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Fig. 1. Tiam1-KO keratinocytes are more susceptible to apoptosis than are wild-type (WT) keratinocytes. (A) Phase-contrast photographs of WT and Tiam1-deficient (KO) keratinocytes cultured on collagen IV-coated tissue culture plates. Cells were grown in normal culture conditions or heat shocked for 90 minutes at 43°C, followed by 6 hours recovery at 37°C. Bar, 50 µm. (B) Quantification of apoptosis induced by GF deprivation (24 hours) or heat-shock treatment in WT and Tiam1-KO keratinocytes. Both adherent and non-adherent cells were collected and subjected to DNA profile analysis. Cells with sub-G1 DNA content were considered to be apoptotic. Error bars represent standard deviation from triplicate samples. (C) Quantification of apoptosis induced by GF deprivation (24 hours) in WT and Tiam1-KO keratinocytes. Both adherent and non-adherent cells were collected, stained with annexin-V and the DNA stain propidium iodide (PI), and analyzed by FACS. Annexin-V-positive cells were considered to be apoptotic. Error bars represent standard deviation from three independent experiments. (D) Rac activity assay performed in lysates from WT and Tiam1-KO keratinocytes. Cells were GF starved for 30 minutes (GF–). Tiam1 and Rac were used as expression and loading controls, respectively. The data presented is a representative example of four independent experiments.
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