Inhibition of cellular senescence by FGF-2 in MSCs in vitro. (A) FGFR1/2⁺ MSCs displayed a characteristic senescent phenotype as early as passage 2 when expanded without FGF-2, including up-regulation of senescence-associated β-Gal (X-Gal staining), increased cell diameter (see also supplemental Figure 4A), morphological changes, disruption of the actin cytoskeleton (phalloidin staining), and growth arrest (decreased Ki67 staining). Scale bars indicate 50 μm for X-Gal and 100 μm for phalloidin and Ki67. (B) Growth curves and Ki67 expression of MSCs culture expanded with or without FGF-2 stimulation for 3 passages, at which point the culture conditions were switched (arrow and double arrow). See “Results” for the numbers of cells counted. (C) Induction of cellular senescence in FGF-2–stimulated MSCs at passage 3 by irradiation at 10 Gy shown by growth arrest (lower Ki67; top panel) and adoption of a senescent phenotype (bottom panel) indicate that MSCs were not immortalized. Scale bars indicate 50 μm for X-Gal and 100μm for phalloidin and Ki67. ND indicates not detected. (D) FGFR1/2⁺ MSCs lost expression of FGFRs when induced into senescence, as shown by decreased p-MDM2(Ser186) by FGF-2 starvation for 48 hours. Scale bar indicates 25 μm. (E) Western blot and densitometry showing that FGF-2 stimulation of FGFR1/2⁺ MSCs resulted in hyperphosphorylation of MDM-2 on Ser186, which could be inhibited by the PI3K/AKT inhibitor LY294002. This experiment was performed in triplicate. (F) Immunofluorescence and confocal analysis confirmed that FGF-2 stimulation of FGFR1/2⁺ MSCs resulted in hyperphosphorylation and increased nuclear localization of MDM2 in a PI3K/AKT–dependent manner (see also supplemental Figure 4C). Scale bar indicates 50 μm.