Figure 3

The effect of CTCF knock-down on apoptosis and proliferation in leukemic cells. (A) sh-CTCF-1 and sh-CTCF-2 are shRNA plasmids specific for CTCF. sh-luc (targeting firefly luciferase) is the shRNA plasmid specific for firefly luciferase and was used as a negative control. CTCF expression levels were semi-quantified by analysis of the Western blot with Gel-Pro Analyzer software. The sh-CTCF-2 plasmid showed better knock-down efficiency than the sh-CTCF-1 plasmid. (B) Knock-down of CTCF resulted in increased early cell apoptosis [Annexin V (+)/PI (-)] and late cell apoptosis [Annexin V (+)/PI (+)]. The percentages of cells in early and late apoptosis are indicated in the figure, respectively. sh-luc was used as a control. (C) The percentage of cell apoptosis (both early and late apoptosis) in each group is shown in the diagram as mean ± SD from three independent experiments (p = 0.001). (D) Effect of knocking down CTCF mRNA on caspase-3 activation in Nalm-6 cells. The expression levels of procaspase-3, cleaved caspase-3 and transfected plasmids were determined using Western blot. (E) Knock-down clones were seeded in a 96-well plate at 1 × 10⁴ cells per well, with three replicates per clone. Viable cells were counted using the CCK-8 assay for 5 days. (F) The percentage of cell viability normalized to nonspecific sh-luc is presented as mean ± SD from three independent experiments.