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Rapamycin inhibits spontaneous and induced RTA expression in a dose-dependent manner regardless of induction pathway.

By Lisa A. Nichols (357612), Laura A. Adang (357613) and Dean H. Kedes (357614)

Abstract

<p>BCBL-1 were treated with 120 nM rapamycin or vehicle for 2 h, and then without (uninduced) or with 0.6 mM VPA (induced). (A) 48 h post-treatment, nuclear extracts were analyzed by immunoblot for RTA expression using the nuclear protein, RCC1, as a loading control. Representative experiment, n = 3. (B) 48 h post-rapamycin, uninduced BCBL-1 cells (top panels) or VPA-induced (bottom panels) were harvested, treated with a dead cell stain, then fixed, stained for intracellular RTA, and analyzed by flow cytometry. Representative plots (n = 7) show RTA expression in live-gated BCBL-1 cells treated with vehicle (left panels) or rapamycin (right panels). (C) Nuclear extracts were analyzed for RTA expression using non-enzymatic infrared detection probes to quantify relative protein levels. Ran (ras-related nuclear protein) was used as loading control. Graph (C, bottom panel) shows immunoblot RTA levels normalized to Ran as a percentage of RTA levels in the vehicle (DMSO) treated control. Representative experiment (n = 2). (D) Induced BCBL-1 cells treated for 48 h with rapamycin at indicated doses were fixed, stained for intracellular RTA and analyzed by flow cytometry. Graph, right, shows percent of RTA<sup>+</sup> cells in population indicated by histogram gate. (E) BCBL1 48 h post-treatment cells were harvested and nuclear extracts immunoblotted for RTA and normalized to Ran. Graph shows quantification of bands using non-enzymatic infrared detection probes. Representative experiment (n = 2). (E) BCBL-1 treated with indicated doses of rapamycin and left uninduced (square, dashed line), or induced with either VPA (triangle, solid line), TPA (triangle, dashed line), or CoCl<sub>2</sub> (open triangle, solid line) were cultured for 48 hrs in presence of rapamycin, then stained with intracellular RTA. Graph shows percentage of RTA<sup>+</sup> cells in live cell population. Mean ± s.e.m.; n for each condition shown in parentheses.</p

Topics: Infectious Diseases, Virology, inhibits, spontaneous, induced, rta, dose-dependent, induction
Year: 2013
DOI identifier: 10.1371/journal.pone.0014535.g002
OAI identifier: oai:figshare.com:article/476783
Provided by: FigShare
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