Inactivation of mTORC1 upregulates mTORC2 and FoxO3a.

<p>(<b>A</b>) Western-blot analyses to detect the indicated signaling molecules, using total cell lysates from WT, p18Rev, p18NΔ5-CAAX, and p18KO cells. (<b>B</b>) Expression levels of mRNAs encoding Rictor, FoxO3a and FoxO1 in p18KO and WT cells were determined by quantitative real-time PCR. Means±SD were obtained from three independent assays. ***<i>P</i><0.001 and **<i>P</i><0.01 (Student's <i>t</i>-test).</p

Nuclear localization of FoxO3a suppresses cell proliferation.

<p>(<b>A</b>) Schematic structures of WT FoxO3a, FoxO3a S314A mutant (S314A), and FoxO3a T32/S252/S314A triple mutant (3A). (<b>B</b>) Immunofluorescence analyses to determine localizations of HA-tagged WT FoxO3a, S314A, and 3A cells to determine FoxO3a (HA) localization. Merged images with PI staining are shown. Scale bars: 10 µm. (<b>C</b>) Western-blot analyses for the indicated molecules in p18KO cells, mock-treated WT cells, and WT cells expressing FoxO3a, S314A, or 3A. (<b>D</b>) Cell proliferation of WT cells and WT cells expressing WT FoxO3a, S314A, or 3A was analyzed by the WST-1 growth assay over the indicated time course. Means±SD were obtained from three independent assays. **<i>P</i><0.01 and *<i>P</i><0.05 (Student's <i>t</i>-test). (<b>E</b>) Expression level of p27^Kip1 mRNA in cells used in (C) was determined by quantitative real-time PCR. Means±SD were obtained from three independent assays. **<i>P</i><0.01, (Student's <i>t</i>-test).</p

Inactivation of mTORC1 induces nuclear accumulation of FoxO3a.

<p>(<b>A</b>) Western-blot analyses to detect the indicated signaling molecules, using total cell lysates from WT, p18Rev, p18NΔ5-CAAX, and p18KO cells. The panels for FoxO3a and FoxO1 are the same panels used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088891#pone-0088891-g003" target="_blank">Figure 3A</a>. Mobility shifts of FoxO proteins are shown by bars. Locations of bands corresponding to long and short forms of SGK1 are indicated by arrows. (<b>B</b>) Schematic structure of FoxO3a. Sites of phosphorylation by the indicated kinases are shown. FH: Fork head domain. (<b>C</b>) HA-tagged FoxO3a constructs with point mutations at the indicated amino-acid positions were transiently expressed in WT cells, and their mobility shifts (indicated by bars) were analyzed by Western blotting. (<b>D</b>) Expression levels of mRNA encoding SGK1 in p18KO and WT cells were determined by quantitative real-time PCR. Means±SD were obtained from three independent assays. n.s.; not significant (Student's <i>t</i>-test). (<b>E</b>) Whole-cell lysates from p18KO and WT cells were separated into cytoplasmic and nuclear fractions, and the indicated proteins were detected by Western-blot analyses. β-tubulin and histone H3 represent control proteins for the cytoplasmic and nuclear fractions, respectively. (<b>F</b>) Immunofluorescence analysis for FoxO3a in p18KO and WT cells. Nuclei were visualized with propidium iodide (PI). Merged images are also shown. Scale bars: 10 µm. Upper graphs show the intensity of signals for FoxO3a obtained by scanning along the yellow dot lines. Right graph shows the statistic data of nuclear intensity of FoxO3a signals in p18KO and WT cells. Means±SD were obtained from 15 cells. ***<i>P</i><0.001 (Student's <i>t</i>-test).</p

Expression of FoxO3a is regulated by DNA methylation.

