Fig. 6: Knocking down SPRY2 or MET induces RMS cell differentiation.

Representative bright field photomicrographs showing elongated, multinucleate myofiber like structures, marked with red arrows, in RD (b, b′, c, and c′) and SJRH30 (f,f′,g and g′) cells transfected with SPRY2 and MET siRNAs, compared to control cells (a, a′, e, and e′). The cells were imaged at 144-h PsiRT. This was further validated by immunofluorescence for myosin heavy chain (MHC) on RMS cells transfected with MET (c″, g″), SPRY2 (b″, f″), or control (a″, e″) siRNAs. Myofiber like structures with nuclei arranged linearly (marked with white arrows) were induced by downregulation of SPRY2 or MET in RD (b″,c″) and SJRH30 (f″, g″) cells, where MHC is labeled in red, F-actin marking the actin cytoskeleton in green and nuclei stained with DAPI in blue. These fiber like structures were absent in control siRNA transfected RD (a″) and SJRH30 (e″) cells. Scale bar in panels (c, g) is 100 µm, (c′,g′) is 50 µm and (c″, g″) is 25 µm. Immunoblots of MET or SPRY2 siRNA transfected RD (d) and SJRH30 (h) cell lysates, prepared at 144-h post transfection, show increased MHC expression as compared to control siRNA transfected cells. This substantiates the phenotypic differentiation observed in RD and SJRH30 cells seen by bright field and immunofluorescent imaging