List of proteins that selectively interact with unphosphorylatable form of rpS6.

<p>Whole cell extract from HEK293 cells infected with pS6^[5S]-GFP, pS6^[5A]-GFP, pS6^[5D]-GFP or pEGFP-N1, was subjected to GFP pull-down, and the bound proteins were size fractionated by SDS-polyacrlamide gel electrophoresis. Mass spectrometric analysis of proteins in each lane was performed as described in “material and Methods” and proteins, selectively bound to pS6^[5A]-GFP in two independent experiments, are presented (numbers separated by slash [/] represent results obtained in each of the two individual analyses).</p

Constitutive expression of Akt induces polyploidy regardless of the rpS6 phosphorylation status.

<p>A. rpS6 deficiency does not affect ploidy in β-cells. Islets were isolated from wild-type (WT) and 2 month-old Akt^tg mice. Dissociated islets were fixed, permeabilized, immunostained for Insulin, HA, Ki67 and Hoechst before FACS analysis. The DNA content of 5,000 to 10,000 quiescent β-cells (insulin-positive, Ki67-negative) was measured by FACS analysis. B. 5,000 quiescent (Ki67-negative) β-cells (insulin-positive) that express myr-Akt (HA-positive) are polyploidy, whereas their HA-negative counterpart shows mostly a diploid profile as wild-type β-cells. C. rpS6 deficiency does not affect the ploidy profile of myr-Akt expressing β-cells. Islets were isolated from 2 month-old mice (three wild-type (WT), two Akt^tg and three rpS6^P-/-;Akt^tg mice) and 5,000 to 10,000 HA-positive β-cells processed as in (A) for FACS analysis.</p

rpS6 phosphorylation deficiency renders Ras^G12D MEFs more sensitive to both genotoxic and proteotoxic stresses.

<p>(A) Immortalized WT, rpS6^P-/-, Ras^G12D and rpS6^P-/-;Ras^G12D MEFs were harvested and their cytoplasmic proteins were subjected to Western blot analysis with the indicated antibodies. (B) Ras^G12D and rpS6^P-/-;Ras^G12D MEFs were seeded in 96-well plates at a density of 4 x10³ per well. Proliferation was monitored by measuring the A₆₅₀ of the methylene-blue dye extracted from stained cells [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149995#pone.0149995.ref032" target="_blank">32</a>]. Absorbance measured 24 h after platting, was set arbitrarily at 1 and absorbance measured at later time points (average ± SEM [n = 6]) for each time point) was normalized to that value. t_d, population-doubling time. (C) Immortalized Ras^G12D and rpS6^P-/-;Ras^G12D MEFs were incubated with the indicated concentrations of etoposide for 24 h, harvested and their cytoplasmic proteins were subjected to Western blot analysis with the indicated antibodies. (D) Immortalized Ras^G12D and rpS6^P-/-;Ras^G12D MEFs were incubated with the indicated concentrations of MG132 for 24 h, harvested and their cytoplasmic proteins were subjected to Western blot analysis with the indicated antibodies.</p

rpS6 phosphorylation deficiency increases proliferation and decreases translational fidelity in fibroblasts.

<p>(A) Immortalized WT, rpS6^P-/-, Akt^tg and rpS6^P-/-;Akt^tg MEFs were harvested and their cytoplasmic proteins were subjected to Western blot analysis with the indicated antibodies. (B) MEFs described in (A) were seeded in 96-well plates at a density of 4 x10³ per well. The proliferation of MEFs described in (A) was monitored as described in (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149995#pone.0149995.g004" target="_blank">Fig 4B</a>). t_d, population doubling time. (C) WT and rpS6^P-/- immortalized MEFs were incubated for 1 h with 1 mM puromycin, harvested and their cytoplasmic proteins were subjected to Western blot analysis with the indicated antibodies. (D) Quantification of signals obtained in 6 experiments similar to that described in (C). (E) WT, rpS6^P-/-, Akt^tg, and rpS6^P-/-;Akt^tg MEFs were cotransfected with Renilla luciferase expression vector and any one of the following firefly luciferase expression vectors: Fluc(WT), Fluc(Stop) and Fluc(R218S). The transfection efficiency of any of the Fluc constructs was normalized to that of the Renilla luciferase. The relative Fluc activity of each mutant was normalized to Fluc (WT). The result obtained for each mutant in WT MEFs (n = 24) was arbitrarily set at 1 and the relative luciferase activity obtained for each Fluc mutant in rpS6^P-/- MEFs (n = 24) was normalized to that value. Similarly, results obtained for each mutant in Akt^tg MEFs (n = 12) were arbitrarily set at 1 and the relative luciferase activity obtained for each Fluc mutant in rpS6^P-/-;Akt^tg MEFs (n = 12) was normalized to that value.</p

