Fig. 1. RNAi depletion of IKAP results in decreased plating efficiency, cell adhesion, spreading and migration. (A) Western blot analysis of IKBKAP-shRNA-transduced MEFs (m1 and m2) and human fibroblasts (h1-h3) after antibiotic selection. % IKAP values represent the quantification of IKAP depletion in human fibroblasts (n=2) and MEFs (n=3). (B) RT-PCR analysis of IKBKAP-shRNA-treated MEFs (means ± s.d.; n=3; triplicates; ^***P<0.001) and human fibroblasts (means ± s.d.; n=1; triplicates; ^***P<0.001) after antibiotic selection. (C) Evaluation of plating efficiency. Cells were inoculated with a density of 1.5x10⁴ cells/ml for MEFs or 2x10⁴ cells/ml for human fibroblasts and analyzed 24 hours later. Calculation of cell numbers (means ± s.d.; n=3; triplicates; ^**P<0.01). (D) The level of cell death in designated MEFs was investigated by measuring LDH release 24 hours after plating and again 24 hours later after a change of media (means ± s.d.; n=1; triplicates; ^*P<0.05). (E) The adhesion of MEFs was analyzed by inoculating 5x10⁴ cells/ml in serum-free media on non-supporting adherent-surface 24-well plates (Sarstedt) for 1.5 hours. Cells were washed with PBS and fixed for 10 minutes with crystal violet stain [CV; 0.5% crystal violet (Sigma) containing 25% methanol]. The excess dye was rinsed off, the cells were re-suspended in Na-citrate buffer (0.1 M Na-citrate, 50% ethanol) and the absorbance (OD) at 570 nm was measured (means ± s.d.; n=3; triplicates; ^**P<0.01). (F) To analyze cell spreading, CV-stained cells were imaged by 20x magnification on an Olympus IX70 inverted microscope with an Olympus C-5050 digital camera. The cell areas were measured using ImageJ software (http://rsb.info.nih.gov/ij/) and divided by the number of cells present (means ± s.d.; n=3; triplicates; ^**P<0.01). (G) For cell migration study, the cells were seeded at a density of 1x10⁵ cells/ml and allowed to adhere over night. A wound was inflicted by scraping a 200 µl sterile tip across the cell layer. The cells were incubated for 17 hours followed by imaging at 10x magnification on an Olympus IX70 inverted microscope with an Olympus C-5050 digital camera. Average wound area was quantified in the picture using ImageJ software (means ± s.d.; n=3; ^**P<0.01).