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Figure 1 | Cancer Cell International

Figure 1

From: The chemotherapeutic agent bortezomib induces the formation of stress granules

Figure 1

Bortezomib induces SGs formation. (A) HeLa cells were treated with 1 μM bortezomib for 3 h, fixed, permeabilized, and processed for immunofluorescence using antibodies against different SG markers. DAPI is used as a marker for nuclei. Pictures were taken using a 100× objective. (B) HeLa cells were treated with 1 μM bortezomib for the indicated times, then incubated with [35S] methionine (50 μCi/ml) for another 30 min. Proteins were resolved on a SDS-polyacrylamide gel, stained with Coomassie Blue (bottom panel), and detected by autoradiography (top panel). (C) HeLa cells were treated with 1 μM bortezomib for the indicated times, and the level of phospho-eIF2α was analyzed by Western blotting using antibodies specific to the phosphorylated form (top panel). Detection of total eIF2α levels is shown in the middle panel and serves as a loading control. The activation of caspase-3 was analyzed using anti-caspase-3 antibodies (bottom panel). (D) HeLa cells were treated with 1 μM bortezomib for 10 h, fixed, permeabilized, and processed for immunofluorescence using antibodies against different SG markers. (E) The indicated histograms represent the percentage of cells harboring SGs (≥5 granules per cell) and is representative of the analysis of five different fields in three independent experiments for a total of 1000 cells. (F) Untreated HeLa cells or cells treated with bortezomib for 24 h were collected, 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 2 independent experiments.

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