Fig. 3. ROCK activity is required for PMA-induced MLC phosphorylation and membrane contraction, which precedes caspase activation. (A) TF-1 cells were either left untreated or pre-incubated with the indicated blocker for 30 minutes: pan-caspase inhibitor (Boc-D-FMK; 50 µM), specific ROCK inhibitor (Y27632; 20 µM, for 1 hour) or F-actin polymerization inhibitor (Latrunculin B; 0.5 µM). Cells were then exposed to PMA for 2 hours and imaged by phase-contrast microscope for photography. The cell viability in each set of cultures was determined by Trypan Blue staining after exposure to PMA for 12 hours. (B) TF-1 cells were pre-treated with Y27632 (20 µM) or ML-7 as indicated, followed by addition of PMA to the medium. Cells were harvested 8 hours later and resolved by SDS-PAGE, followed by immunoblotting with antibodies specific for phosphoMLC, MLC and phosphoERK1/2. After 12 hours exposure to PMA, viability was determined using the Trypan Blue exclusion method. (C) The effects of Y27632 (20 µM) or ML-7 (20 µM) on PMA-induced apoptosis were assessed by DNA laddering. NT, not treated.

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