Figure 4. The effect of GNC–siRNA complex on cells in vitro.

(a) Viability of Panc-1 cells after 24 h incubation of GNC–siRNA versus the identical siRNA concentration coupled with Lipofectamine 2000 transfection agent (Lipo2000-siRNA). Mean±s.d. (n=3). (b) Cells were pre-incubated with GNC–siRNA or GNC–nsRNA (100 nM siRNA equivalent) for 24 h. Then the proliferation of Panc-1 cells within 3 days was evaluated by CCK-8 assay. Mean±s.d. (n=3). (c) Scheme of the microfluidic chip for cell co-culture, the right panel is the cross-sectional view of the chip. (d) On-chip migration assay of Panc-1 cells, which were pretreated with GNC–siRNA or GNC–nsRNA for 48 h (100 nM siRNA equivalents). The Panc-1 cells were seeded and adhered in the channels for 6 h, then the polydimethylsiloxane (PDMS) cover were peeled off (t=0 h) and the migration of Panc-1 cells was monitored by microscope. Scale bars, 100 μm. (e) Extent of gap closure (%) at 24 and 48 h, respectively. The quantification was conducted from at least 10 fields for each condition (mean±s.d.). (f) On-chip co-culture of DRG neurons and Panc-1 cells to assess the neurite sprouting. The Panc-1 cells were pretreated with GNC–siRNA or GNC–nsRNA for 48 h (100 nM siRNA). The DRG neurons and Panc-1 cells were seeded into the left and right channels of the chips, and adhered for 6 h before the removal of the cover. The neurite sprouting of DRG neurons from the neuron channel was evaluated. Neurites were stained with Tuj1 antibody and observed by confocal microscope. Scale bars, 100 μm. (g) Density of DRG neurite sprouting in the microfluidic chips. Mean±s.d. (n=5). (h) Average length of DRG neurite sprouting in the microfluidic chips. Mean±s.d. (n=12). Significant difference was from the nontreated control. *P<0.01 compared with the nontreated control; Student's t-test.