Figure 1.

Restoration of p53 expression in the human Saos-2 osteosarcoma cell line upregulates the expression of Notch1. (A) Subconfluent cultures of Saos^Arg72 cells were either incubated at 39°C (lane 1), incubated at 32°C for 16 hours (lane 2), or incubated at 32°C for 24 hours (lane 3). After incubation, total cell lysates were analyzed by immunoblotting using antibodies specific to the indicated proteins. (B) Saos-2 cells were nucleofected with either an empty vector pCMV (lane 1) or the plasmid pCMV-p53 encoding wild-type p53 (lane 2). Sixty hours after the nucleofection of cells, cells were lysed, and cell lysates containing an equal amount of proteins were processed for immunoblotting using antibodies specific to the indicated proteins. (C) Subconfluent cultures of Saos^Arg72 cells were incubated either at 39°C (lane 1) or at 32°C (lane 2). Twenty-four hours after incubation, total RNA was isolated, and steady-state levels of Notch1, p21, and actin mRNA were analyzed by semiquantitative reverse transcription-polymerase chain reaction. (D and E) Two sets of Saos^Arg72 cell cultures (in 60-mm plates) were transfected with p21-luc, CBF1-luc, or Hes1-luc reporter plasmid (1.8 µg) along with pRL-TK plasmid (0.2 ag; plasmids in a 9:1 ratio) using FuGene6 transfection reagent. One set of plates for each reporter was incubated at 39°C, and the other sets of plates were incubated at 32°C. Forty-four hours after incubation, cells were processed for dual luciferase reporter activity assays, as described in Materials and Methods. Firefly luciferase reporter activity was normalized to Renilla luciferase activity to control for variations in nucleofection efficiencies. Luciferase activity for p21-luc (C) and for CBF1-luc and Hes1-luc (D) in control cells is shown.