A Microplate-Based Nonradioactive Protein Synthesis Assay: Application to TRAIL Sensitization by Protein Synthesis Inhibitors
Fig 3
Effects of protein synthesis inhibitors on protein synthesis, levels of cFLIP protein, and TRAIL- induced apoptosis.
(A) ACHN cells were treated for 4 h with 1 μM of the indicated compound at which point cells were processed for ICW detection and quantitation of GAPDH (open bars), puromycin (light grey), or cFLIP (dark grey). In a parallel experiment, TRAIL was added after 4 h with compounds and cell survival assessed after 24 h by the XTT assay (black bars). Signals were normalized to control on the same plate (vehicle control = 100%). Error bars represent sd (n = 4 for puromycin and GAPDH; n = 3 for cFLIP; and 3 plates, duplicate wells per plate for compounds + TRAIL). (B) Caspase 8 activity was measured after 4 h treatment with 1 μM of the indicated compound followed by 4 h in the absence (open bars) or presence (black bars) of TRAIL and normalized (vehicle control = 1.0). Error bars represent sd (n = 3). compounds: ANS: anisomycin, CHX: cycloheximide, EME: emetine, GLA: glaucarubinone, VA: verrucarin A).