Figure 2

Cytokine-induced apoptosis in human keratinocytes is suppressed by SOCS1 and SOCS3 molecules. (a) HaCaT cells were stably transfected with myc-tagged socs1, socs2 and socs3 genes or control plasmids. MOCK (, n=6), SOCS1 (, n=6), SOCS2 (, n=6) and SOCS3 (, n=6) clones were left untreated or stimulated with TNF-α for 48 h and their apoptosis was examined by measuring Annexin/PI fluorescent staining or expression of active caspase 9 through FACS analysis. Data were expressed as F.I. between TNF-α-treated samples and untreated samples, to which a value of 1 was given. Ectopic SOCS1, SOCS2 and SOCS3 expression in different clones was determined by immunoblotting with an anti-c-myc Ab. (b) Transient RNA interference was performed by transfecting cultured healthy keratinocytes with SOCS1, SOCS3 or irrelevant (NC) siRNA, and then by treating cells with IFN-γ/TNF-α for 48 h. Apoptosis was evaluated as described in (a). SOCS1 and SOCS3 mRNA levels in siRNA-transfected cells were measured by real-time PCR. SOCS3 mRNA levels were monitored to validate the specificity of SOCS1 siRNA, and vice versa. All data were obtained from three independent experiments of RNA interference. Values of P<0.05 (*) and P<0.01 (**) were considered significant