Team:Nanjing high school/Results


Figure 1. SDS-PAGE assay
        First of all, 6×His-PPM1A was purified with a Ni-NTA column followed by AKTA FPLC according to the protocol. Then we used SDS-PAGE to test the purity of 6×His-PPM1A. As shown in Figure 1, the 6×His-PPM1A was purified successfully.
Figure 2. PPM1A enzyme activity
        Next, we screened the PPM1A activator in Lab in-house compound library by phosphatase enzyme activity assay. The effect of compounds 5 (0.01, 0.1, 1, 5, 10, 20, 40, 100 μM) on the PPM1A was detected by phosphatase activity assay with pNPP as the substrate. All data were presented as mean ± S.E.M (*P<0.05, **P< 0.01, ***P<0.001).
        As indicated in Figure 2, among the compounds, Compound 5 was finally selected for its highest enzymatic activity against PPM1A. Besides, it could also tell that compound 5 dose-dependently enhanced PPM1A enzyme activity. These results thus implied that compound 5 was a PPM1A enzymatic activator.
Figure 3. Compound 5 suppressed inflammation in BV-2 cells
        Finally, the qPCR assay was further carried out to verify the inhibitive effect of compound 5 against PPM1A. BV-2 cells were co-incubated with LPS and different concentrations (5, 10, 20 μm) of compound 5 for 24 h. Then the mRNA level of IL-1β and IL-6 were detected by the qPCR assay. All data were presented as mean±S.E.M (*P<0.05, **P< 0.01, ***P< 0.001).
As shown in Figure 3, LPS effectively increased the mRNA level of IL-1β and IL-6, and compound 5 suppressed this increase effectively. Thus, these results confirmed that the suppressive effect of compound 5 against inflammation in BV-2 cells.
        In this project, Compound 5 was demonstrated as a PPM1A activator and its anti-inflammatory effect was determined. We will investigate the regulation of PPM1A against inflammation and the diseases related to inflammation in future scientific research work.