TGF-β–associated replicative senescence of caspase-3–deficient BMSSCs. (A) Replicative senescence assessed by β-gal staining. Representative pictures of β-gal–positive cells induced by TGF-β are shown in the upper panel (arrows). Original magnification, ×200. Replicative senescence was increased in Casp3^–/– BMSSCs compared with WT BMSSCs (lower panel; n = 6; *P < 0.001). TGF-β accelerated senescence in caspase-3–deficient BMSSCs ( P < 0.01; ^#P < 0.05) but not in WT mice. (B) Annexin V staining of BMSSCs. The number of annexin V–positive cells was found to be similar among each genotype under the regular culture condition (–, upper panels). However, TGF-β treatment reduced the number of annexin V–positive cells in Casp3^–/– BMSSCs (+, lower panels). Annexin V–positive cells appear in green. Original magnification, ×200. (C) Population doubling of BMSSCs. BMSSCs were continuously passaged at the same cell density after confluency. Fifty days after the culture was started, Casp3^–/– BMSSCs stopped proliferating and showed enlarged cell body and nuclei, although WT BMSSCs continued proliferating (upper panels). Caspase-3–deficient BMSSCs showed decreased population doubling (lower panel) (n = 6; *P < 0.001; P < 0.01). (D) Western blot analysis of BMSSCs. Casp3^–/– BMSSCs showed upregulated expression of TGF-βRI, Smad2, p21, and p53 along with downregulated expression of Cdc2 compared with WT. After TGF-β treatment, expression of TGF-βRI, Smad2, p-Smad2, p21, and p53 was further upregulated accompanying with downregulated expression of Cdk2 and Cdc2. Smad3 and TGF-βRII expressions were not changed in Casp3^–/– BMSSCs even with TGF-β treatment. Ten micrograms of protein was applied to each lane, and HSP90 was used as an additional control for protein loading.