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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 S6. cAMP does not modify the phospho-SYK (Tyr348) levels (JPG, 245 KB)
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Elevation of cAMP levels (Forskolin 40 µM for 1 to 5 hours) in PDE4B-low DLBCL cell lines did not modify the phosphorylation of SYK (Tyr348), as defined by flow cytometry. The lack of cAMP activity was unrelated to BCR engagement, which increased in SYK Tyr348 phosphorylation in both cell line models – compare top and bottom panels.
- Figure S7. Expression of PDE4D and PDE7A in DLBCL (JPG, 137 KB)
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Quantitative real-time RT-PCR was used to determine the expression of additional cAMP-specific PDEs in DLBCL. PDE4D (left panel) and PDE7A (right panel) expression did not correlate with the intra-cellular levels of cAMP (Fig. S1) and the inhibition of SYK/BLNK (Fig. 1 in the article). In particular, these PDEs were markedly expressed in the cell lines that show the highest level of cAMP and most sensitive to its inhibitory effects (DHL6, DHL10 and WSU-NHL). Together, these data indicate a minimal, if any, contribution of these PDEs to the cAMP hydrolyzes in DLBCL. PDE7B was not expressed in these DLBCL cell lines.
- Figure S8. Functional validation of PDE4B constructs (JPG, 125 KB)
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Intra-cellular cAMP levels were measured in DHL6 and DHL10 DLBCL cell lines stably expressing a PDE4B phosphodiesterase inactive (PI) mutant or the WT enzyme. In both cell line models, expression of PDE4B-WT led to cAMP hydrolysis and significantly abrogated induction of this second messenger by the adenylyl cyclase activator Forskolin. Conversely, as predicted, PDE4B-PI did not limit cAMP elevation. These data further support the concept that PDE4B is the dominant PDE in DLBCL.
- Figure S9. Reconstitution of PDE4B-WT expression blocks cAMP inhibitory effects towards SYK/BLNK (JPG, 210 KB)
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The PDE4B-null DLBCL cell line DHL6 was reconstituted with PDE4B-WT or with a phosphodiesterase inactive (PI) mutant. Treatment with the adenylyl cyclase activator Forskolin (40 µM for 3h), which enhances cAMP levels, led to a marked inhibition of phospho-BLNK levels in PI cells (right panel) but had limited effects on PDEB-WT expressing cells (left panel). These results confirmed the central role of PDE4B in controlling cAMP activities towards SYK/BLNK in DLBCL.
- Figure S10. Functional validation of PDE4B knockdown (JPG, 132 KB)
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Intra-cellular cAMP levels were measured in the OCI-Ly3 cell line following transient transfection of two independent PDE4B-specific siRNA oligonucleotides or a siRNA control (targeting GFP). PDE4B knockdown resulted in significant elevation (2.5- to 3-fold) of intracellular cAMP whereas minimal changes were noted in the siRNA-control transfected cells.
- Figure S11. Stable PDE4B knockdown in OCI-Ly3 cell line (JPG, 108 KB)
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Quantitative real-time RT-PCR–based measurement of PDE4B levels in three clonal populations expressing two independent PDE4B-specific shRNA sequences (#2 and #5). 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.
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