Fig. 1. PMA-induced pro-apoptotic signals are associated with early detection of membrane blebbing and MLC phosphorylation, followed by caspase-3 activation. (A) TF-1 cells (1x10⁶) were treated with PMA (32 nM) in 10% (v/v) heat-inactivated FBS-containing RPMI medium in the presence of GM-CSF. Cell lysates obtained from cells that remained in suspension were analyzed for caspase-3 activity. Results are expressed relative to those obtained in the untreated control, and are the mean±s.d. of two independent experiments. (B) TF-1 cells were pre-incubated with cycloheximide (CHX, 50 µg/ml), or staurosporine (STS, 200 nM) as indicated for 30 minutes prior to PMA treatment. Viability was determined by Trypan Blue exclusion method and was expressed relative to untreated cells. Results are the mean±s.d. (n=3). (C) TF-1 cells treated with PMA for the indicated time points were imaged by phase-contrast microscopy and harvested for immunoblot analysis. (D) Whole cell lysates (50 µg) were resolved by SDS-PAGE and blotted with antibodies specific for phosphoERK1/2, phosphoMLC, MLC, caspase-3 and ROCKI.