<i>VHL</i> deletion induces accelerated hepatic glucose uptake and storage of PAS-positive substances.

<p>(<b>A</b>) H&E stained liver sections from <i>VHL-KO</i> mice showed characteristic appearances of hepatocytes (top panel); translucent hepatocytes with conserved cellular architectures. These translucent hepatocytes were strongly positive for PAS staining (middle panel) compared to those cells from WT Tamoxifen+and <i>VHL-KO</i> Tamoxifen −, which suggested marked glycogen accumulation. VHL immunostaining showed mosaic loss of VHL expression in the liver in <i>VHL-KO</i> mice (bottom panel). (<b>B</b>) In <i>VHL-KO</i> mice, 2-NBDG fluorescence intensity in the liver was considerably increased compared to control mice (<i>VHL-KO</i> Tamoxifen −).</p

IGF-IR expression and IGF-IR interaction with RACK1 are upregulated in <i>VHL-KO</i> livers.

<p>(<b>A</b>) <i>VHL-KO</i> livers resulted in downregulation of VHL expression (top panel). <i>VHL-KO</i> livers had significantly higher levels of IGF-IR compared to control livers. p-Akt expression was also enhanced in <i>VHL-KO</i> livers. No significant effects of <i>VHL</i> deletion were observed for the expression levels of RACK1 and IR. (<b>B</b>) IGF-IR immunoreactivity was increased in <i>VHL-KO</i> livers. (<b>C</b>) Immunoprecipitation (IP) of <i>VHL-KO</i> liver cell lysates using an anti-IGF-IR antibody were followed by immunoblotting with an anti-RACK1 antibody. In the <i>VHL-KO</i> liver lysates, the interaction between IGF-IR and RACK1 was markedly enhanced. (<b>D</b>) However, immunoprecipitated hepatocyte lysates from both <i>VHL-KO</i> and control mice using an anti-IR antibody did not contain RACK1.</p

NO production due to <i>VHL</i> deletion does not contribute to hypoglycemia in <i>VHL-KO</i> mice.

<p>There were no significant increases in blood glucose levels (BS) in both _L-NAME-treated <i>VHL-KO</i> mice (n = 5) and <i>eNOS</i>-deficient <i>VHL</i>-<i>KO</i> mice (n = 5). The hypoglycemic levels of both these mouse groups were not different from those of <i>VHL-KO</i> mice (n = 6–7) (_L-NAME-treated <i>VHL-KO</i> mice vs. <i>VHL-KO</i> mice, <i>p</i> = 0.210: N.S.; <i>eNOS</i>-deficient <i>VHL</i>-<i>KO</i> mice vs. <i>VHL-KO</i> mice, <i>p</i> = 0.523: N.S.). ΔBS was determined by subtracting BS before the tamoxifen injection from BS after the injection. Data were used to determine ΔBS values: ΔBS = BS_after – BS_before.</p

<i>VHL-KO</i> mice exhibit hypoglycemia despite normal glucose tolerance and intact pancreatic β cells.

<p>(<b>A</b>) <i>VHL-KO</i> mice had significant decreases in blood glucose levels (BS) after tamoxifen injection (4 mg/mouse; n = 10). (B) <i>VHL-KO</i> mice were treated with streptozotocin (STZ) before or after <i>VHL-</i>knockdown (n = 4 per group). Before tamoxifen injection, STZ treated mice (blue line) had significant increases in BS compared with their pre-STZ-blood glucose levels. After tamoxifen injection, their BS gradually decreased (day 0 vs. day 7, *<i>p = </i>0.0057; day 7 vs. day 17, **<i>p = </i>0.036). The mice treated with STZ after tamoxifen injection (red line) did not show any significant increases in blood glucose levels throughout the experiment. (<b>C</b>) <i>VHL-KO</i> mice (<i>VHL-KO</i> tamoxifen +) had glucose tolerance results comparable to that of other mice (WT tamoxifen +/− and <i>VHL-KO</i> tamoxifen −): WT tamoxifen −, n = 11; WT tamoxifen +, n = 10; <i>VHL-KO</i> tamoxifen −, n = 11; <i>VHL-KO</i> tamoxifen +, n = 7. (<b>D</b>) Histopathological images of pancreatic tissues showed that the immunoreactivity patterns for insulin and glucagon and morphological changes were not affected by <i>VHL</i> deletion with comparable maximum diameters of islets between control (<i>VHL-KO</i> tamoxifen −) and <i>VHL-KO</i> mice. (<b>E</b>) Glucose tolerance tests showed similar trends in insulin concentrations between <i>VHL-KO</i> and control mice (n = 5 per group, Time 0 min, <i>p</i> = 0.773:N.S.; 30 min, <i>p</i> = 0.294: N.S.; 60 min, <i>p</i>>0.9999: N.S.). (<b>F</b>) Basal glucagon levels were comparable between <i>VHL</i>-<i>KO</i> and control mice (<i>p</i> = 0.465: N.S.).</p

