Loss of PKC-alpha-mediated Rac activation in the absence of Rictor.

<p><b>A-D.</b><i>Rictor</i>^<i>FL/FL</i> PMECs and organoids were infected with the indicated adenoviruses. <b>A-B.</b> Western analysis of PMEC lysates. Quantitation was performed using Image J software and numbers represent P-PKC-alpha bands normalized to total PKC-alpha levels. <b>C</b>. Organoids photographed after 10 days in Matrigel culture. Average number of branches/organoid ±S.D. indicated below panels. N = 3 independent organoid isolates/condition analyzed in triplicate, Student’s T-test. <b>D.</b> Average colony size of organoids measured in pixel area ± S.D. N = 3 independent organoid isolates/condition, analyzed in triplicate, Student’s T-test<b>. E.</b> Western analysis of MCF10A parental and Rictor^ZFN lysates. Quantitation was performed using Image J software and numbers represent P-PKC-alpha band normalized to actin for each sample. <b>F-I.</b> MCF10A parental and Rictor^ZFN cells were infected with Ad.RFP or Ad.PKC-alpha. <b>F.</b> Western analysis of MCF10A parental and Rictor^ZFN lysates, 48 hours post-infection. Quantitation was performed using Image J software and numbers represent P-PKC-alpha band normalized to actin for each sample. <b>G.</b> GST-Pak-PBD effector pull-downs followed by western analysis for Rac performed 48 hours post-infection. Quantitation was performed using Image J software. Numbers in upper panel represent Rac-GTP levels in MCF10A-Rictor^ZFN compared to MCF10A parental control. Lower numbers represent Rac-GTP levels normalized to total Rac for each sample. <b>H.</b> Cells were assessed for invasion through Matrigel-coated transwell filters. Cells were stained with crystal violet after 24 hours, and then imaged. Number of cells invading was quantitated in Image J. Values shown represent the average ± S.D., Student’s T-test. N = 2 independent experiments, analyzed in triplicate. <b>I.</b> Cells were assessed for Annexin V-FITC. Number of Annexin V-FITC+ per total number of cells was quantitated in Image J, Student’s T-test. N = 2 independent experiments, analyzed in triplicate. <b>J.</b> MCF10A parental cells were assessed for invasion through Matrigel-coated transwell filters in the presence of PKC-alpha inhibitor GO6976. Cells were stained with crystal violet after 24 hours, and then imaged. Number of cells invading was quantitated in Image J. Values shown represent the average ± S.D., Student’s T-test. N = 2 independent experiments, analyzed in quadruplicate.