Blockade of Rho activation mimics the effects of PKA.
LNCaP cells were plated into 6-well plates and incubated for 6 h with either vehicle (2% (v/v) PBS) (Panel A) or 4 μg/ml C3T (Panel B) prior to stimulation with either vehicle (0.1% (v/v) EtOH) or 10 μM Fsk for 1 h. Images were captured as described at 0 h (0 h), 6 h post-stimulation (6 h) and following stimulation with Fsk (post-stimulation). Panel C: Changes in LNCaP morphology consistent with NE-like differentiation were assessed via increases in mean dendrite length. Results are represented as mean values ± SEM for n = 3 separate experiments. ***p < 0.001 vs. 0 h, ###p < 0.001 vs. vehicle. Panel D: Protein sequences corresponding to the known human Rho family members retrieved from the UniProt knowledge base were aligned using ClustalW (http://www.clustal.org/). The N-terminus of RhoA was then compared to other family members in order to assess whether they showed similar motifs to those implicated in the binding of C3T to RhoA and subsequent ADP ribosylation (47). = site of ADP-ribosylation, equivalent to N41 = residues involved in C3T recognition, equivalent to R5, K6, E47 and E54 = residues involved in correct ternary complex formation between Rho, C3T and NAD+, corresponding to E40 and V43. All amino acid positions refer to the position of these residues in RhoA.