mTORC2 associates with Rit and is required for Rit-mediated Akt phosphorylation.

<p>(A) HA-Rit^Q79L or HA-K-Ras^Q61L containing complex was immunoprecipitated by HA antibody and blotted for Sin1 and Biotin-HA. (B) GFP-Sin1 containing complex was immunoprecipitated by GFP antibody and blotted for HA-Rit^Q79L and Sin1. (C) Lysates from serum-starved WT or Rictor^-/- MEFs overexpressing Flag-EV or Flag-Rit^Q79L were analyzed by immunoblotting with indicated antibodies. (D) Relative phosphorylation of p38 MAPK in WT and Rictor^-/- MEFs overexpressing Flag-EV or Flag-Rit^Q79L. The results are presented as mean ± SEM (One-way ANOVA, n = 3).</p

p38 MAPK controls mTORC2 kinase activity in response to oxidative stress.

<p>(A) HeLa cells were treated with 10 µM SB203580 for 30 min prior to H₂O₂ (1 mM, 15 min) or IGF-1 (100 ng/mL, 10 min) stimulation. mTORC2 complex was pulled down by anti-Rictor antibody and subjected to <i>in vitro</i> kinase assay using inactive His-Akt as the substrate (<i>Top</i>). The specificity and equal loading of Rictor immunoprecipitates were confirmed by immunoblotting with indicated antibodies (<i>Middle</i>). The whole cell lysates were also analyzed by immunoblotting with indicated antibodies (<i>Bottom</i>). (Arrowhead, 82 kDa) (B) Quantification of relative mTORC2 kinase activity. The results are presented as mean + SEM. (One-way ANOVA followed by Newman-Keuls <i>post-hoc</i> test when appropriate; * p<0.05, ** p<0.01 vs corresponding control groups, ## p<0.01 vs corresponding non-SB203580 treated groups, n = 3) (C) HeLa cells transfected with Flag-EV or dominant negative p38α along with GST-Akt were treated with H₂O₂ (1 mM, 15 min). The whole cell lysates were prepared and analyzed by immunoblotting using indicated antibodies. (*, non-specific band)</p

Rit mediates mTORC2 activation following oxidative stress.

<p>(A–C) Lysates from primary WT or Rit^-/- MEFs were prepared following H₂O₂ (100 µM, 15 min), EGF (100 ng/mL, 10 min) or IGF-1 (100 ng/mL, 10 min) stimulation and analyzed by immunoblotting with the indicated antibodies. (D) mTORC2 complex was immunoprecipitated using anti-Rictor antibody and subjected to <i>in vitro</i> kinase assay using inactive His-Akt as the substrate following H₂O₂ (1 mM, 15 min) or IGF-1 (100 ng/mL, 10 min) stimulation (<i>Top left</i>). The specificity and equal loading of Rictor immunoprecipitates was confirmed by immunoblotting with the indicated antibodies (<i>Bottom left</i>). Whole cell lysates were also analyzed by immunoblotting with the indicated antibodies (<i>Right</i>). (Arrowhead, 82kDa) (E) Anti-Rictor immunoprecipitation was used to isolate the mTORC2 complex from primary WT or Rit^-/- MEFs following H₂O₂ (100 µM, 15 min) exposure and subjected to <i>in vitro</i> kinase assay using inactive His-Akt as the substrate (Arrowhead, 82 kDa). (F) Fold induction of mTORC2 kinase activity in WT and Rit^-/- MEFs after stimulation relative to basal. The results are presented as mean ± SEM (<i>t</i>-test; * p<0.05, n = 3).</p

mTORC2 is required for H₂O₂-induced phosphorylation of Akt at Ser⁴⁷³.

<p>(A) Lysates from HeLa cells infected with lentivirus expressing shRNA against Rictor, Sin1 or Raptor were analyzed by immunoblotting with indicated antibodies following H₂O₂ (1 mM, 15 min) exposure (Arrowhead, 82 kDa). (B) Relative phosphorylation of signaling proteins following H₂O₂ stimulation. The results are presented as mean ± SEM. (<i>t</i>-test; * p<0.05, ** p<0.01, *** p<0.001 vs shCTR, n = 4). (C) Lysates from wild-type or Rictor^-/- MEFs were prepared following H₂O₂ (100 µM) treatment for indicated times and analyzed by immunoblotting with the indicated antibodies.</p

Influence of VPA on DICER protein expression levels.

<div><p><b>A</b>. Effect of VPA on DICER protein level in different cell lines. The cell lines indicated were treated with 20 mM VPA for 16 hours and the expression of DICER was detected using Western blot. Beta actin was used as a loading control in each cell line. </p> <p><b>B</b>. Time course of DICER expression in HEK293 cells treated with 20 mM VPA. DICER protein level is significantly reduced after 12 hours of VPA treatment. To quantify the effect, we calculated the intensity of DICER protein signal normalized to ß-actin, shown below, n=3.</p> <p><b>C</b>. Concentration dependency of DICER degradation in HEK293 cells during 16 hours of treatment. </p></div

VPA changes gene expression.

<div><p><b>A</b>. Overview of array analysis. HEK293 cells were treated for 16 hours with 20 mM Valproic Acid and compared with untreated cells using Affymetrix Exons Junction Arrays. About 11% of all human genes show changes on the transcriptional level, as indicated in the pie chart.</p> <p><b>B</b>. Chromatin immunoprecipitations using an H3K27ac antiserum on HEK293 cell lysates after six hours of VPA treatment. Promoter regions of genes influenced by VPA (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082895#pone.0082895.s002" target="_blank">Figure S2</a>) were tested.</p> <p><b>C</b>. Statistical analysis of three independent experiments.</p></div

VPA regulates miR-129 containing reporter genes in cis and trans.

