Figure 4.

cy(WPHPY) inhibited the TIMP2–proMMP2 interaction and prevented proMMP2 activation. (a) Principle and results of the ELISA-based competitive binding assay. Surface-immobilized recombinant TIMP2 can pull down MMP2 from the solution, while cy(WPHPY) binds to MMP2 and blocks this interaction. Phosphate-buffered saline (PBS) was used in place of cy(WPHPY) in the control sample. (b–f) Schematic illustration and results of the in vitro bioactivity assay evaluating the function of cy(WPHPY). (b) Media from WM115 cell culture were collected and incubated with varying concentrations of cy(WPHPY). The corresponding MMP2 activities were analyzed using an MMP2-specific fluorogenic substrate. The assay buffer was used in place of cy(WPHPY) in the control sample. (c) WM115 culture media were incubated with TIMP2 and cy(WPHPY), and the corresponding MMP2 activities were evaluated. For the control samples, the assay buffer was used in place of cy(WPHPY) and TIMP2. (d–f) WM115 cells were cultured with varying concentrations of cy(WPHPY), and the corresponding media were collected for subsequent analyses. Serum-free media without cy(WPHPY) were used for the control samples. (d) Gelatin zymography and western blotting results demonstrate that cy(WPHPY) inhibited the MMP2 activity in the WM115 cell culture. (e, f) Enzymatic activity assays show that cy(WPHPY) was highly specific to MMP2 activities and did not affect MMP9. (g, h) Confocal microscopy images of WM115 cells showing membrane-bound MMP2 immunofluorescence. The scale bars are 10 μm. Media without cy(WPHPY) were used in the control sample. (i) Comparison of the single-cell MMP2 immunofluorescence signals extracted from the confocal images. A total of 30 single cells were analyzed for each image. The boxes denote the middle two quartiles, and the horizontal blue bars represent median values. The whiskers show the standard deviation values.