Figure 5. Effects of IRS1, S668T, D674H and double mutants: (a) On proliferation of 293T cells; 293T cells were transiently transfected with expression vectors for 72 h, and cell viability was measured via MTT assay; (b) on proliferation of A549 cells; A459 cells were transiently transfected with expression vectors for 72 h, and cell viability was measured via MTT assay; (c) on migration of 293T cells; 293T cells were transiently transfected with expression vectors for 24 h, trypsinized, counted and 5000 cells were placed in upper chamber BD Biocoat Insert Systems with 8-micron pore size, serum-containing DMEM was added to lower chamber. After overnight incubation, medium in upper chamber was removed, cells were fixed with methanol, and cells in upper chamber were scraped off with cotton swabs. Migrated cells were stained with hematoxylin and eosin. Images of migrated cells were obtained with an Olympus IX51 microscope. (d) Effects of S668T, D674H and double mutants of IRS1 on insulin-induced glucose uptake in A549 cells. A Cayman Glucose Uptake Cell Based Assay Kit was used to determine the level of glucose uptake. (CTCF: Corrected Total Cell Fluorescence).