Fig. 3: Exposure of MC-3129 activates RhoA/ROCK1/PTEN and inactivates of PI3K/Akt.

a U937 cells were treated with various MC-3129 concentrations for 24 h or with 10 μM of MC-3129 for different time intervals, as indicated. Whole cell lysates were prepared and subjected to western blot analysis using antibodies against against ROCK1, phospho-PTEN (p-PTEN), PTEN, phospho-PI3K (p-PI3K), PI3K, phospho-Akt (Ser473, p-Akt), Akt, and GADPH. b Active RhoA-GTP was pulled down by glutathione S-transferase linked to the RhoA-binding domain of Rhotekin (GST-RBD); bead/protein complexes and total RhoA were detected by immunoblotting with anti-RhoA. U937 cells were pretreated with the caspase inhibitor Z-VAD-fmk (20 μM) for 2 h, followed by treatment with 10 μM of MC-3129 for 24 h. c Apoptosis was determined by flow cytometry using Annexin V/PI staining, the percentage of apoptotic cells was analyzed for three separate experiments (mean ± SD, **P < 0.01). d Total protein lysates were analyzed by immunoblotting using antibodies against cleaved-Caspase 3 (C-Caspase 3), ROCK1, cleaved-ROCK1 (ROCK1 CF), and GAPDH. e U937 cells were stably transfected with lentivirus containing RhoA-siRNA (shRhoA) or a scrambled control siRNA (shCon), cells were treated with or without 10 μM of MC-3129 for 24 h. Total protein lysates were analyzed by immunoblotting using antibodies against RhoA, ROCK1, cleaved-ROCK1 (ROCK1 CF), and GAPDH