FIG. 9.

Overexpression of CRT blocks C/EBPα-mediated growth arrest. (A) Overexpression of CRT in HT1 cells inhibits the expression of C/EBPα. HT1 cells were transfected with HA-CRT plus GFP (10:1) (top) or with GFP alone (bottom). C/EBPα was induced by the addition of 10 mM IPTG, and C/EBPα levels were determined by immunostaining with specific antibodies (14AA). Two fields for CRT-plus-GFP transfections are shown. DAPI (nuclear) staining (blue) shows all of the cells in the field. CRT-plus-GFP cells are green, and C/EBPα expression is shown in red. The table below shows a summary for 84 cells transfected with CRT plus GFP and for 30 cells transfected with GFP alone. (B and C) Overexpression of CRT abolishes C/EBPα-mediated growth arrest. HT1 cells were cotransfected with β-Gal plasmid and an empty vector (pcDNA) or with plasmid expressing HA-CRT. C/EBPα was induced by the addition of 10 mM IPTG, and the cells were stained for β-Gal activity on days 1 and 3 after C/EBPα induction. (B) Typical picture of colonies on day 3 after transfection. CRT-overexpressing cells proliferate and form colonies containing three or more cells, while cells transfected with the empty vector do not proliferate and stay as single cells. (C) Summary of three independent experiments. The error bars indicate standard deviations.