Figure 3.

Cellular depletion of the individual CK2 catalytic subunits enhances gemcitabine-mediated cell death. A. PANC-1 cells were left untreated (CT), treated with transfection reagent (TR) or siRNAs against the catalytic α- and α'-subunits of CK2 for 96 h. One day after transfection, cells were cultured for 72 h in the presence of 50 nM gemcitabine. Total cell lysates (50 μg) were analyzed by Western blot using the indicated mouse monoclonal antibodies. β-actin was used as control for equal loading. Insert shows Western blot analysis (long autoradiographic exposure) of whole extracts from cells treated with transfection reagent, CK2α-siRNA or CK2α'-siRNA, using anti-CK2α/α' antibody. Data shown are representative of three independent experiments. B. Cells were treated as described in A. Total lysates (15 μg) were subjected to a radioactive CK2 kinase assay with a specific CK2 peptide substrate as described in [14]. The average +/- STD of three independent experiments are shown. C. Cells treated as described above were fixed, stained with propidium iodide and analyzed by flow cytometry. The fraction of dead cells expressed in percentage (average from four independent experiments +/- STD) is reported on the ordinate axis after subtraction of the average sub-G1 value (i.e. 17.5%) of cells treated with transfection reagent. Stars denote statistically significant differences in the percentage of cell death after treatment with CK2α-siRNA and gemcitabine or CK2α'-siRNA alone or in combination with gemcitabine as compared to the treatment with the sole gemcitabine (Student's t-test, P < 0.05). CK2α'-siRNA treatment was tested against CK2α'-siRNA in combination with gemcitabine (no statistically significant difference). Statistically significant difference was found when comparing gemcitabine treatment versus control (results not shown).