Fig. 3: SPRY2 colocalizes and interacts with MET in RMS cells.

Immunocytochemical staining shows that SPRY2 (red) and MET (green) colocalize with each other in RD (a–a″) and SJRH30 (b–b″) cells, suggesting that SPRY2 might interact with MET in embryonal and alveolar sub-types of RMS. Panels a′′′ and b′′′ represent magnified images (×10) of regions marked by white squares in a′′ and b′′. Nuclei are stained with DAPI (blue) and the scale bar is 50 µm and 5 µm in panels b″ and b′′′, respectively. Quantification of SPRY2-MET colocalization was performed as described in materials and methods. Representative scatter plots showing SPRY2 (red) and MET (green) colocalization signal in the entire cell in RD (a″″) and SJRH30 (b″″) cell lines. Pearson’s correlation co-efficient of the representative RD (a″″) and SJRH30 (b″″) scatter plots are 0.6608 and 0.6878, respectively. Bar graph showing the mean Pearson’s correlation coefficient for SPRY2-MET colocalization in RMS cells (c) (n = 17), where perfect correlation=1, no correlation=0 and error bar represents ± SEM. Equal concentrations of whole cell lysates from RD (d) and SJRH30 (e) cells were immunoprecipitated (IP) using anti-MET antibody. The immunocomplexes were probed with anti-MET and anti-SPRY2 antibodies by immunoblotting. IP was also performed using IgG isotype control. SPRY2 immunoprecipitated with MET in both RMS cell lines (d, e)