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. Author manuscript; available in PMC: 2014 Jun 10.
Published in final edited form as: Cancer Cell. 2013 Jun 10;23(6):826–838. doi: 10.1016/j.ccr.2013.05.002

Figure 2. SYK and PI3K-dependent BCR signaling in DLBCLs with high baseline NF-κB.

Figure 2

(A) NF-κB (p65) activity was determined in vehicle and R406-treated DLBCL cell lines. Pos. Ctrl.: Manufacturer’s positive control. BCR-dependent NF-κB-low lines, purple; BCR-dependent NF-κB-high lines, blue; BCR-independent OxPhos lines, green. (B) GSEA of NF-κB targets was performed in vehicle- vs. R406-treated DLBCL cell lines with high baseline NF-κB activity (HBL-1 and U-2932). The 19K genes in the genome were sorted from highest (left, red) to lowest (right, blue) relative expression in vehicle- vs. R406-treated lines (horizontal axis). Note that the positions of the NF-κB targets (Set #1, (Davis et al., 2010) Set #2, (Schreiber et al., 2006)) were significantly skewed toward the right end of the sorted list, reflecting their statistically significant downregulation in R406-treated lines. (C) Relative abundance of NF-κB target genes in vehicle- vs. R406-treated DLBCL cell lines with high baseline NF-κB activity was displayed with a colometric scale. NF-κB target genes derived from the GSEA leading edge, Set #1 (Davis et al., 2010). (D) BCL2A1 transcript abundance in DLBCL cell lines treated with vehicle or R406 (24 hr) was determined by qRT-PCR relative to PPIA. (E) BCL2A1 protein abundance in vehicle- and R406-treated DLBCL cell lines was assessed by immunoblotting. β-actin, loading control. (F) BCL2A1 transcript abundance in SYK-depleted DLBCL cell lines (72 hr following completion puromycin selection) was determined by qRT-PCR relative to PPIA. (G) NF-κB (p65) activity was determined in DLBCL cell lines treated with the PI3K inhibitor LY294002 or vehicle. (H) BCL2A1 transcript abundance in DLBCL cell lines treated with LY294002, R406, or vehicle (24 hr) was assessed by qRT-PCR relative to PPIA. In (A), (D), (F), (G) and (H), p-values for control vs. treated were determined with a one-sided Welch t-test. ***, p <0.0001; **, p <0.001; *, p <0.01. Error bars represent the SD of 3 independent assays in a representative experiment. See also Figure S2 and Table S1.