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. Author manuscript; available in PMC: 2016 May 18.
Published in final edited form as: Leukemia. 2015 Oct 27;30(3):617–626. doi: 10.1038/leu.2015.302

Figure 2. Genetic and pharmacological modulation of PDE4B in DLBCL regulates VEGF levels and HUVEC tube formation.

Figure 2

a) Top - VEGF quantification in the supernatant of the PDE4B-low SU-DHL6 cell line expressing an empty vector (MSCV), wild-type PDE4B (WT) or a phosphodiesterase inactive (PI) mutant. PDE4B-WT abolished Forskolin (cAMP) effects on VEGF (p<0.0001, ANOVA; p<0.05 Bonferroni’s multiple comparison post-test for MSCV and PDE4B-PI and non-significant for PDE4B-WT). Data shown are the mean ± SD of four biological replicates. Expression of PDE4B was confirmed by WB. Concordantly, conditioned media from Forskolin-exposed PDE4B-WT cells did not impair the tube-forming capacity of HUVEC (bottom graph). Data shown are the mean ± SD of three biological replicates (p<0.01, two-tailed Student’s t-test for each measure in MSCV and PDE4B-PI CELLS; non-significant for PDE4B-WT). Tube formation images are shown below the graph. b) Top - VEGF quantification in the supernatant of the PDE4B-high cell line OCI-Ly18 transfected with PDE4B-specific siRNA oligonucleotides. Knockdown (KD) of PDE4B (confirmed by WB) decreased VEGF secretion (p<0.0001, two-tailed Student’s t-test, si-ctrl vs. si-PDE4B). Conditioned media from Forskolin-exposed PDE4B-KD cells impaired tube-forming capacity of HUVEC (bottom graph) (p<0.001, two-tailed Student’s t-test, for each tube formation measure). Data shown are mean ± SD of six data point derived from two biological replicates. Tube formation images are at the bottom. c) Top - VEGF quantification in the supernatant of the PDE4B-high DLBCL cell lines exposed to vehicle or Roflumilast. PDE4 inhibition suppressed VEGF secretion in all models analyzed (p<0.01 ANOVA for all cell lines; p<0.05 Bonferroni’s multiple comparison post-test). Data represent the mean ± SEM of three biological replicates. Concordantly, conditioned media from Roflumilast-treated cell lines (30 μM) impaired the tube formation capacity of HUVEC (p≤0.01 two-tailed Student’s t-test for each measure in each cell line) (bottom graph). Data shown are mean ± SD of an assay performed in triplicate; two biological replicates were completed. Representative tube formation images are shown at the bottom. The conditioned media used in these assays do not contain Forskolin or Roflumilast, these agents were washed-off and the cells cultured in drug-free media for 24h before collection.