Detection of the expression levels of three major types of MDR proteins in cultured cell lines using immunostaining with monoclonal antibodies and a flow cytometry assay.

<p>Average D values (± SEM) for three representative experiments are presented.</p

Examples of multiplex (triple) probe treatment for MDR activity profiling in the CHO K1 cell line.

<p>(Average D values for three representative experiments are provided as characteristics of MDR, with SD not exceeding 10% for each value).</p

Comparison of MDR activity detection in model cell lines using MDR probes and inhibitors of different specificity.

<p>Average MAF values for three representative experiments are provided, with SD not exceeding 10% for each value.</p

Concentration-dependent inhibitory effect of various general and specific inhibitors on MDR probes accumulation in CHO K1 (panel A), HCT-15 (panel B), HepG2 (panel C) and HL-60/MX1 (panel D) cells.

<p>The following MDR probes were used: eFluxx-ID® Green (filled squares), eFluxx® Gold (filled triangles), calcein AM (grey circles), DiOC₂(3) (open triangles), CMFDA (open diamonds), pheophorbide A (filled diamonds). Cells were stained with the indicated probes in the presence of the various concentrations of the appropriate inhibitors. EC₅₀ values are defined as the concentration of the inhibitor resulting in half-maximum inhibition of dye accumulation.</p

A–D. CHO K1 and A549 cell lines display increased chemoresistance toward the cytotoxic drugs that are mostly associated with MDR: doxorubicin (panel A), taxol (panel B), vincristine (panel C), and mitoxantrone (panel D).

<p>The U-2 OS cell line demonstrates moderately increased chemoresistance to a few drugs associated to MDR, such as doxorubicin (panel A) and mitoxantrone (panel D). The HeLa cell line was used as a non-chemoresistant control specimen. Cells were seeded in 96 well plates, treated with the different doses of indicated drugs, and a standard MTT viability test was performed 1 and 2 days post-treatment. Results are presented as a ratio of the OD₅₉₅ of treated cells to the OD₅₉₅ of the untreated cells.</p

Examples of multiplex (dual) probe treatment for MDR activity profiling in the CHO K1 cell line.

<p>(Average D values for three representative experiments are provided as characteristics of MDR, with SD not exceeding 10% for each value).</p

Comparison of MDR activity detection in model cell lines overexpressing a single ABC transporter pump using MDR probes and inhibitors of different specificity.

<p>Average MAF values + SEM for three representative experiments are provided. Negative MAF values have been replaced with 0.</p

eFluxx-ID® MDR probes detect all three major types of ABC transporters in a similar manner as doxorubicin and mitoxantrone probes, but are significantly brighter, providing much higher sensitivity compared with the other dyes.

<p>Model cell lines (CHO K1, panel A, and A549, panel B) were trypsinized, washed with PBS, aliquoted at 5×10⁵ cells/sample, and treated in triplicates with different inhibitors (5 µM of cyclosporin A, 20 µM of verapamil, 50 µM of MK-571, or 0.05 µM of novobiocin) or left untreated. Tested probes (eFluxx-ID® Green, eFluxx-ID® Gold dyes, doxorubicin or mitoxantrone) were added to every sample. The cells were incubated with the dye(s) in the presence or absence of inhibitors for 30 min at 37°C. Then cells were immediately analyzed by flow cytometry. Population comparison was performed using Kolmogorov-Smirnov statistics <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022429#pone.0022429-Young1" target="_blank">[26]</a>. Clear histograms represent sample fluorescence in the presence of the inhibitor, shaded – without the inhibitor. The numbers indicate average D-values for each sample from at least three independent experiments, with SD not exceeding 10% for each value.</p

Ni-Assisted Transformation of Graphene Flakes to Fullerenes

Transformation of graphene flakes to fullerenes assisted by Ni clusters is investigated using molecular dynamics simulations. The bond-order potential for Ni–C systems is developed. The potential reproduces the experimental and first-principles data on the physical properties of pure Ni as well as on relative energies of carbon species on Ni surfaces and in Ni bulk. The potential is applied for molecular dynamics simulations of the transformation of graphene flakes consisting of 50–400 atoms with and without Ni clusters attached. Free fullerenes, fullerenes with Ni clusters attached from outside, and fullerenes encapsulating Ni clusters (Ni endofullerenes) are observed to form in the presence of Ni clusters consisting of 5–80 atoms. Moreover, a new type of heterofullerenes with a patch made of a Ni cluster is found to form as an intermediate structure during the transformation. The Ni clusters are shown to reduce the activation energy for the graphene–fullerene transformation from 4.0 eV to 1.5–1.9 eV, providing the decrease of the minimal temperature at which such a transformation can be observed experimentally from about 1400 K for free graphene flakes to about 700–800 K. While the transformation of free graphene flakes is found to occur through formation of chains of two-coordinated carbon atoms at the flake edges, the mechanism of the Ni-assisted graphene–fullerene transformation is revealed to be based on the transfer of carbon atoms from the graphene flake to the Ni cluster and back. The way of controlled synthesis of endofullerenes with a transition metal cluster inside and heterofullerenes with a transition metal patch is also proposed

Ni-Assisted Transformation of Graphene Flakes to Fullerenes

Transformation of graphene flakes to fullerenes assisted by Ni clusters is investigated using molecular dynamics simulations. The bond-order potential for Ni–C systems is developed. The potential reproduces the experimental and first-principles data on the physical properties of pure Ni as well as on relative energies of carbon species on Ni surfaces and in Ni bulk. The potential is applied for molecular dynamics simulations of the transformation of graphene flakes consisting of 50–400 atoms with and without Ni clusters attached. Free fullerenes, fullerenes with Ni clusters attached from outside, and fullerenes encapsulating Ni clusters (Ni endofullerenes) are observed to form in the presence of Ni clusters consisting of 5–80 atoms. Moreover, a new type of heterofullerenes with a patch made of a Ni cluster is found to form as an intermediate structure during the transformation. The Ni clusters are shown to reduce the activation energy for the graphene–fullerene transformation from 4.0 eV to 1.5–1.9 eV, providing the decrease of the minimal temperature at which such a transformation can be observed experimentally from about 1400 K for free graphene flakes to about 700–800 K. While the transformation of free graphene flakes is found to occur through formation of chains of two-coordinated carbon atoms at the flake edges, the mechanism of the Ni-assisted graphene–fullerene transformation is revealed to be based on the transfer of carbon atoms from the graphene flake to the Ni cluster and back. The way of controlled synthesis of endofullerenes with a transition metal cluster inside and heterofullerenes with a transition metal patch is also proposed