Figure 6

Lamin A/C suppression in primary ovarian epithelial cells results in growth retardation. Primary HOSE cells were transfected with control or shRNA to suppress lamin A/C expression. The cells were analyzed for DNA content and bromodeoxyuridine (BrdU) incorporation simultaneously by flow cytometry. (A) Primary HOSE cells were analyzed by flow cytometry for DNA content following treatment with control or lamin A/C shRNA for three days. Lamin A/C-suppressed cells have > 2n DNA in 75% of cells, compared to 23% of control cells. (B) The same cell populations were incubated with BrdU for one hour in culture, and then fixed and stained with FITC-anti-BrdU prior to flow cytometry to determine BrdU incorporation as an indicator of proliferation. (C) The control cells show a high percentage of mitotic nuclei as shown by immunofluorescence microscopy of beta-tubulin staining of the mitotic spindles. (D) In comparison, beta-tubulin-immunostained mitotic spindles are rare in lamin A/C-suppressed cells. (E) The HOSE cells were treated with siRNA-p53 and/or siRNA-lamin A/C for three days and then analyzed for p53 and p21 by Western blot.