<p>(<b>A</b>) WT cells were treated with trichostatin A (TSA), 5-aza-deoxycytidine (5-Aza), or a combination of TSA and 5-Aza for 48 h, and FoxO3a mRNA levels were determined by quantitative real-time PCR. Fold change in mRNA levels was calculated after normalization against β-tubulin mRNA (internal control). **<i>P</i><0.01 and *<i>P</i><0.05 (Student's <i>t</i>-test). (<b>B</b>) DNA methylation status of <i>FoxO3</i> CpG islands in p18KO, p18Rev, and WT cells was analyzed by bisulfite sequencing. The methylation status in the gray boxed region was determined for ten clones obtained from each cell line. Individual clones are indicated by lines with circles. Methylated and non-methylated cytosines are indicated by closed and open circles, respectively. (<b>C</b>) A series of fragments from the intronic region of the mouse FoxO3a gene were subcloned upstream of a luciferase reporter gene. Each construct was transfected into MEFs, and luciferase activity was measured and normalized against pRLTK activity. Normalized luciferase activity is expressed as means±SD (<i>n</i> = 3). ***<i>P</i><0.001 and **<i>P</i><0.01 (Student's <i>t</i>-test).</p

Inactivation of lysosomal mTORC1 causes growth arrest.

<p>(<b>A</b>) Cell proliferation of WT, p18Rev, p18NΔ5-CAAX, and p18KO cells analyzed by the WST-1 growth assay over the indicated time course. (<b>B</b>) WT and p18KO cells were analyzed for DNA content by flow cytometry. M1-4 indicates the following areas: M1; sub-G1, M2; G1, M3; S, M4; G2/M. (<b>C</b>) Quantitative data from FACS analysis. Means±SD were obtained from three independent assays. ***<i>P</i><0.001, **<i>P</i><0.01, and *<i>P</i><0.05 (Student's <i>t</i>-test).</p

Localization of Dnmt1 at the replication region in ESC.

<p>ESC (<i>Dnmt1</i>-F/F), and ESC expressing no Dnmt1 (<i>Dnmt1</i>-F/F +OHT), or ectopic full-length Dnmt1 (FL), oocyte-type Dnmt1 (oocyte), Dnmt1(291–1620) (291), Dnmt1(602–1620) (602), or full-length Dnmt1 with the mutation of H168R (PBDm) treated with OHT were labeled with EdU, and detected the EdU (green) and Dnmt1 (red), and the images were merged. White bars indicate 5 μm.</p

Global DNA methylation of <i>Dnmt1</i> and <i>Uhrf1</i> double-knockout, or TKO ESC expressing ectopic Dnmt1.

<p>(<b>A</b>) Genome DNA prepared from <i>Dnmt1</i> and <i>Uhrf1</i> conditional double-knockout ESC expressing either no ectopic Dnmt1 (Double F/F), or full-length Dnmt1 (FL), oocyte-type Dnmt1 (oocyte), Dnmt1(291–1620) (291), or Dnmt1(602–1620) clone #1 (602#1) treated without (-) or with OHT (+) for ten days was immuno-blotted with anti-methylated cytosine antibody (5mC, upper panel) or stained with methylene blue (DNA, lower panel). The amounts of DNA are shown at the left. (<b>B</b>) The genome DNA in panel A and Dnmt1(602–1620) clone #5 (602#5) were fragmented by sonication, and then precipitated with MBD1. The precipitated DNA was quantitated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137509#pone.0137509.g001" target="_blank">Fig 1D</a>, averages ± S. D. being shown. ***, p<0.001. (<b>C</b>) Genome DNA prepared from <i>Dnmt1</i> conditional-knockout ESC expressing either no ectopic Dnmt1 (<i>Dnmt1</i>-F/F), Dnmt1(602–1620), Dnmt1(602–1620) with C1229S clones#1 (602-C1229S #1), or clone #11 (602-C1229S #11) treated without (0) or with OHT for four days (4) or ten days (10) was immuno-blotted with anti-methylated cytosine antibody (5mC, upper panel), or stained with methylene blue (DNA, lower panel). The amounts of DNA are shown at the left. (<b>D</b>) Genome DNA prepared from <i>Dnmt1</i> conditional-knockout ESC expressing either no ectopic Dnmt1 (<i>Dnmt1</i>-F/F), or Dnmt1(602–1620) with C1229S clone #1 (602-C1229S #1) or clone #11 (602-C1229S #11) treated without (blue) or with OHT for four days (red) or ten days (green) was fragmented by sonication, and then precipitated with MBD1. The precipitated DNA was quantitated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137509#pone.0137509.g001" target="_blank">Fig 1D</a>, averages ± S. D. being shown. (<b>E</b>) Genome DNA prepared from parent ESC (J1), TKO ESC (TKO), TKO ESC expressing full-length Dnmt1 (FL), Dnmt1(291–1620) (291), Dnmt1(602–1620) (602), full-length Dnmt1 and Dnmt3a2-TAP (FL+Dnmt3a2), or (602–1620) and Dnmt3a2-TAP (602+Dnmt3a2) was immuno-blotted with anti-methylated cytosine antibody (5mC, upper panel), or stained with methylene blue (DNA, lower panel). The amounts of DNA are shown at the left. (<b>F</b>) Genome DNA prepared from J1, TKO ESC (TKO), TKO ESC expressing no ectopic Dnmt1 (TKO), or full-length Dnmt1 (FL), Dnmt1(291–1620) (291), Dnmt1(602–1620) (602), full-length Dnmt1 and Dnmt3a2-TAP (FL+Dnmt3a2), or Dnmt1(602–1620) and Dnmt3a2-TAP (602+Dnmt3a2) was fragmented by sonication, precipitated with MBD1, and then analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137509#pone.0137509.g001" target="_blank">Fig 1D</a>. The value obtained for TKO was considered as no DNA methylation and subtracted from each measurement. Averages ± S. D. are shown.</p

