Figure 4. Ror2 regulation of MMP2 expression in RCC cells is dependent upon an intact kinase domain.

Quantitative RT-PCR for 786-0 cells shows a significant correlated decrease in A) Ror2 and B) MMP2 mRNA levels in both shRNA knockdown cell lines relative to control. C) Additionally, induction of wildtype Ror2 and Ror2-DM is evident following treatment with doxycycline (500 ng/ml). D) But a matching increase in MMP2 is only seen with overexpression of wildtype Ror2. For all quantitative RT-PCR assays, transcript values were normalized to β-actin RNA internal standard with fold change calculated in reference to either 786-0 control or unstimulated GFP expressing cells. Error bars represent SEM across triplicates of a representative duplicated experiment. Gelatin Zymography shows increased levels of both the precursor pro-MMP2 and its cleaved active form with overexpression of wildtype Ror2 relative to GFP vector control in both E) 786-0 and F) HEK293T cells. Quantification of the levels of active MMP2 normalized to the media only control (Con) is shown below each gel. G) Invasion of 786-0 cells seeded in Boyden matrigel coated chambers under normal culture conditions show a significant decrease upon treatment with MMP2 inhibitor (OA-Hy - 60 uM) in both independent shRNA knockdowns and Ror2 overexpression in comparison with vehicle control. The percentage of area covered was quantitated from random fields using ImageJ. Error bars represent stdev across duplicates of a representative duplicated experiment. P-values were calculated using an Anova one-way analysis (*<.05, **<.001).