(A) 7 × 104 IOMM-Lee-GFP cells were cultured in 96-well low attachment plates and spheroids were allowed to grow for 3 to 4 dayswith shaking at 40–60 rpm at 37°C. The spheroids were then transfected with shRNA plasmids pSV, pUR, pM and pUM. Untreated spheroids were also maintained to serve as the control (mock). 48 h after transfection, the spheroids were transferred to vitronectin-coated (50 mg/mL) 8-well chamber slides and maintained for another 72 h in serum-free media. Migration of spheroids was analyzed after taking pictures under a fluorescent microscope. (B) Migration was quantified as the distance spheroid cells moved away from spheroids on vitronectin-coated substrate. Values are mean ± S.D. from three different experiments (p<0.001). (C) IOMM-Lee cells were transfected with pSV, pUR, pM and pUM. Untransfected cells (mock) were also maintained to serve as a control. 48 h later, the cells were trypsinized, counted and 1×105 cells were cultured in the upper chamber of a Transwell insert coated with matrigel (1 mg/mL) and processed per manufacturer’s instructions. (D) Number of cells was counted in three different fields for each sample and the percentage invasion of cells treated with shRNA plasmids was analyzed in comparison with the untreated (mock) cells. The graph represents the percentage invasion shown by the cells transfected with pSV, pUR, pM and pUM in comparison with untreated cells (mock). Values are mean ± S.D. from three different experiments (p<0.001). (E) 7 × 104 IOMM-Lee cells were cultured in 96-well low attachment plates and allowed to grow for 3 to 4 days with shaking at 40–60 rpm at 37°C. The spheroids were later transfected with pSV, pUR, pM and pUM. 48 h after transfection, the cells were labeled with Dil (red fluorescent dye). Untreated spheroids (mock) were maintained as control under similar conditions. Simultaneously fetal rat brain aggregates (FRBA) were grown from 16 to 17 day old fetal rat brain cells and labeled with DiO (green fluorescent dye). Later, both the IOMM-Lee spheroids and spheroids from fetal rat brain cells were co-cultured and maintained in serum-free media. Invasion of fetal rat brain spheroids by tumor cell spheroids was recorded at 24, 48 and 72 h using a fluorescent microscope and quantified. (F) The percentage invasion of the untransfected spheroids and spheroids treated with shRNA plasmids was quantified using image analysis software and plotted as the fetal rat brain aggregates remaining uninvaded against different time intervals. Values shown are the mean ± S.D. from three different experiments (p<0.001).