<div><p><b>A</b>. Schematic diagram of the reporter construct indicating the location of the miRNA binding sites (small box) in the UTR of the hRluc gene.</p> <p><b>B</b>. HEK293 cells were transfected with dual luciferase reporter genes containing miR-129 binding sites (1, 2). Renilla luciferase activity was subject to the regulation of miR-129 binding site. Firefly luciferase activity from the same reporter construct was used to normalize and to generate relative activity of Renilla luciferase. Ctl. is a luciferase construct without the miR-129 binding sites. The fold-change of relative luciferase activities Renilla/Firefly is shown. The transfected cells were treated with 20 mM VPA for 16 hours. The binding sequences for miR-129 were: 1: CACACAGCAAAAA; 2: GCAAAAA; Control sequence: TTTATTT.</p></div

VPA leads to a degradation of the RNAse III DICER.

<div><p><b>A</b>. Influence of VPA on DICER over expressed as a cDNA. HEK293 cells were transfected with RFP-tagged DICER and treated for 16 hours with 20 mM VPA. Proteins were separated on SDS page and analyzed by western blot using anti-DICER antisera.</p> <p><b>B</b>. Proteasome inhibitors block the action of VPA on DICER.</p> <p>HEK293 cells were treated with 20 mM VPA in the presence of the proteasome inhibitor MG132 (10 µM), as well as the lysosome inhibitor bafliomycin A1 (200 nM) for 16 hours. Expression of endogenous DICER was detected using Western blot (top) and compared to ß-actin expression (bottom). The quantification is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082895#pone.0082895.s005" target="_blank">Figure S5</a>.</p> <p><b>C</b>. Ubiquitination of DICER: HEK293 cells were transfected with a HA-tagged ubiquitin expression vector, in the presence of 10 µM MG132. Endogenous DICER was immunoprecipitated using anti-DICER antiserum, and analyzed with an anti-HA antiserum. VPA treatment was for 16 hours. The western blot with anti-DICER antiserum is shown below. </p> <p><b>D</b>. Effect of VPA on cells after inhibition of translation. Cells were treated with cyclohexamide (100 mg/l) and 20 mM VPA for 12 hrs and DICER protein levels detected by western blot. ß-actin was used to demonstrate loading. The quantification underneath shows the fold change relative to the water control, set to 1, n=3.</p> <p><b>E</b>. HEK293 cells were treated with 20 mM VPA, 10 µM MG132 and 200 nM bafliomycin A1 and DICER was detected by western blot. ß-actin was used as a loading control. The signal for DICER relative to actin was set to 1, VPA:0.36; MG132:0.93; Bafliomycin: 1.</p> <p><b>F</b>. Response of LC3 protein levels to bafliomycin, MG132 and VPA treatment. Cells were treated for 12 hours with the drugs and the conversion of LC3-I to LC3-II was monitored by Western blot.</p></div

Validation of VPA induced changes.

<div><p><b>A</b>. Effect of VPA on dicer and LSM8 mRNA expression after 6 and 12 hours of treatment, determined by RT-PCR (*: p < 0.05, from three independent experiments). H₂O was used as negative control (ctl).</p> <p><b>B</b>. Real time PCR analysis of DICER mRNA expression after VPA treatment for six and 12 hrs.</p> <p><b>C</b>. Changes on protein level determined by Western Blot. HEK293 cells were treated for 16 hours with 20 mM VPA. Total cell lysates were separated by SDS page and analyzed using the antisera indicated. Beta-actin was used as a loading control. The mRNA changes of these genes are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082895#pone.0082895.s003" target="_blank">Figure S3</a>, VAMP8: downregulated, STX7: upregulated; STX2: downregulated; LSM8: downregulated.</p></div

Farnesyl Diphosphate Analogues with Aryl Moieties Are Efficient Alternate Substrates for Protein Farnesyltransferase

Farnesylation is an important post-translational modification essential for the proper localization and function of many proteins. Transfer of the farnesyl group from farnesyl diphosphate (FPP) to proteins is catalyzed by protein farnesyltransferase (FTase). We employed a library of FPP analogues with a range of aryl groups substituting for individual isoprene moieties to examine some of the structural and electronic properties of the transfer of an analogue to the peptide catalyzed by FTase. Analysis of steady-state kinetics for modification of peptide substrates revealed that the multiple-turnover activity depends on the analogue structure. Analogues in which the first isoprene is replaced with a benzyl group and an analogue in which each isoprene is replaced with an aryl group are good substrates. In sharp contrast with the steady-state reaction, the single-turnover rate constant for dansyl-GCVLS alkylation was found to be the same for all analogues, despite the increased chemical reactivity of the benzyl analogues and the increased steric bulk of other analogues. However, the single-turnover rate constant for alkylation does depend on the Ca₁a₂X peptide sequence. These results suggest that the isoprenoid transition-state conformation is preferred over the inactive E·FPP·Ca₁a₂X ternary complex conformation. Furthermore, these data suggest that the farnesyl binding site in the exit groove may be significantly more selective for the farnesyl diphosphate substrate than the active site binding pocket and therefore might be a useful site for the design of novel inhibitors