Figure 2.

Prep1 overexpression induces a strong apoptotic response in murine cells (A; left) Total extracts from murine F9 teratocarcinoma cells stably transfected with a Prep1-expressing or control vector (16) were resolved in an 8% SDS-PAGE and transferred to a PVDF membrane. The endogenous amount of Prep1 was checked by immunoblotting using a specific monoclonal antibody and normalized to β-actin content. (Middle) Cells were treated with etoposide as indicated or (Right) UV irradiated (UVC 254 nm; 60 J/m²). After 24 h of treatment or 24 h post-UV irradiation the number of AnnexinV-positive (apoptotic) cells was determined by FACS and plotted for overexpressing and control cells. Values are expressed as percentages of total events. C, untreated cells. (B) After 12 h of etoposide treatment (as indicated) or 12 h post-UV irradiation (UV C 254 nm; 60 J/m²), total cell extracts from Prep1-overexpressing or control F9 cells were resolved on 12% SDS-PAGE, transferred to a PVDF membrane and active caspases 9 levels were determined by immunoblotting and densitometric analysis, using tubulin for normalization. The densitometric analysis of the blot (Supplementary Figure S1A; one of three independent immunoblots is shown as representative experiment) is shown in the graph. (C) MEF from Prep1^+/+ and Prep1^i/i embryos infected with a retroviral vector (pBABE) either empty or containing the cDNA for human Prep1 were previously described [(5); Figure 6D]. Infected MEF were treated (or not) with etoposide (200 µM) for 24 h or irradiated with UV light (UV C 254 nm; 60 J/m² or 1000 J/m²). Crude extracts were prepared after 24 h of drug treatment or 12 (1000 J/m²) or 24 h (60 J/m²) post-UV irradiation, fractionated in 12% SDS-PAGE, transferred to a PVDF membrane and the level of active caspase-3 determined by densitometric analysis (shown in the graph) of immunoblots (Supplementary Figure S1B). The experiment was repeated three times and the results from a representative experiment are shown.