Fig 2. Generation and validation of RMS cells with stable modulation of furin.

Rh30 and RD cells were stably transfected with pcDNA3.1(+) full length furin (fur) or α1-AT Portland (pdx). shRNA lentiviral particles were employed for stable furin silencing (shFA, shFE). A) Furin mRNA levels were quantified by qRT-PCR. Expression levels were normalized to GAPDH and to furin expression of the respective control cells (empty vector or scrambled shRNA). B) Levels of furin protein in stable Rh30 and RD cell lines were assessed by immunoblotting with antibody MON-152. C) Assessment of furin activity in furin overexpressing or silenced cell lines. The results were normalized over the respective wt cells on a log₁₀ scale. Data represent three independent experiments. Depicted are mean values ± SD. Two-way ANOVA, p<0.05.