Figure 3. PACAP exhibits age-dependent effects on DNA synthesis and promotes G1 cyclins and G1/S-phase progression in vitro. a, Effects of PACAP exposure on DNA synthesis at 24 h in rat and mouse precursors from different ages. The timeline above the graphs details the experimental paradigm. DNA synthesis was assessed using [3H]thymidine incorporation. Data were obtained from five experiments for each species, using three wells (25,000 cells per well) per group for each experiment. Data are expressed as percentage control: control (Con) values ranged from 4000 from 12,000 cpm. *p < 0.05, **p < 0.01. b, PACAP treatment increases the proportion of E10.5 rat precursor cells engaged in S phase. BrdU-positive cells (green) and total cells (phase) were counted in 10 randomly selected fields. Scale bar, 10 μm. PACAP exposure increased the BrdU LI from 36% in control to 45% in the PACAP group at 24 h. c, Flow cytometric analysis of BrdU immunolabeling of control and PACAP-treated E10.5 rat precursors. PACAP treatment increased S-phase labeling from 43 to 50% at 24 h. d, Flow cytometric analysis of DNA content using PI indicates that more cells were engaged in S phase in response to PACAP exposure at 24 h. e, Real-time PCR analysis indicates that PACAP treatment increased cyclin D1 mRNA by 221%, cyclin mRNA D2 by 116%, and cyclin mRNA D3 by 118%, but not cyclin E (data not shown), at 24 h. Data were obtained using total RNA (50–75 embryos for each isolation) obtained from three separate experiments. f, PACAP treatment increased protein levels of cyclin D1 by 58% and D2 by 74% at 24 h, as defined by Western blot analysis. Quantifications were performed on three to four separate blots for each protein, cyclins D1, D2, D3, and E.