E bound to CHO-K1/VPAC1 cells by displaying the VP2 peptide. When the concentration of exogenous VP2 peptide was increased, the number of positive VP2 phages binding to CHO-K1/VPAC1 cells decreased, and the rate ofinhibition increased gradually. When the peptide concentration was increased above 0.001 mg/ml, significant inhibition occurred, and the IC50 was approximately 18.5 mg/L (13.2 nM) (Figure 4). A control peptide (an unrelated peptide displayed by an unrelated phage) had no effect on the binding of VP2 phage to CHO-K1/ VPAC1 cells.Binding specificity of the VP2 peptide to the VPAC1 receptorTo investigate the effect of the positive phage clone and its corresponding peptide VP2 on the binding of the VPAC1 receptor to its native ligand VIP, two competitive inhibition experiments were performed. The Eltrombopag (Olamine) results of a competitive inhibition ELISA showed that with an increase in the concentration of VIP, the number of VP2 phages binding to CHO-K1/VPAC1 cells decreased, the rate of inhibition increased gradually, and theScreening of a VPAC1-Binding Peptidewas significantly inhibited, indicating that VIP had a negative effect on FITC-VP2 binding to CHO-K1/VPAC1 cells (Figure 5B). These results further confirmed that VIP and VP2 peptides could compete for the same binding site, and VP2 specifically bound to the VPAC1 receptor. When an unrelated peptide was incubated with CHO-K1/VPAC1 cells, it had no effect on the binding of FITC-VP2 to these cells (Figure 5B).Binding of VP2 to CHO-K1/VPAC1 and colorectal cancer cell linesThe results of the experiments described above demonstrate that the VP2 peptide can specifically bind to the VPAC1 receptor. To directly observe the binding of VP2 to CHO-K1/VPAC1 cells and further investigate whether VP2 could bind to CRC cells that express VPAC1 receptors at high levels, a fluorescence microscopy assay using FITC-conjugated VP2 (FITC-VP2) was performed. After CHO-K1/VPAC1, HT29, SW480, SW620 and CHO-K1 cells were incubated with FITC-VP2, specific fluorescence was observed on the membrane and in the perinuclear cytoplasm of CHO-K1/VPAC1, HT29, SW480 and SW620 cells using a fluorescence microscope. In contrast, there was no significant green fluorescence in the control CHO-K1 cells, and negative results were MedChemExpress EHop-016 obtained in all cell types when a FITC-conjugated control peptide was used in place of FITC-VP2 (Figure 6). Flow cytometry analysis indicated that the fluorescence intensities of CHO-K1/VPAC1, HT29, SW480, and SW620 cells incubated with FITC-VP2 were 87.164.1 (Figure 7A), 68.963.1 (Figure 7B), 63.463.5 (Figure 7C), and 77.864.2 (Figure 7D), respectively, and the corresponding fluorescence intensities observed when the cells were incubated with a FITC-labeled unrelated peptide (FITCURp) were 3.460.4 (Figure 7A), 3.960.4 (Figure 7B), 4.360.5 (Figure 7C), and 4.860.7 (Figure 7D), respectively (p,0.01). TheFigure 2. Specific enrichment of recovered phages. A specific enrichment of phages binding to CHO-K1/VPAC1 cells was seen after four rounds of panning. The titers of the recovered phages from each round were evaluated by the blue plaque-forming assay on LB/IPTG/Xgal plates. Here, Mp represents phages recovered from an acid elution fraction, INp represents phages recovered from a lysate fraction and CHO-K1 denotes phages recovered from CHO-K1 cells. doi:10.1371/journal.pone.0054264.gIC50 was approximately 9.1 mg/ml (2.7 mM) (Figure 5A). Because the positive phage clone bound to CHO-K1/VPAC1 cells through the.E bound to CHO-K1/VPAC1 cells by displaying the VP2 peptide. When the concentration of exogenous VP2 peptide was increased, the number of positive VP2 phages binding to CHO-K1/VPAC1 cells decreased, and the rate ofinhibition increased gradually. When the peptide concentration was increased above 0.001 mg/ml, significant inhibition occurred, and the IC50 was approximately 18.5 mg/L (13.2 nM) (Figure 4). A control peptide (an unrelated peptide displayed by an unrelated phage) had no effect on the binding of VP2 phage to CHO-K1/ VPAC1 cells.Binding specificity of the VP2 peptide to the VPAC1 receptorTo investigate the effect of the positive phage clone and its corresponding peptide VP2 on the binding of the VPAC1 receptor to its native ligand VIP, two competitive inhibition experiments were performed. The results of a competitive inhibition ELISA showed that with an increase in the concentration of VIP, the number of VP2 phages binding to CHO-K1/VPAC1 cells decreased, the rate of inhibition increased gradually, and theScreening of a VPAC1-Binding Peptidewas significantly inhibited, indicating that VIP had a negative effect on FITC-VP2 binding to CHO-K1/VPAC1 cells (Figure 5B). These results further confirmed that VIP and VP2 peptides could compete for the same binding site, and VP2 specifically bound to the VPAC1 receptor. When an unrelated peptide was incubated with CHO-K1/VPAC1 cells, it had no effect on the binding of FITC-VP2 to these cells (Figure 5B).Binding of VP2 to CHO-K1/VPAC1 and colorectal cancer cell linesThe results of the experiments described above demonstrate that the VP2 peptide can specifically bind to the VPAC1 receptor. To directly observe the binding of VP2 to CHO-K1/VPAC1 cells and further investigate whether VP2 could bind to CRC cells that express VPAC1 receptors at high levels, a fluorescence microscopy assay using FITC-conjugated VP2 (FITC-VP2) was performed. After CHO-K1/VPAC1, HT29, SW480, SW620 and CHO-K1 cells were incubated with FITC-VP2, specific fluorescence was observed on the membrane and in the perinuclear cytoplasm of CHO-K1/VPAC1, HT29, SW480 and SW620 cells using a fluorescence microscope. In contrast, there was no significant green fluorescence in the control CHO-K1 cells, and negative results were obtained in all cell types when a FITC-conjugated control peptide was used in place of FITC-VP2 (Figure 6). Flow cytometry analysis indicated that the fluorescence intensities of CHO-K1/VPAC1, HT29, SW480, and SW620 cells incubated with FITC-VP2 were 87.164.1 (Figure 7A), 68.963.1 (Figure 7B), 63.463.5 (Figure 7C), and 77.864.2 (Figure 7D), respectively, and the corresponding fluorescence intensities observed when the cells were incubated with a FITC-labeled unrelated peptide (FITCURp) were 3.460.4 (Figure 7A), 3.960.4 (Figure 7B), 4.360.5 (Figure 7C), and 4.860.7 (Figure 7D), respectively (p,0.01). TheFigure 2. Specific enrichment of recovered phages. A specific enrichment of phages binding to CHO-K1/VPAC1 cells was seen after four rounds of panning. The titers of the recovered phages from each round were evaluated by the blue plaque-forming assay on LB/IPTG/Xgal plates. Here, Mp represents phages recovered from an acid elution fraction, INp represents phages recovered from a lysate fraction and CHO-K1 denotes phages recovered from CHO-K1 cells. doi:10.1371/journal.pone.0054264.gIC50 was approximately 9.1 mg/ml (2.7 mM) (Figure 5A). Because the positive phage clone bound to CHO-K1/VPAC1 cells through the.