Expression of SMAD4 in BxPC3 does not impact invasiveness but decreases migration. A, Expression of SMAD4 has no effect on invasiveness of BxPC3. Invasiveness was measured by Matrigel invasion assay with invasion ratio calculated by dividing the number of invading cells in experimental Matrigel-coated inserts by the number of invading cells in the control inserts. Untreated BxPC3-pINS4c5 cells were compared with BxPC3-pINS4c5 cells treated with 1-μg/mL doxycycline 24 hours before the invasion assay. B, Expression of SMAD4 decreases migration of BxPC3 by 71%. Migration was measured by wound healing assay. Confluent wells of BxPC3-pINS4c5 ± 3-hour pretreatment with 1-μg/mL doxycycline were scratched with a P200 pipette tip and the scratches were photographed at 0- and 21.5-hour time points; % wound closure was calculated for triplicate wells by measuring the change in the area of the scratch using ImageJ. Error bars are ±SEM. Comparisons are Student's 2-tailed paired t test with ***, P ≤ .001; **, P ≤ .005; *, P ≤ .05.