Figure 6

Inhibition of PI3K or MEK but not mTOR disrupts IGFIR acinar development. (a) Confocal sections of 20-day control (pCLXSN, panels a, d, g) and IGFIR (panels b, c, e, f, h, i) acinar structures grown in the presence of the MEK inhibitor, UO126 (10 μm; panels a-c), the PI3K inhibitor LY294002 (50 μm; panels d-f) and the mTOR inhibitor rapamycin (40 nM; panels g-i). Fixed structures were labeled with the nuclear counterstain TO-PRO-3 (blue) and with antibodies to Ki-67 (green; panels a, b, d, e, g, h) or cleaved caspase-3 (green; panels c, f, i). Scale bars = 50 μm. The arrow in panel b indicates Ki-67-labeled cells. (b) Whole-cell lysates of 16d IGFIR acinar structures were isolated after overnight serum starvation followed by 30 minute pretreatment with DMSO (vehicle) or 40 nM rapamycin and subsequent 30-minute incubation with 10 μg/ml insulin. Lysates were immunoprecipitated with antibodies to p70S6K, separated by 12.5% SDS-PAGE, transferred to PVDF and immunoblotted with antibodies to p70S6K or phosphospecific antibodies to Thr389 of p70S6K as indicated.