Fig. 5.

FOXM1/Rb-dependent expression of β-galactosidase in myeloma cells. a Co-immunoprecipitation (Co-IP) result indicating physical interaction of Rb and FOXM1 in CAG and XG1 myeloma cells. Immunoblots using a specific IgG antibody (Ab) to FOXM1 (following IP with Ab to Rb) or Rb (following IP with Ab to FOXM1) are shown on top of each other. The IgG isotype control is labeled “IgG.” Whole cell lysates not subjected to Co-IP (“Input”) were included as an additional control. b Shown on top is a Western blot of total Rb and its activated, phosphorylated form (pRb) in FOXM1^Hi and FOXM1^N CAG and XG1 myeloma cells. A similar blot containing samples of FOXM1^KD and FOXM1^N H929 and ARP1 myeloma cells is presented at bottom. The abundance of Rb and pRb, relative to β-actin, was determined using densitometry. The ratios are indicated below the blots. c The upper panel illustrates the elevation of β-galactosidase (β-gal) activity, a classic phenotype of cellular senescence, in FOXM1^KD H929 cells (bottom) relative to FOXM1^N controls (top). Cells were not treated with drug. This result was confirmed using paired FOXM1^KD / FOXM1^N ARP1 samples (not shown). Depicted in the lower panel is the increased proportion of β-gal⁺ XG1 cells following treatment with Dox. FOXM1^Hi cells exhibited a lesser increase than FOXM1^N cells (not shown). Cells were evaluated using an Olympus BX-51 Light Microscope equipped with an UPLSAPO objective (Olympus) of 40x magnification and 0.95 numerical aperture. The imaging medium was air. The light temperature of the microscope bulb varied between 3000 and 3400 K. Images were acquired with the help of a DP2 digital camera (Olympus) and DP2-BSW imaging software (Olympus), saved as TIF data files, and enhanced—with respect to brightness, contrast, and color balance—using the Adobe Photoshop CS2 Version 9.0.2 software (Adobe Systems, Inc)