A. Ror2 expression is suppressed in 786-0 cells by shRNA. Whole cell protein extracts from RCC cells (786-0) were infected with a scramble short hairpin retrovirus, a pRS control virus or Ror2 short hairpin retroviruses and immunoblotted with polyclonal Ror2 antibody, Twist1 antibody or Ku80 antibody as a loading control (LC).
B. MMP2 expression is suppressed when Ror2 levels are knocked down. Quantitative RT-PCR analysis of two 786-0 Ror2 knockdown cell lines demonstrates MMP2 suppression coordinate with the degree of suppression of Ror2. Significant differences were observed in MMP2 (**p<0.001 for both comparisons) in the 786-0 Ror2 suppressed cell lines compared to the Ror2 expressing cell lines (**p<0.001 for both comparisons). Transcript values are normalized to β-actin RNA internal standard and are shown relative to 786-0 scramble RNA. Error bars represent SEM.
C. Ror2 knockdown decreases cell migration. RCC cells (786-0) infected with a control virus pRS or Ror2 short hairpin RNA retroviruses were plated and allowed to grow overnight. Cells were scratched and the length of the scratch was observed for both 0 hr and 16hr timepoints (left). Significant differences were observed when comparing the % invasion of the Ror2 knockdown cell lines compared to the empty vector pRS control (right) - (**p<0.0001, *p=0.009). Error bars represent SEM.
D. Ror2 knockdown inhibits anchorage independent growth. RCC cells (786-0) infected with a scramble short hairpin RNA retrovirus, a control virus pRS or Ror2 short hairpin RNA retroviruses were allowed to grow in soft agar over a period of 3-4 weeks. Ror2 knockdown cells have inhibited growth in comparison to the scramble short hairpin retrovirus and the empty vector pRS control. Multicellular colonies >150 uM were counted from triplicate plates. Data shown is from the combination of two representative experiments (**p<0.0001). Error bars represent SEM. Inset - Cells were stained with MTT dye, and pictures taken at 10x magnification. Black bar represents 67.3 um.