Figure 3. PRIP-1 is a survival factor in undifferentiated HESCs.

A, Three independent primary cultures were transfected with either NT or PRIP-1 siRNA. After 48 hours, some cultures were decidualized for 96 hours, whereas others remained untreated. PRIP-1 mRNA and protein levels were measured in parallel cultures by qRT-PCR (upper panel) and Western blotting (lower panel), respectively. Transcript levels were normalized to expression in undifferentiated cells transfected with NT siRNA; ***, P < .001. B, Cell viability as measured by trypan blue exclusion assay in 3 independent primary cultures first transfected with either NT or PRIP-1 siRNA. After transfection, the cultures remained either untreated or were decidualized for 96 hours. Data normalized to untransfected control (dotted line); **, P < .01. C, In parallel experiments, caspase 3/7 activity was measured and expressed in fluorescent intensity units (F.I.U.). The data represent mean (±SEM) activity in 3 independent cultures; **, P < .01 and ***, P < .001. D, Real-time monitoring of the growth of undifferentiated HESCs over 100 hours after transfection with NT or PRIP-1 siRNA. E, Protein lysates from undifferentiated HESC transfected with either NT or PRIP-1 siRNA were subjected to proteome profiler MAPK array membranes and analyzed by densitometry; *, P < .05 and ***, P < .01. F, Western blot analysis of total protein lysates from undifferentiated HESCs 48 hours after transfection with either NT or PRIP-1 siRNA. G, Nuclear accumulation of FOXO1 was confirmed by Western blot analysis of cytoplasmic and nuclear cell fractions. VINCULIN and LAMIN A/C confirmed enrichment of the cytoplasmic and nuclear proteins, respectively. H, Confocal microscopy showing FOXO1 immunoreactivity in primary HESCs transfected with either NT or PRIP-1 siRNA. Arrowheads indicate cells characterized by marked nuclear FOXO1 accumulation.