Figure 4

The formation of SGs correlates with resistance to bortezomib-mediated apoptosis. HeLa, Calu-1, and Hs578T cells were treated with bortezomib for 3 h (A) or 24 h (B to D). (A) Cells were processed for immunofluorescence to detect SGs using anti-FMRP and anti-G3BP antibodies. (B) Cells were stained with annexin V-FITC and PI, and analyzed by flow cytometry. The percentage of total dead or dying cells (indicated at the top of each panel) was defined as the sum of early (lower right box) and late (upper right box) apoptosis and is presented as the means ± SEM from two independent experiments. (C) Cells were harvested and protein extracts analyzed by Western blot for the activation of caspase-3 using anti-caspase-3 antibodies. G3BP serves as a loading control. (D) Following bortezomib treatment for 24 h, cells were trypsinized, replated in the absence of drug, and incubated for 10 d. Before colony counting, cells were fixed and dried. Populations > 50 cells were counted as one surviving colony. Data were calculated as the percentage of surviving colonies relative to untreated plates. The results are presented as the mean of triplicate measurements.