Figure 3
From: The chemotherapeutic agent bortezomib induces the formation of stress granules
Reducing HRI levels by siRNA promotes bortezomib-mediated apoptosis. (A) HeLa cells were transfected for 48 h with HRI-1 or control siRNA, treated with bortezomib for 24 h, and total extracted proteins were analyzed by Western blot using anti-caspase-3 antibodies (top panel). CP: cleaved product. FMRP serves as a loading control (bottom panel). (B) Following treatment with HRI-1 or control siRNA, HeLa cells were incubated with bortezomib for 24 h, then 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 mean ± SEM from two independent experiments. (C) Following treatment with HRI-1 or control siRNA, HeLa cells were incubated with bortezomib for 24 h, trypsinized, counted, 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 control (untreated) plates. The results are expressed as the mean of triplicate measurements.