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Effect of Fe-SP or Cu-SP on viability, ROS generation, and induction of apoptic markers and MAPK expression.

By Kyu Kwang Kim (352288), Rakesh K. Singh (173687), Robert M. Strongin (285461), Richard G. Moore (173622), Laurent Brard (173668) and Thilo S. Lange (173627)

Abstract

<p>(<b>A</b>) <b>Cytotoxicity of Fe-SP or Cu-SP in NB cells.</b> The viability assay was carried out after 24 h treatment of SMS-KCNR NB cells with 0–3 µM of Fe-SP, Cu-SP or respective metal salts. Experiments were performed in triplicates; data are expressed as the mean of the triplicate determinations (X±SD) of a representative experiment in % cell viability of untreated cells [ = 100%]. (<b>B</b>) <b>Generation of intracellular Reactive Oxygen Species (ROS) after Fe-SP treatment.</b> Generation of intracellular ROS following SMS-KCNR NB cells (large panels) or SKOV-3 OC control cells (small panels) after treatment for 4 h with 1.6 µM of Fe-SP or Cu-SP was measured by flow cytometry (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019049#s4" target="_blank">Materials and Methods</a>). Data are presented as relative fluorescence intensity in a 2-dimensional FACS profile. Standardized gating was used for all samples. (<b>C</b>) <b>Fe-SP cytotoxicity is blocked by antioxidant ascorbic acid.</b> Cells were treated with with1.6 µM Fe-SP alone or in combination with antioxidant ascorbic acid. Viability of SMS-KCNR NB cells is presented as bar diagram and in percentages and of SKOV-3 OC control cells in percentages (insert). (<b>D</b>) <b>Inhibiton of ROS generation in NB cells after Fe-SP treatment.</b> Generation of intracellular ROS in SMS-KCNR NB was measured after treatment with 1.6 µM Fe-SP alone or in combination with antioxidant ascorbic acid (200 µM). (<b>E</b>) <b>Expression of apoptotic markers and MAPK in NB cells after Fe-SP treatment with and without inhibiton of ROS generation.</b> SMS-KCNR NB cells were treated with 0.8 µM Fe-SP in the absence (3, 6, 14, 24 h treatment, left panel) or presence of ascorbic acid (200 µM, 24 h treatment, right panel). Immunoblotting was carried out with primary antibodies against PARP-1, caspase-3, -7, pro-survival marker XIAP (inhibitor of effector caspases), and pro-apoptotic MAPK SAP/JNK or p38 in the active (phosphorylated) or inactive form. As an internal standard for equal loading blots were probed with an anti-GAPDH.</p

Topics: Cancer, fe-sp, cu-sp, ros, induction, apoptic, markers, mapk
Year: 2013
DOI identifier: 10.1371/journal.pone.0019049.g004
OAI identifier: oai:figshare.com:article/449125
Provided by: FigShare
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