Clusterin restrains pituitary cell proliferation by inducing Cdk inhibitors.

<p>Western blot analysis of Cdk inhibitors and proliferation markers <b>A</b>) in LβT2 cells, <b>B</b>) in αT3 cells 48 h after transfection with m<i>Clu</i>; <b>C</b>) Confocal images of immunofluoprescence of histone H3 methylation on lysine 9 (H3-K9M) (red) in vector and <i>Clu</i>-expressing αT3 cells 48 hours after transfection; <b>D</b>) Quantification of positive H3-K9M foci. Cells were fixed, stained with H3-K9M antibody, and one thousand cells/field counted in three randomly chosen visual fields; <b>E</b>) Percentage of BrdU positive cells 48 h after transfection with m<i>Clu</i>. Triplicate samples were pulsed with BrdU for 30 min and analyzed by flow cytometry, *, p<0.05; <b>F</b>) αT3 cells stably overexpressing <i>mClu</i> or vector were synchronized in 0.1% fetal bovine serum for 18 hours, and then cultured in 10% fetal bovine serum. At the indicated times, duplicate samples were pulsed with BrdU for 30 min, analyzed by flow cytometry, and cells in S-phase identified by staining with BrdU antibodies.