Fig. 2: MET protein levels depend on SPRY2 expression in ERMS and ARMS cells. | Cell Death & Disease

Fig. 2: MET protein levels depend on SPRY2 expression in ERMS and ARMS cells.

From: SPRY2 is a novel MET interactor that regulates metastatic potential and differentiation in rhabdomyosarcoma

Fig. 2

Relative quantification of MET and SPRY2 transcripts by qPCR at 48 and 144-h post MET, SPRY2, or control siRNA transfection in RD (a, d) and SJRH30 (g, j) RMS cells. The transcript levels were normalized to control genes, GAPDH and HPRT, and compared to control siRNA treated RMS cells. MET and SPRY2 siRNAs show >90% knockdown of their specific transcripts, upper and lower bar graphs respectively, in RD cells at 48-h (a) and 144-h (d) post transfection. MET siRNA shows similar knockdown efficiency of >90% in silencing MET transcripts (upper bar graphs) in SJRH30 cells at 48-h (g) and 144-h (j) post transfection. A knockdown of >90% and ~82% was observed in SPRY2 transcript levels (lower bar graphs) in SJRH30 cells at 48-h (g) and 144-h (j) post transfection respectively. Representative western blots of MET or SPRY2 siRNA transfected RD (b, e) and SJRH30 (h, k) cell lysates show efficient knockdown of MET (upper panels) and SPRY2 (middle panels), as compared to control siRNA transfected cells, at 48-h (b, h) that is maintained at 144-h (e, k) post-transfection in both the cell lines. Densitometry shows significant decrease in MET protein levels in SPRY2 siRNA transfected RD cells at 48-h (c) and 144-h (f) post transfection, whereas the SPRY2 protein expression is unchanged in MET siRNA transfected RD cells at both the time points. MET levels are also significantly downregulated in SPRY2 siRNA transfected SJRH30 cells, but only at 144-h (l) post transfection. Notably, unlike RD cells, SPRY2 expression is significantly reduced in MET siRNA transfected SJRH30 cells at both 48-h (i) and 144-h (l) post transfection. RD and SJRH30 cells transfected with SPRY2 or control siRNA for ~96-h were treated with Bafilomycin A1, MG132, or vehicle and cell lysates were analyzed for MET stabilization (m to t). Representative western blots show levels of MET and SPRY2 proteins in siRNA transfected and Bafilomycin A1, MG132 or vehicle treated RD (m,q) and SJRH30 (o,s) cell lysates. Densitometry was performed using β-ACTIN as the loading control. In SPRY2 silenced RD cells, MG132 treatment (r) stabilizes MET levels significantly but not upon treatment with Bafilomycin A1 (n). Conversely, in SJRH30 cells, MET levels upon SPRY2 siRNA treatment are stabilized significantly by Bafilomycin A1 (p) and are not significantly stabilized by MG132 (t). The graphical data represent the mean ± SEM of a minimum of three independent experiments

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