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. 2012 Apr 10;22(9):1374–1389. doi: 10.1038/cr.2012.57

Figure 3.

Figure 3

DHPS upregulated WDR83 mRNA and protein expression through the 3′UTRs. (A) Knockdown of DHPS by siRNAs in MGC803 cells reduced WDR83 mRNA levels by 31.4% (n ≥ 3, P = 0.0039). (B) Western blot analysis demonstrated that the siRNAs knockdown of DHPS decreased WDR83 protein expression. (C) Quantification of WDR83 mRNA levels after overexpression of three different plasmids in MGC803 cells. qPCR analysis indicated that overexpression of DHPS-full length or DHPS-3′UTR resulted in a significant increase in WDR83 mRNA levels (n ≥ 3, P = 0.0110 and 0.0080, respectively). (D) Western blot showing that WDR83 protein levels were significantly increased in MGC803 cells transfected with DHPS-full length or DHPS-3′UTR plasmids. (E) WDR83 mRNA levels were measured by qPCR in MGC803 cells where endogenous DHPS were first knocked down by siRNA for 24 h and then restored by exogenous expression of DHPS-3′UTR (pIRES2-DHPS-3′UTR) for further 24 h. (F) The luciferase reporter assay showed that the WDR83 3′UTR-containing construct induced a 24.4% increase in luciferase activity in cells that were cotransfected with the DHPS 3′UTR plasmid (P = 0.0020), and the WDR83 full-length construct also showed an increase in luciferase activity (P = 0.0310). The DHPS CDS-containing construct did not have an obvious effect on pmirGLO-WDR83-3′UTR (P = 0.7845). Meanwhile, silencing DHPS decreased luciferase activity (P = 0.0158). Relative firefly luciferase activity over renilla luciferase activity: FL/RL, n ≥ 3, ANOVA; *P< 0.05, **P< 0.01, ***P< 0.001.