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Fig. 7. Partial recovery of the delayed differentiation observed in TRAP1^OE cells. (A) TRAP1^OE cells grown at 1-3x10⁶ cells/ml were harvested, and shaken in BSS for 6 hours with or without 50 nM cAMP pulses externally applied at 6-minute intervals, followed by incubation in 24-well plates. The incubation times after starvation are shown. Bar, 150 µm. (B) Effect of exogenously applied cAMP pulses on the induction of car1 expression in TRAP1^OE cells. Total RNAs were extracted from TRAP1^OE and Ax-2 cells shaken with (+) or without (–) cAMP pulses for the indicated times. Northern blots were performed using the cDNA probe of car1 gene. (C) TRAP1^OE cells grown up to a high cell density (>1x10⁷ cells/ml) exhibit no delay of differentiation. TRAP1^OE and Ax-2 cells grown at the indicated cell densities were harvested, starved and developed under submerged conditions. These cells acquired aggregation competence after the indicated times of starvation, assuming elongated cell shape. The results of two independent experiments are presented. (D) The localization of Dd-TRAP1 in TRAP1^OE and Ax-2 cells. Ax-2 cells (a) and TRAP1^OE cells (b) grown at 1-2x10⁶ cells/ml were separately harvested and shaken in BSS for 6 hours, followed by immunostaining with the Dd-TRAP1 antibody. A significant number of Dd-TRAP1 molecules were found to be retained in the cell cortex of TRAP1^OE cells (arrowhead). By contrast, when TRAP1^OE cells were harvested at the early stationary phase (>1x10⁷ cells/ml) and starved for 6 hours, most of Dd-TRAP1 were translocated to mitochondria (c). Bar, 5 µm.

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