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. 2005 Dec;25(24):10875–10894. doi: 10.1128/MCB.25.24.10875-10894.2005

FIG. 4.

FIG. 4.

Early-passage Foxm1−/− MEFs express high levels of senescence-associated β-galactosidase, p16INK4A, and p19ARF proteins. Passage 3 Foxm1−/− MEFs express high levels of senescence-associated β-galactosidase enzyme (A) and increased nuclear levels of CDKI p16INK4A (B) and p19ARF tumor suppressor (C) proteins. Passage 3 Foxm1+/+ (WT), Foxm1+/−, or Foxm1−/− MEFs were stained for senescence-associated β-galactosidase enzyme activity with X-Gal substrate (55, 70) or immunofluorescently stained with antibody specific to either p16INK4A or p19ARF protein. In panels B and C, the MEF nuclei were counterstained with DAPI and merged with the immunofluorescent staining. β-Galactosidase enzyme and immunofluorescent staining micrographs are taken at 200× and 400× magnification, respectively. (D) Foxm1−/− MEFs express significantly higher levels of p19ARF mRNA compared to control WT and Foxm1+/− MEFs. RNAs isolated from Foxm1−/−, Foxm1+/−, and WT MEFs were analyzed for p19ARF mRNA levels by quantitative real-time RT-PCR analysis using primers specific to the mouse p19ARF gene, and Foxm1−/− MEFs displayed a statistically significant increase in p19ARF mRNA expression compared to control MEFs (*, P = 0.02).