Increased dependence on Wnt signaling by CLL samples harboring Wnt pathway mutations. Silencing of BCL9, RYK, and CSNK1E protein in normal CD19⁺ B cells 48 hours after NW-mediated delivery of gene-specific siRNAs compared with nontargeting control siRNAs (“control”) was confirmed by gene-specific immunofluorescence staining (red) and visualized by confocal microscopy (A,C,D) (top panels). Nuclei were probed with DAPI (blue). Protein level silencing efficiency was estimated using Image J software. Per gene, 2 different targeting siRNAs were tested and the representative results are shown. (B) (Top) Silencing of DKK2 in HEK293T cells using gene-specific siRNAs detected by quantitative Taqman RT-PCR of complementary DNA derived from HEK293T cells that were either untreated (white bar) or treated with control nontargeting siRNA (“control”, gray bars) or with siRNA specific for DKK2 (black bars). (A-D) (Lower panels) Cell survival rate was normalized to nontargeting control in normal B cells (n = 4 for BCL9 and DKK2; n = 2 for RYK and CSNK1E), the CLL B cells with either mutated BCL9 (P48), DKK2 (P46), RYK (P35), or CSNK1E (P42) (all n = 1) or CLL-B samples without Wnt pathway mutations (n = 7 for BCL9 and DKK2; n = 5 for RYK and CSNK1E), using the Cell-Titer Glo assay 48 hours after NW-mediated siRNA delivery. Three replicates per independent sample were performed.