DNA methylation of ESC expressing full-length Dnmt1 with a mutation in the PBD.

<p>(<b>A</b>) DNA methylation activity of the recombinant full-length Dnmt1 (FL) and that with the H168R mutation (PBDm) was determined. The specific activities (mol/h/mol Dnmt1) towards hemi-methylated (hm) and un-methylated (um) DNA are shown as averages ± S. D. (n = 3). (<b>B</b>) Genome DNA prepared from the cells before and after deletion of the endogenous <i>Dnmt1</i> gene with OHT for ten days was immuno-blotted with anti-methylated cytosine antibody (5mC, upper panel) or stained with methylene blue (DNA, lower panel). The amounts of DNA are shown at the left. (<b>C</b>) Genome DNA prepared as in B was sonicated and precipitated with MBD1, and then quantitated, averages ± S. D. (n = 3) being shown as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137509#pone.0137509.g001" target="_blank">Fig 1D</a>. The values for the parent genome before and after the OHT-treatment were taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137509#pone.0137509.g001" target="_blank">Fig 1D</a>. ***, P<0.001. (<b>D</b>) Genome DNA prepared as in B was analyzed as to the methylation state at the <i>IAP</i> and DMR of three imprinted genes, <i>Rasgrf1</i>, <i>Peg3</i>, and <i>Kcnq1ot1/Lit</i>, in PBDm cells as in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137509#pone.0137509.g002" target="_blank">2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137509#pone.0137509.g004" target="_blank">4</a>. The percentages of methylation are indicated at the top.</p

DNA methylation analyses of the <i>gag</i> of <i>IAP</i>.

<p>The methylation state of the genome DNA prepared from ESC expressing no Dnmt1 (<i>Dnmt1</i> F/F), or full-length Dnmt1 (FL), oocyte-type Dnmt1 (oocyte), Dnmt1(291–1620) (291), or Dnmt1(602–1620) (602) four or ten days after addition of OHT. The methylation states of the <i>gag</i> of <i>IAP</i> are shown with that of the parent cells before (<i>Dnmt1</i>-F/F) and after the OHT treatment (<i>Dnmt1</i>-F/F+OHT). Each horizontal line indicates the CpGs in one analyzed clone. Each circle indicates one CpG site, methylated (filled circles) or un-methylated (open circles). The percentages of methylation are indicated at the top.</p

DNA methylation analyses of the DMR of three imprinted genes.

<p>The methylation states of the DMRs of <i>Rasgrf1</i>, <i>Peg3</i>, and <i>Kcnq1ot1/Lit</i> are shown with that of the parent cells before and after ten days OHT treatment (+OHT 10d). Each horizontal line indicates the CpGs in one analyzed clone. Each circle indicates one CpG site, methylated (filled circles) or un-methylated (open circles). The percentages of methylation are indicated at the top.</p