Fig. 1
Overexpression of EGFRvIII or EGFRwt induced anchorage-independent growth of NIH3T3 cells. a Anchorage-independent (3D) growth of NIH3T3/EGFRvIII or NIH3T3/EGFRwt cells. Cells (2 × 105 cells/mL) were seeded and observed by phase-contrast microscopy after cultivation for 3 days. Images were acquired using an Olympus DP71 microscope camera and processed by Adobe Photoshop. b Growth curves of NIH3T3 cells overexpressing EGFRvIII or EGFRwt in 3D cell cultures. Cells (1 × 105 cells/mL, 100 μl) were seeded on ULAS 96-well plates on day 0. Viable cells were counted at the indicated times by CellTiter-Glo Luminescent Cell Viability Assay. c EGFR-siRNA inhibited EGFRvIII and EGFRwt protein expression in NIH3T3 cells overexpressing each protein. Cells were lysed and 6 μg protein was applied to 10 % SDS-PAGE gel. Cells were cultured on ULAS plate for 3 days (3D) or on a normal cell-attachment plate for 1 day (2D) after EGFR-siRNA transfection. d EGFR-siRNA inhibited NIH3T3/EGFRvIII and NIH3T3/EGFRwt anchorage-independent 3D growth. Viable cell counts were measured by CellTiter 96 AQueous One Solution Cell Proliferation Assay 3 days after transfection with EGFR-siRNA (NM005228_stealth_2438). Cells were cultured with (50 ng/mL) or without EGF on ULAS plates (upper, 3D) or on normal tissue culture plate (lower, 2D)