Figure 3. The interaction of EGFR-ligand-CTFs and PLZF, and high-throughput screening for inhibitors blocking the interaction.
(A) Direct interaction between EGFR-ligand-CTF and PLZF (GST-tagged-ZnF5-8) with the SPR system. The recombinant biotin-EGFR- ligand-CTFs were individually immobilized, by SA sensor chips. GST-Zn5-8 with concentrations ranging from 0.03 to 1.0 µM was then injected with running buffer at 25°C and a flow rate of 30 µL/min for 2 min. (B) The expression vector encoding CFP-PLZF was co-transfected into wild-type HT1080 cells and HT1080 cells stably expressing either proHB-EGF, proTGF-α, proAR or proEPR. The cells were cultured for 24 h and then pretreated in serum-free medium with KB-R7785. Cells were then treated in the serum-free conditioned medium with TPA, and the subcellular localization of the CFP fusion protein was observed. To determine the percentage of cells (mean±SD) demonstrating nuclear localization of CFP-PLZF, cells were counted in at least two transfections, and at least 200 cells expressing CFP-PLZF were examined in each experiment. *P<0.05 for the stimulus effect during TPA treatment vs. no treatment, and **P<0.05 for the inhibitory effect during KB-R7785 treatment vs. TPA treatment. (C) A schematic of the high-throughput Alphascreen system. Upon excitation at 680 nm, ambient oxygen is converted to singlet oxygen (1O2) by a photosensitizer present in the donor beads. If the acceptor beads are in close proximity (<200 nm), 1O2 transfers its energy to thioxene derivatives present in the acceptor beads leading to emission of light at 520–620 nm. One complex consists of streptavidin-coated donor beads and biotinylated EGFR ligands-CTF. Another consists of anti-GST antibody-conjugated acceptor beads and GST-ZF5-8. If the association between EGFR ligand-CTF and ZnF5-8 occurs, the beads are close enough to allow detection of a signal. Any inhibitors of this interaction would increase the distance between the beads, and the signal would be lost. (D) Alphascreen signals of GST or GST-Zn5-8 incubated with various concentrations of biotin-HB-EGF-CTF. (E) Alphascreen signals of biotin-HB-EGF-CTF incubated with various concentrations of GST or GST-Zn5-8. (E) The binding abilities of HB-EGF, TGF-α, amphiregulin (AR), and epiregulin (EPR), to PLZF with Alphascreen system.