Figure 5. SINE compounds specificity analysis.

MCF-7 cancer cells with and without empty vector puro, wild type or mutant-XPO1 (lacking SINE target site Cys528 amino acid. See methods section for transfection procedures) were seeded at 3 × 10³ cells per well in 96 well-plates for MTT and 50,000 cells per well for Histone DNA ELISA. After 24 hrs, cells were exposed to 100 nM of selinexor (72 hrs). Cell death was evaluated by MTT and apoptosis by Histone DNA ELISA. (A) Protein isolated from different XPO1 construct harboring MCF-7 cells (grown at a density 1 × 10⁶ in petri dish 100 mm size) was subjected to western blotting as described above. Blots were probed for FBXL5, XPO1/CRM1 and loading control β-actin. Note: The different cells show normal expression of FBXL5 and XPO1. (B) MTT assay. (C) Apoptosis. Note: Selinexor is ineffective in mut-XPO1 harboring cells (black bars). Graphs representative of two independent experiments (D) 3000 cells were seeded in 4 well chambered slides followed by exposure to selinexor at a concentration of 1 μM for 24 hrs as described in methods section. Immunofluorescence images (40×) showing lack of snail degradation or FBXL5 nuclear localization in mut-XPO1 cells. Conversely, snail is found to be degraded in cells harboring wild type XPO1 or empty puro vector (E). Images are representative of two independent experiments.