FIGURE 2.

Expression and cleavage of HuR during CoCl₂-induced hypoxia. A, total protein (50 μg) from UM74B oral cancer cells was grown under 250 μm CoCl₂ used for Western blot analysis. Cells were treated and harvested at the indicated time points to identify HuR cleavage. The blots were probed for HuR and β-actin (used as loading control). B, active caspases are required for the appearance of the 24-kDa HuR fragment. Cells were incubated with DMSO (first lane 1), CoCl₂ (second lane), and CoCl₂ + benzyloxycarbonyl-Val-Ala-Asp (zVAD; third lane) followed by Western blotting for HuR, performed as described above and probed with antibodies to active caspases-3 and -7. β-Actin serves as a loading control. C, UM74B cells treated with siRNA are described under “Experimental Procedures” in the presence of 250 μm CoCl₂ for 36 h were analyzed by staining with annexin V-FITC and propidium iodide by flow cytometry. The percentage of apoptotic cells (top boxes) upon CoCl₂ treatment was determined for HuR (siRNA-HuR) or control siRNA (siRNA-SCR)-treated HeLa cells. The values were normalized to control untreated cells. The graph represents the number of apoptotic cells after treatment as described in C. Values are the means ± S.E. (error bars) from three independent experiments. D, immunofluorescence detection of HuR in UM74B cells, either untreated or treated with CoCl₂ as indicated. Distribution of cytoplasmic HuR (merged panel) was observed after 24 h. Blue indicates DAPI staining used to detect nuclei, red indicates β-actin staining to detect cytoplasm, and green indicates HuR immunofluorescence. The scale bar denotes 5 μm.