Figure 4. Lipofection of top broccoletti-miR candidates does not influence basal and induced apoptosis.

(A) BxPc-3 and Bx-Gem cells were transfected as described in Figure 3. Seventy-two hours later, the cells were stained with Annexin V-PE and 7-AAD, followed by FACS analysis. The percentage of Annexin V- and 7-AAD-positive cells is shown. (B) Lipofected BxPc-3 and Bx-Gem cells were stained with an antibody specific for the proliferation marker Ki-67 (green) or the apoptosis marker cleaved fragment of caspase-3 (red), which indicates apoptosis. Representative images at ×100 magnification are shown. The percentage of Ki-67- or caspase-3-positive cells was counted in 18 visual fields, and the means ± SD are shown in the diagram below. (C) BxPc-3 and Bx-Gem were lipofected as described above, and at 24 h later, the cells were treated with gemcitabine (10 nM) or were left untreated. Ninety-six hours after gemcitabine treatment, viability was determined by MTT assay. The data are presented as the means ± SD (^** P < 0.01).