Treatment with jasplakinolide increases expression of CCN growth factors in HeLa cells.

<p>A. Transcript levels for CCN growth factors increased after jasplakinolide treatment. HeLa cells were treated with 0.5 µM jasplakinolide for 10 min. and then cultured for indicated times periods. Representative data from two independent experiments is shown. Error bars represents mean ± SEM, n = 3. *<i>P</i><0.05, **<i>P</i><0.001 and ***<i>P</i><0.0005. B. CYR61 and CTGF protein levels after jasplakinolide treatment. HeLa cells were treated with 0.5 µM jasplakinolide for 10 min. and then cultured for indicated time periods. Protein levels for CYP61 and CTGF were measured from both medium and cells as described in Materials and Method. C. Quantitative estimation of CYR61 and CTGF antigens from both cells and medium fractions. For CYR61, the values of medium fractions are plotted on right axis.</p

Actin polymerization lead to YAP de-phosphorylation and nuclear localization.

<p>A. The levels of pYAP are decreased after treatment with jasplakinolide. HeLa cells were treated with 0.5 µM jasplakinolide as described in Materials and Methods. Representative data from three to five independent experiments is shown. B. Changes in the ratio of pYAP/tYAP after jasplakinolide treatment. Densitometric analysis of immunoblotting analyses was done using ImageJ. Error bars represents mean ± SEM, n = 3. *<i>P</i><0.05. C. Nuclear localization of YAP after Jasplakinolide treatment. HeLa cells were treated with 0.5 µM jasplakinolide and the cells were stained with the YAP antibody as described in Materials and Methods. D. Quantification of number of cells with nuclear YAP signals after jasplakinolide treatment at indicated time points.</p

Jasplakinolide induces actin polymerization.

<p>A. HeLa cells were treated with different doses of jasplakinolide (Jasp). G-actin (G) and F-actin (F) fractions were separated as described in Materials and Methods. Equal amounts of G-actin and F-actin fractions were loaded onto SDS-PAGE gels and immunoblotting was done using the actin antibody. Representative data from three independent experiments is shown. B. Changes in the ratio of G-actin and F-actin from immune-blots were quantified using ImageJ. Data are from three independent experiments.</p

Knockdown of YAP suppresses CCN growth factor expression after actin polymerization.

<p>CYR61 and CTGF protein levels after YAP knockdown. HeLa cells were transfected with control and YAP siRNAs for 48-PAGE analyses. Immunoblotting was performed using specific antibodies.</p

Generation of mutant mice with oocyte-specific deletion of <i>Pten</i>.

<p>A schematic representation of deletion of <i>Pten</i> exon 5 in oocytes of primary and further developed follicles by using the <i>Zp3</i> promoter-mediated Cre transgenic mice. The developmental stages at which the <i>Gdf-9</i> promoter and the <i>Zp3</i> promoter become active are indicated above the illustration of follicles in the figure.</p

Characterization of <i>Pten</i> deletion by western blot and PCR.

<p>(A) Oocytes were prepared and lysed for western blot as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006186#s4" target="_blank"><i>Materials and Methods</i></a>. PTEN expression was found to be completely absent in <i>Pten^loxP/loxP</i>; <i>Zp3-Cre+</i> oocytes. For each lane, 150 oocytes were used. β-actin was used as internal control. (B) PCR analysis showing the complete deletion of <i>Pten</i> exon 5 (<i>Pten</i> Δ5) in one allele of the genomic DNA of pups from <i>Pten^loxP/loxP</i>; <i>Zp3-Cre+</i> females.</p

Enhanced Akt signaling in oocytes of <i>Pten^loxP/loxP</i>; <i>Zp3-Cre+</i> mice.

<p>Oocytes were prepared from ovaries of 3–4 week old mice that were treated with PMSG, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006186#s4" target="_blank"><i>Materials and Methods</i></a>. Signaling studies in <i>Pten^loxP/loxP</i>; <i>Zp3-Cre+</i> oocytes showed elevated levels of p-Akt (Ser473), p-Akt (Thr308), and p-Tsc2 (Thr1462) as compared to <i>Pten^loxP/loxP</i> oocytes. Levels of total Akt, Tsc2, and β-actin were used as internal controls. 100–150 oocytes were used for each lane. All experiments were repeated at least three times and representative results are shown.</p

Normal follicular development in <i>Pten^loxP/loxP</i>; <i>Zp3-Cre+</i> mice.

<p>Morphological analysis of ovaries from 13- and 23-day-old, and 16-week-old <i>Pten^loxP/loxP</i>; <i>Zp3-Cre+</i> mice, <i>Pten^loxP/loxP</i>; <i>GCre+</i> mice, and control <i>Pten^loxP/loxP</i> mice. Ovaries were embedded in paraffin and sections of 8-µm thickness were prepared and stained with hematoxylin. Note the overactivation of primordial follicles in <i>Pten^loxP/loxP</i>; <i>GCre+</i> ovaries (C, F, and I, arrows) and the normal follicular development and CL in <i>Pten^loxP/loxP</i>; <i>Zp3-Cre+</i> ovaries (B, E, H and K), which is comparable to the control <i>Pten^loxP/loxP</i> ovaries (A, D, G, and J). CL, corpora lutea.</p