Figure 1. Cyclic-AMP-mediated PDE4B-dependent suppression of VEGFA in DLBCL.

a) Quantitative real-time RT-PCR shows that VEGFA mRNA levels are significantly reduced following increase in the intracellular levels of cAMP (10 μM Forskolin, Frsk, for 6hrs) exclusively in DLBCL cell lines expression low levels of PDE4B (p<0.0001, ANOVA; p<0.05 Bonferroni’s multiple comparison post-test for PDE4B-low cell lines, non-significant for PDE4B-high cells). Data shown are the mean ± SD of an assay performed in triplicate. Three biological replicates were completed. In all instances, VEGFA expression was normalized to that of a housekeeping gene TBP, and relative quantification achieved by calculating ΔΔCt, and expression defined as 2^−ΔΔCt, where cells exposed to vehicle represent the baseline. b) VEGF quantification in the supernatant of DLBCL cell lines assay shows a significant cAMP-mediated suppression in DLBCL cell lines expressing low PDE4B but not in those with high levels of this enzyme (p<0.0001, ANOVA; p<0.05 Bonferroni’s multiple comparison post-test for PDE4B-low cell lines, non-significant for PDE4B-high cells). Data shown are the mean ± SD of three independent biological replicates. c) Top - relative quantification of three output measures (tube length, branch point and loops) for the HUVEC tube formation assay is shown. Conditioned media from PDE4B-low DLBCL cell lines exposed to Forskolin were significantly less efficient in promoting tube formation, while no changes were detectable in DLBCL cell lines expressing high PDE4B (p<0.05, two-tailed Student’s t-test for each measure in PDE4B-low cell lines; non-significant in the PDE4B-high).. Data shown are the mean ± SD of two independent biological replicates, each performed in triplicate. Note that the conditioned media used in b) and c) do not contain Forskolin, this drug was washed-off from the culture 24h before supernatant collection.