Fig. 2

Effect of CIGB-300 on CK2-dependent signaling pathways. a Nuclear, cytoplasmic or whole cell extracts prepared from H125 cells treated with low-lethal doses of CIGB-300 in combination or not with an activating stimulus, were resolved on 10% SDS-PAGE and blotted with p65/RelA and P-p65 (Ser529) antibodies (50 μg protein/lane). ACTIN expression level was used as protein loading control. For the determination of p65 levels PMA was used as the activating stimulus, while TNFα was used for P-p65 determinations. Results are representative of three independent experiments. b Whole cell extracts prepared from H125 cells treated with CIGB-300, were resolved on 10% SDS-PAGE and blotted with CYCLIN D1, CYCLIN E and BAX antibodies (50 μg protein/lane). ACTIN expression level was used as protein loading control. c H125 cells were transiently co-transfected with pNF-κB-RE-luc and pRL-TK-luc vectors, treated with low-lethal doses of CIGB-300 in combination or not with PMA and luciferase activity was determined. Data were normalized to the constitutive Renilla luciferase activity and expressed as the mean ± SD. **p < 0.01 vs. PMA treated cells and *p < 0.05 PMA + CIGB-300 treated cells vs. PMA treated cells (one-way ANOVA test). Results are representative of three independent experiments. d H125 cells were treated with low-lethal doses of CIGB-300 in combination or not with PMA. Cellular distribution of p65 was visualized by immunofluorescence microscopy. Figure shows representative images of three independent experiments, scale bar 50 μm. White arrows indicate highly positive nucleus. Representative insets of cells treated with PMA in combination or not with CIGB-300 are shown, scale bar 5 μm (left side). Relative p65 nuclear intensity was measured and represented as a percentage of control cells (light gray bars). **p < 0.01 vs. CIGB-300 treated cells (one-way ANOVA test). The percentage of those cells with highly positive nucleus (white arrows) was also measured (dark gray bars). **p < 0.01 vs. PMA treated cells and *p < 0.05 PMA + CIGB-300 treated cells vs. PMA treated cells (one-way ANOVA test) (right side). e H125 cells were incubated with CM containing the wnt3a factor and then treated with low-lethal doses of CIGB-300 or TBB. Cytoplasmic protein content was separated by SDS-PAGE 10% and the membrane was blotted with anti β-CATENIN antibody (50 μg protein/lane). ACTIN expression level was used as protein loading control. Results are representative of three independent experiments