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Blood, Vol. 113, Issue 24, 6153-6160, June 11, 2009
Rational combined targeting of phosphodiesterase 4B and SYK in DLBCL
Blood Kim et al.
113: 6153
Supplemental materials for: Kim et al
Files in this Data Supplement:
- Figure S1. Correlation between PDE4B expression and activity in DLBCL (JPG, 159 KB)
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(A) Quantitative real-time RT-PCR based measurements define two groups of DLBCL cell lines with significantly distinct levels of PDE4B expression. The assays were performed in triplicate and the PDE4B expression was normalized by the expression of the TATA-binding protein (TBP) gene. Data are displayed as relative expression determined using the ΔΔCT method and reported as 2−ΔΔCT. (B) Intracellular measurement of cAMP levels following exposure to the adenylyl cyclase activator Forskolin (40µM, for 1 hour) shows robust induction (~30 fold) in PDE4B-low but not in PDE4B high cells. As PDE4B hydrolyzes and inactivates cAMP, these data confirm that PDE4B is the main cAMP phosphodiesterase in DLBCL cell lines and that its expression correlates with activity.
- Figure S2. Inhibition of phospho-BLNK expression by 8-Br-cAMP (JPG, 234 KB)
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PDE4B-low DLBCL cell lines were treated with a cell permeable, synthetic cAMP isoform (8-bromo-cAMP, 1 mM for 6 hours), or vehicle, followed by BCR engagement with anti-IgM +IgG (10 µg for 10 minutes). Cells were fixed, permeabilized and intracellular phospho-BLNK (Tyr84) levels determined by flow cytometry. Elevation of cAMP levels resulted in inhibition of intracellular phospho-BLNK level; 69% in WSU-NHL and 26% in DHL10 (mean fluorescence ratio of cAMP and vehicle treated cells).
- Figure S3. PDE4B expression defines the sensitivity to cAMP growth inhibitory effects in DLBCL (JPG, 168 KB)
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DLBCL cell lines were exposed to the adenylyl cyclase activator Forskolin (10–40µM for 48 hours) and cell proliferation determined with the MTS assay. DLBCL cells expressing low PDE4B levels were consistently more sensitive to cAMP effects than the PDE4B high cell lines. Data shown is mean −∕+ SEM of experiments performed in triplicate.
- Figure S4. Western blot analyses of phospho-LYN (Tyr396) and phospho-SRC (Tyr416) in DLBCL cell lines (JPG, 172 KB)
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Elevation of intracellular levels of cAMP did not modify LYN and SRC phosphorylation, irrespective of PDE4B levels.
- Figure S5. The cAMP negative effects on phospho-BLNK are independent of BCR activation (JPG, 215 KB)
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Phospho-BLNK levels were determined at baseline (i.e. without BCR activation) in DLBCL cell lines with null/low PDE4B expression. cAMP elevation with Forskolin (40 µM for 1 to 5 hours) consistently inhibited phospho-BLNK confirming that the effects of this second messenger are not directed at the antigen-mediated activation of the B-cell receptor but rather at SYK/BLNK. These studies are necessary because the levels of SYK Tyr525/26 at baseline are below detection level with western blotting.
Additional supplemental figures can be found here.
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