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Fig. 4. Early prestarvation response observed in TRAP1^OE cells. (A) The localization of Dd-TRAP1 in growing TRAP1^OE cells. Vegetatively growing TRAP1^OE cells and parental Ax-2 cells were harvested at a density of 0.5-1.0x10⁶ cells/ml and fixed for immunostaining with the Dd-TRAP1 antibody. Bar, 5 µm. (B) Expression patterns of early developmental genes discoidin I, car1, dia1 and dia3 in TRAP1^OE cells and parental Ax-2 cells. TRAP1^OE cells and parental Ax-2 (clone MS) cells were separately harvested at the indicated cell density of exponential growth phase (0.25-3x10⁶ cells/ml), followed by extraction of total RNAs to detect the expression of discoidin I (upper two panels). In another experiment, cells were harvested at the exponential growth phase (1-5x10⁶ cells/ml) or stationary growth phase (1.5-2.0x10⁷ cells/ml, S), followed by extraction of total RNAs. The RNAs (40 µg for each) were size-fractionated and analyzed by northern blots using the cDNA probe of each gene. (C) TRAP1^OE cells assemble into 3D aggregates, even in the presence of nutrients. Bar, 100 µm. (D) Early acquisition of EDTA-resistant adhesion in TRAP1^OE cells. Ax-2 and TRAP1^OE cells were separately starved and shaken gently in 16 mM phosphate buffer (pH 6.4) at a density of 1x10⁷ cells/ml. After 3 hours of shaking, 10 mM EDTA was added to prevent the EDTA-sensitive cell-cell adhesion. Under this condition, only TRAP1^OE cells form many aggregates. Bar, 100 µm.

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