Ibuprofen destabilizes Tat2p without triggering other TOR pathway outputs.

<p><b>A</b>, Steady-state Tat2p-TAP levels are reduced upon ibuprofen treatment. Exponentially proliferating cells expressing from its chromosomal location TAP-tagged Tat2p were exposed to ibuprofen or rapamycin at the indicated concentration and for the times shown. Tat2p levels were evaluated by SDS-PAGE and immunoblotting. From the same samples, steady-state levels of untagged Cdc28p are shown for comparison. <b>B</b>, Ibuprofen destabilizes Tat2p-TAP. The experiment was performed as in A, except that the cells were treated with cycloheximide at time 0. Tat2p-TAP band densities were quantified with Image J software. These values were then normalized for loading and fitted on an exponential decay function to obtain the half-life values shown on the right of each blot. <b>C</b>, Ibuprofen does not trigger gene expression of downstream effectors of the TOR pathway. <i>Left</i>, we examined three sets of targets of the TOR pathway, whose expression is known to be affected by rapamycin. <i>Right</i>, expression of the targets genes shown was evaluated by qPCR (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004860#s4" target="_blank">Materials and Methods</a>) from exponentially growing cells after addition of ibuprofen (at 0.2 mM) or rapamycin (at 50 ng/ml) at the indicated times. The targets were grouped based on the diagram shown on the left. We also included <i>TAT1</i> and <i>TAT2</i> expression in this experiment. The average values from at least three experiments in each case were log₂-transformed and displayed on the heatmap shown using the open-source <i>pheatmap</i> package for the R language. <b>D</b>, Ibuprofen does not trigger Gln3p dephosphorylation. Cells expressing TAP-tagged variants of two downstream effector of TOR, Npr1p and Gln3p, were treated with ibuprofen or rapamycin for 60 min, at the same concentrations as in A. Npr1p-TAP and Gln3p-TAP levels and mobility, evaluated by immunobloting. <b>E</b>, Ibuprofen does not extend the RLS of a strain that carries the stable <i>2HA-TAT2-5KR</i> allele. Survival curves of the strains shown treated with ibuprofen at 0.2 mM compared to experiment-matched untreated cells. For comparison, wild type cells carrying a similarly tagged wild type <i>TAT2</i> allele (<i>2HA-TAT2</i>) were also included in this experiment and they were either untreated (shown in black) or treated (shown in red) with ibuprofen. Mean lifespans are shown in parentheses, along with the number of cells assayed.