Bcl-2 mRNA (A) and VIP up-regulated Bcl-2 mRNA in CE cells in corneoscleral explants (B and C). Agarose gel (2%) electrophoresis of RT-PCR product of Bcl-2 mRNA isolated from fresh bovine corneas (A) and that of semi-quantitative RT-PCR products of Bcl-2 and 18S (as the internal standard) mRNA isolated from VIP-pre-treated CE cells (B and C). For (A), -RT: PCR negative control (RNA not subjected to RT); H2O: reagent negative control (No RT products, H2O instead); M: DNA size markers; bp: base pair. For B and C: CE cell Bcl-2 mRNA level increased in a VIP-concentration dependent manner. Bcl-2 and 18 S mRNA levels reflected in the electrophoresed (2% agarose) semi-quantitative RT-PCR products (B). The ratio of normalized CE cell Bcl-2 cDNA (against the18S internal standard) levels over that averaged from control CE cells of the same experiment (Y-axis) was shown as a function of VIP concentration (C). Data were combined from three separate experiments. The difference between CE cells in the control (N=6) vs VIP-treated corneal cups was significant at the levels of p =0.01 (10−12 M; N=3), p=0.0001 (10−10 M; N=3), p=0.005 (10−8 M; N=5), and p=0.03 (10−6 M; N=4). The difference among various treatment groups was significant (P = 0.01, ANOVA):