FIGURE 5:

The absence of ZO-2 stimulated the cross-talk between Hippo and mTOR signaling pathways. (A) ZO-2 KD MDCK cells displayed a lower amount of PTEN than parental cells and instead showed increased phosphorylation of Akt at S473 and T308. Left, representative Western blots of three independent experiments done with a specific antibody against PTEN, pAkt-T308, and pAkt-S473. Right, densitometric analysis. Statistical analysis was done on three independent experiments with Student’s t test; *p < 0.05. (B) The cross-talk between YAP and PTEN, mediated by a small RNA, is critical for the increase in cell size observed in MDCK ZO-2 KD cells. Top, in ZO-2 KD cells, the expression of PTEN increased only after transfection with siRNA against Dicer and not with the sole transfection of PTEN. Statistical analysis done on three independent experiments with a one-way ANOVA followed by Bonferroni’s multiple comparison test; **p < 0.01, ***p < 0.001. Bottom, cell size measured by flow cytometry. Treatment of ZO-2–depleted cells with siRNA against Dicer decreased cell size to a value similar to that of parental cells (left), whereas no effect was observed after PTEN transfection (right). (C) The amount of PIP₃ present in ZO-2 KD cells is higher than in parental cells. PIP₃ was measured in parental and ZO-2 KD cells using a competitive enzyme-linked immunosorbent assay. Results from three independent experiments. Statistical analysis with Student’s t test; **p < 0.01