Figure 1.

Bcl2L12 inhibits passage-induced senescence and p53-dependent apoptosis. (A) MEF immortalization assays. pBabe and Bcl2L12-infected (wild-type) MEF cultures were serially passaged and cell numbers were determined. Shown is one representative growth curve documenting unabated growth of Bcl2L12-infected MEF cultures at >30 passages. (B) Staining for and quantification of senescence-associated β-galactosidase (SA-β-Gal) in representative pBabe and Bcl2L12 MEF lines. Histogram shows quantification of β-Gal-positive cells in pBabe versus Bcl2L12 transfectants; three high-power fields (HPFs) were counted. Data are represented as mean ± SD. (C) Molecular analysis of p53–Rb signaling components. pBabe and Bcl2L12 MEFs of indicated passages were treated with Dox (0.4 μg/mL) for 16 h to assess p53, p19^Arf, and Rb protein levels. p53 proteins levels were quantified densitometrically. (D) Bcl2L12 potently blocked Dox-induced effector caspase activation. Cultures were treated with Dox (5 μg/mL) and STS (0.5 μM) for the indicated periods of time, and post-mitochondrial caspase-3 and caspase-7 activation was followed by Western blot analysis. Of note, p53 protein was induced in pBabe (pB), Bcl2L12 (L12^V5), and EGFRvIII (vIII) astrocytic cultures upon Dox, but not STS, treatment. The migration positions of cleaved caspase-3 and caspase-7 species ([LS] large subunits; [LS+N] large subunits with N-peptide) are indicated. Hsp70 is shown as a loading control.