The expression of core Wnt pathway components is required for CLL survival. (A) Scanning electron micrographs (SEMs) of normal CD19⁺ B (left panels) and CLL-B cells (right panels) atop NWs taken 24 hours after plating. (B) Normal CD19⁺ B cells can grow and divide on NWs. Normal CD19⁺ B cells isolated from peripheral blood of healthy adult volunteers were stimulated with IL4 (2 ng/mL) and CD40L (0.1 mg/mLl)²⁸  either in vitro (“culture”) or atop NWs (“SiNWs”) for 48 hours. Cell proliferation was measured using an adenosine triphosphate (ATP)-dependent Cell-Titer Glo assay. Cell growth rate was calculated based on the measurement of ATP amount after 48 hours of stimulation, normalized relative to the day 0 value. (Inset) Scanning electron microscope image showing that proliferating B cells on NWs grow in clusters. (C) Confocal scanning images of Alexa Fluor 546-labeled human anti-vimentin siRNA delivered into CLL-B cells. (Upper panel) siRNA delivery is calculated by manually counting the number of cells that have higher levels of fluorescent siRNAs compared with untransfected controls (not shown here). Alexa Fluor 546-labeled siRNA is shown (orange), whereas cell membranes are shown outlined (gray). (Lower panel) Viability was calculated as a percentage of the number of live cells in the total cells using a live–dead cell staining method. Intact cells (stained with Calcein-AM) are shown (green), whereas the nuclei of dead cells are shown in magenta (stained with EthD-1). (D) Core Wnt pathway components can be silenced in HEK293T using siRNA delivery. Gene expression of Wnt pathway members DVL1, CTNNB1, and LEF1 (relative to glyceraldehyde-3-phosphate dehydrogenase expression) were analyzed by quantitative Taqman RT-PCR using complementary DNA derived from HEK293T cells that were either untransfected (“untreated,” white bars) or transfected for 48 hours with control nontargeting siRNA (“control,” pink bars) or siRNA specific for DVL1, CTNNB1, or LEF1 (dark red bars). (E) Efficient knockdown of protein expression of Wnt pathway members in normal CD19⁺ and CLL-B cells via NW-mediated siRNA delivery. Representative images of target protein expression, detected by immunofluorescence 48 hours after siRNA delivery using gene-specific antibodies against the target proteins are shown. (F) Median decrease in cell survival (measured by CellTiter Glo) 48 hours after NW-mediated delivery of siRNAs against LEF1, DVL, and CTNNB1 in normal CD19⁺ (n = 3) and CLL-B (n = 3) compared with silencing using nontargeting siRNA controls (2 different siRNAs per target gene). Percentage of cell survival was normalized to ATP amount at day 0.