Synergistic effects of CCR4-NOT mutants on the <i>gtb1 mad2</i> double mutant.

<p>The KYPD+TBZ plate represents a permissive condition for the <i>gtb1 mad2</i> double mutant. Chromosome instability of the double mutant was enhanced on the YE plate, particularly at lower temperature. The indicated double and triple mutants were streaked on the plates and incubated at 30°C or 33°C for 3 d.</p

Synergistic effects of DNA repair-related mutants on the <i>gtb1 mad2</i> double mutant.

<p>See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002776#pgen-1002776-g002" target="_blank">Figure 2</a> legend for details. *Enlarged black and white image of this portion is shown on the right side.</p

Colony formation from spores produced in triploid fission yeast.

<p>(A) Heterogeneously sized colonies. Colonies were incubated on YE medium at 30°C for 5 d. (B) Representative morphologies of C1- (left) and C2- (right) type microcolonies. Colonies were incubated on YE medium at 30°C for 48 h.</p

Segregation analysis of chromosome 3 disome.

(a)<p>P219 (<i>h</i>⁻<i>leu1 ade6-M210/ade6-M216</i>) was crossed with a haploid strain that was <i>h</i>⁺ with <i>ade6-M216</i> (or <i>ade6-M210</i>) and one of the indicated alleles (except <i>not2</i>, which is mapped on chromosome 3). Strain 56-1 (<i>h</i>⁻<i>leu1 ade6-M210 not2::kan/ade6-M216 not2</i>⁺) was crossed with <i>h</i>⁺<i>ade6-M216 not2::kan</i>. Ade⁺ segregants were selected on an EMM2 plate at 30°C.</p>(b)<p>Ade⁺ colonies were randomly selected and tested for drug resistance. For the <i>not2</i> mutant, see (d).</p>(c)<p>All tested 12 Ade⁺ segregants had the “unstable Ade⁺” phenotype, indicating a chromosome 3 disome. Note, wild-type did not produce drug-resistant segregants.</p>(d)<p>For this cross, 24 of 26 Ade⁺ (disomic for chromosome 3) were G-418 resistant. Of these 24 segregants, 15 were homozygous for the <i>not2::kan</i> mutant, while 9 were heterozygous (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002776#s4" target="_blank">Materials and methods</a>).</p>(e)<p>G-418 resistant Ade⁺ segregants in this mutant were generally small, and upon restreaking on YE plates only stable Ade⁺ colonies (probably diploids) and Ade⁻ haploid colonies were produced. Chromosome 3 disome was hardly recovered thereafter.</p

Comparison of C1-type microcolony size in wild-type and <i>not3</i> mutant.

<p>Pictures are representative images of C1 type microcolonies from aneuploid spores. The difference was statistically significant for aneuploid spores (Mann-Whitney U-test; p = 4×10⁻⁷), while for the control haploid spores there was no significant difference (p = 0.41). The size estimation procedure is described in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002776#s4" target="_blank">Materials and Methods</a>.</p

Characterization of chromosome 3 disomes of the CCR4-NOT mutants.

<p>(A) Temperature sensitivity of chromosome 3 disomes. 5-fold serial dilutions of the disomes with the indicated mutations were spotted on a selective EMM medium (-adenine) at 30°C for 4 d or at 36°C for 5 d. For all strains, the target cell number for the last dilution was 25. All or most of the large colonies forming after incubation at 36°C were diploid. (B) Stability of chromosome 3 disomes. The indicated strains were spotted on EMM with a low concentration of adenine. Colonies were incubated at 30°C for 4 d. Red colonies represent haploids that lost one copy of chromosome 3. Enlarged images of the third spots (*) on the right show the presence of sectored colonies in the mutants.</p