Figure 3

Overexpression of TIMP-2 inhibited the MMP-2 activity of ameloblastoma cells. Ameloblastoma cells were transfected with mock, lipofectamine, pcDNA-neg, pcDNA-TIMP-2 (1 μg), pcDNA-TIMP-2 (2 μg), or pcDNA-TIMP-2 (3 μg) for 72 h. GAPDH was included as an internal control to normalize the amounts of protein or mRNA. The relative protein or mRNA level of TIMP-2 was depicted as the ratio of the density of TIMP-2 to GAPDH for the same time point. The results are a representative experiment among three. *Compared with the mock, lipifectamine or vector controls, respectively, p < 0.05. (A) Western blotting (WB) was used to detect TIMP-2. TIMP-2 protein levels increased in ameloblastoma cells after transfection with pcDNA-TIMP-2. (B) RT-PCR was used to detect the transcription of TIMP-2 after pcDNA-TIMP-2 transfection. Transcript levels of TIMP-2 in TIMP-2-transfected cells were significantly higher compared with cells transfected with mock and lipofectamine and pcDNA-neg. (C) Zymographic analysis for MMP-2 and TIMP-2 activity in the conditioned medium of ameloblastoma cells transfected with various doses of pcDNA-TIMP-2. The zymography assay revealed that pcDNA-TIMP-2 transfection decreased MMP-2 activity. The inhibition rates were 31.88, 67.87, and 43.57% for 1, 2, and 3 μg, respectively. Reverse gelatin zymography showed that pcDNA-TIMP-2 transfection increased TIMP-2 activity.