Figure 5.

REN induces cell growth arrest. (A) Cell growth arrest in P19, N2a, and ST14A cells transfected with expression vector pCXN2-REN-myc or with GFP expression vector. BrdU was added 24 h after transfection and detected after 2–24 h of incorporation. The percentage of BrdU incorporating cells in the REN⁺ or in the GFP⁺ population, was determined by coimmunostaining with anti-myc epitope and anti-BrdU antibodies or with anti-GFP and anti-BrdU antibodies and immunofluorescence microscopy. Average (± SEM) percentages of BrdU positive cells from three independent experiments performed in triplicate are shown. (B) Expression of p27^Kip1 in control (GFP-transfected cells, GFP) and REN cDNA-transfected cells (REN) 48 h after transfection into P19, N2a, and ST14A cells. The percentage of p27^Kip1 positive cells in the REN⁺ and in the GFP⁺ population, was determined by coimmunostaining with anti-myc epitope or anti-GFP antibodies and by immunofluorescence microscopy. Average (± SEM) percentages of p27^Kip1 positive cells from three independent experiments performed in triplicate are shown. (C) Western blot analysis of p27^Kip1 and α-tubulin expression in control (GFP-transfected, GFP) and REN cDNA-transfected N2a cells (REN), 48 h after transfection. (D) Exogenous expression of REN enhances p27^Kip1 expression in N2a and ST14A cells. Confocal microscopy of coimmunostaining of p27^Kip1 (red) and either myc epitope (green, REN) or GFP (green, GFP), in either GFP- (GFP) or REN-transfected (REN) cells, 48 h after transfection.