SINEs-specifically inhibit nuclear export. (A) Confocal fluorescence microscopy for p53, FoxO3a, and IκB show time-dependent increases in nuclear levels of these proteins in KPT-185 treated cells compared with vehicle control. Results shown are representative of 5 experiments. Z stacks were collected (0.4 μm per slice) and images were chosen from the middle of nuclei. Side views (across bottom and side of figures) are also shown. (B) Nuclear and cytosolic fractions were isolated from KPT-185 treated CLL cells (12 and 24 hours) and analyzed by immunoblot for AKT, FoxO3a, IκB, p53, and BRG1. Results shown are from 1 representative patient sample. (C) CD19⁺ cells from CLL patients (N = 3) were incubated with 1μM KPT-185 for 12 hours. EMSA was done with nuclear extract using a radio-labeled oligonucleotide containing a consensus NF-κB binding site. KPT-185–treated samples were also incubated with antibodies specific to the p65 or p50 subunits of NF-κB. The p65/p50 complex is indicated by arrows. Results are shown from 3 of 3 experiments. (D) Nuclear and cytosolic fractions were isolated from KPT-185 treated CLL cells (12 and 24 hours) and analyzed by immunoblot for p50 and p65, and BRG-1. Results shown are from 1 representative patient sample. (E) Real-time RT-PCR for Mcl1, Bcl-2, and Bcl-xL after 12 hours (0.5μM) KPT-185 treatment. Data are normalized to 18S transcript and represented as fold change in expression of KPT-185 treated relative to the vehicle control. Squares represent individual patient samples, and horizontal bars represent the average. (F) Whole cell expression of Mcl1 and Bcl-2. Results shown are from 2 representative patient samples.