellular uptake and nuclear delivery. Concurrently, our finding is relevant to the still-unresolved question of cytotoxicity of metallocenes because it excludes binding and/or damage to the DNA as a mechanism of metallocene cytotoxicity. This finding is confirmed by a combined yeast cytotoxicity/genotoxicity assay, which also shows very little toxic effects for all organometal-NLS conjugates that were tested.

Glass-based microfluidic device fabricated by parylene wafer-to-wafer bonding for impedance spectroscopy

The paper reports the realization of a glass-based microfluidic device for characterization of cells in suspensions using impedance spectroscopy. The device consists of two glass wafers: a bottom wafer comprising a microfluidic channel with two electrodes added for impedance measurement, and a top glass wafer in which inlets and outlets are realized. The main focus of this work is the original fabrication process of this device, which combines three key techniques: firstly, successfully applying a through-wafer wet etching method in order to pattern the inlets and outlets in the top glass wafer; secondly, patterning the electrodes not only on flat surfaces but also in the microfluidic channel etched in the bottom wafer; and thirdly, employing indirect wafer to wafer bonding using an intermediate polymer layer. No external pumping is required as capillarity enables direct suction of a sample droplet in the channel. The device can be reusable if a thorough cleaning procedure is carried out. Devices with three different electrode geometries were successfully tested in electrochemical impedance spectroscopy (EIS) measurements using DI water and HepG2 cells. Although clear differences between DI water and live HepG2 cells have been observed in all cases, different results were obtained for various electrode geometries, highlighting the critical importance of the device design in performing EIS measurements and especially when making comparisons with other reported results.

Mathematical Analysis of Hepatic High Density Lipoprotein Transport Based on Quantitative Imaging Data

Hepatocytes internalize high density lipoprotein (HDL) at the basolateral membrane. Most HDL is recycled while some is shuttled to the canalicular membrane by transcytosis. Here, transport of HDL was analyzed by mathematical modeling based on measurements in polarized hepatic HepG2 cells. Recycling of HDL from basolateral sorting endosomes was modeled by applying the rapid equilibrium approach. Analytical expressions were derived, which describe in one model the transport of HDL to the subapical compartment/apical recycling compartment, the biliary canaliculus (BC), and to late endosomes and lysosomes (LE/LYS). Apical endocytosis of HDL predicted by the model was confirmed for rhodamine-dextran and fluorescent asialoorosomucoid, markers for LE/LYS in living HepG2 cells. Budding of endocytic vesicles from the BC was directly observed by time lapse imaging of a fluorescent lipid probe. Based on fitted kinetic parameters and their covariance matrix a Monte Carlo simulation of HDL transport in hepatocytes was performed. The model was used to quantitatively assess release of HDL-associated free cholesterol by scavenger receptor BI. It is shown that only 6% of HDL-associated sterol reaches the BC as a constituent of the HDL particles, whereas the remaining sterol is rapidly released from HDL and shuttled to the BC by non-vesicular transport.

Development of a Bioluminescence Resonance Energy-Transfer Assay for Estrogen-Like Compound in Vivo Monitoring

A new bioluminescence resonance energy transfer (BRET) homogeneous assay to evaluate the presence of estrogen-like compounds has been developed and optimized. The assay is based on the direct evaluation of estrogen alphareceptor (ERalpha) homodimerization as a result of estrogen-like compound binding. ERalpha monomer was genetically fused either to Renilla luciferase (Rluc) or to enhanced yellow fluorescent protein (EYFP). In the presence of estrogens, ERalpha dimerization brings Rluc and EYFP molecules close enough for an energy transfer. An in vitro BRET assay was first developed using purified fusion proteins (ERalpha-Rluc and ERalpha-EYFP) expressed in Escherichia coli to evaluate and optimize the analytical performances of the assay in the presence of 17-beta estradiol. The "in vivo" BRET quantitative assay was then developed by coexpressing the two fusion proteins in live HepG2 cells. The assay can be performed in 96-well microplate format with a 30-min incubation and allows detection with adequate accuracy and precision of as low as 1 nM of 17-beta estradiol. This new "in vivo" BRET assay allows evaluating the estrogen-like activity and synthetic xenoestrogens from biological and environmental samples.

Complex formation and submembranous localization of annexin 2 and S100A10 in live HepG2 cells

The Ca 2+ and membrane binding protein annexin 2 can form a heterotetrameric complex with the S100A10 protein and this complex is thought to serve a bridging or scaffolding function in the membrane underlying cytoskeleton. To elucidate which of the subunits targets the complex to the subplasmalemmal region in live cells we employed YFP/CFP fusion proteins and live cell imaging in HepG2 cells. We show that monomeric annexin 2 is targeted to the plasma membrane whereas non-complexed S100A10 acquires a general cytosolic distribution. Co-expression of S100A10 together with annexin 2 and the resulting complex formation, however, lead to a recruitment of S100A10 to the plasma membrane thus identifying annexin 2 as the membrane targeting subunit.