Figure 5
siRNA-mediated reduction of SOCS1 and SOCS3 enhances IFN-responsiveness of melanoma cells. Real Time PCR was used to validate reduced expression of (a) SOCS1 and (b) SOCS3 48 hours following transient transfection of 1259 MEL cells with empty pSilencer vector (Control) or pSilencer expressing SOCS-specific siRNA. Data were normalized to β-actin (housekeeping gene) and expressed as mean expression values relative to cells transfected with control vector. (c) P-STAT1 levels were measured following a 15 minute stimulation of melanoma cells with IFN-α (104 U/mL) or IFN-γ (1 ng/mL) by flow cytometry. PBS (vehicle) treated cells served as negative controls. Data represent the mean Fsp values (± standard deviation) from n = 2 experiments with similar results. (d) IFN-stimulated gene expression was evaluated in response to a 4 hour treatment with IFN-α (104 U/mL) 48 hours post-transfection with empty vector (negative control) or vector expressing SOCS1- or SOCS3-specific siRNA. Data were normalized to β-actin and expressed relative to PBS-treated cells. * Denotes p < 0.05 as compared to empty vector transfected cells.