Rescue of rpS6 phosphorylation deficient mice from myr-Akt1-induced insulinoma is associated with decreased cell proliferation and increased p27 nuclear localization.

<p>(A) Pancreatic sections of islets from 12-month-old WT and Akt^tg mice were stained for phosphorylated rpS6 and (green) and for β-cells nuclei by PDX1 antibodies (red). (B) Pancreatic sections from WT, rpS6^P-/-, Akt^tg and rpS6^P-/-;Akt^tg 12-month-old mice were stained by haematoxylin-eosin. All images presented at the same magnification (see size bar [100 μm] in WT). Arrows point to islets and the black line (lower left panel) marks the right margin of the insulinoma. (C) The number of mice with readily detectable insulinoma (dashed-line box) is presented within the total number of examined Akt^tg and rpS6^P-/-;Akt^tg mice (open box). (D) Pancreatic sections from1, 2 and 10 to 15-mo-old mice of the indicated genotypes were stained as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149995#pone.0149995.g001" target="_blank">Fig 1D</a> (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149995#pone.0149995.s003" target="_blank">S3 Fig</a> for representative islets from all 4 genotypes). The proliferation is presented as % Ki67 positive within insulin-positive cells. Values are presented as a mean ± SEM of counts derived from 20 to 50 islets identified in 3 to 4 mice of each genotype at each time point. (E) Pancreatic sections from 10 to 15-mo-old mice were stained for p27 and for insulin (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149995#pone.0149995.s004" target="_blank">S4 Fig</a> for representative islets from all 4 genotypes). Values are presented as a mean ± SEM of counts derived from 12 to 42 islets identified in 2 to 4 mice of each genotype. (F) The size of β-cells in 10 to 15-mo-old mice was analyzed and calculated as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149995#pone.0149995.g001" target="_blank">Fig 1C</a>. Values are presented as a mean ± SEM of counts derived from 215 to 1252 squares (2,500- μm²) within 21 to 31 islets identified in 2 to 7 mice of each genotype.</p

rpS6 phosphorylation deficiency renders WT or Akt^tg MEFs more resistant to a proteotoxic and genotoxic stress.

<p>(A) Immortalized WT, rpS6^P-/-, Akt^tg, and rpS6^P-/-;Akt^tg MEFs were incubated with the indicated concentrations of MG132 for 16 h, harvested and their cytoplasmic proteins were subjected to Western blot analysis with the indicated antibodies. (B) Viability of cells described in (A) was assessed by the Trypan blue exclusion assay, as described in Material and Methods. The percentage of viable cells (average of six wells for each time point) is presented as mean ± SEM (the size of the symbol for most measurements was larger than the respective SEM). (C) Primary WT and rpS6^P-/- MEFs (passage 3) were analyzed as described in (A). (D) Immortalized WT, rpS6^P-/-, Akt^tg, and rpS6^P-/-Akt^tg MEFs were incubated with the indicated concentrations of etoposide for 24 h, harvested and their cytoplasmic proteins were subjected to Western blot analysis with the indicated antibodies.</p

The phosphorylation status of rpS6 plays no role in autophagy.

<p>(A) Immortalized Akt^tg and rpS6^P-/-;Akt^tg, MEFs were either untreated, incubated with 20 nM rapamycin, 50 μM chloroquine or both for 16 h. Cells were harvested and their cytoplasmic proteins were subjected to Western blot analysis with the indicated antibodies. (B) The signals of LC3-II, obtained in Western blot analyses of two independent experiments, as described in (A), were quantified and normalized to that tubulin. The fold change in the expression of LC3-II upon addition of rapamycin, chloroquine or both, was related to that of untreated cells, which was arbitrarily set at one.</p