Fig. 2. Jak1, SOCS3 and PIAS3 in v-Src-mediated STAT3 activation. (A) Parental and Jak1-deficient human fibrosarcoma cells were transiently transfected with STAT3-YFP. Cells were stimulated with 20 ng/ml IL-6 and 1 µg/ml sIL-6R for 30 minutes (middle panel) or left unstimulated (upper panel). Cells transiently co-transfected with v-Src were left unstimulated (lower panel). Cells were fixed with paraformaldehyde and, as a nuclear marker, lamin A/C was stained. v-Src was stained with an antibody recognizing pY⁴¹⁶-Src. Cells were analyzed by confocal laser-scanning microscopy. Bars, 10 µm. (B) HepG2 human hepatocellular carcinoma cells were transiently transfected with v-Src or with an empty vector. A STAT3-responsive luciferase reporter gene construct under control of the 2M-promoter was co-transfected. Cells were incubated with 20 ng/ml IL-6 or left unstimulated for 24 hours in the presence or absence of 100 ng/ml Jak inhibitor 1 as indicated. Reporter gene assays were performed in triplicate and standard deviations were calculated. (C) HepG2 cells were transiently transfected with YFP-SOCS3 and STAT3-CFP (upper and middle panel) or with YFP-SOCS3, STAT3-CFP and v-Src (lower panel). Stimulation was performed with 20 ng/ml IL-6 for 30 minutes. Fixed cells were stained for lamin A/C or pY⁴¹⁶-Src, respectively. Images were taken by confocal laser-scanning microscopy. Bars, 10 µm. (D) HepG2 cells were transiently transfected with SOCS3, v-Src and a 2M reporter gene construct as indicated. Reporter gene assays of IL-6 stimulated (+) and unstimulated (–) cells were performed as described in B. (E) HepG2 cells were transiently transfected with FLAG-PIAS3 and STAT3-CFP (upper and middle panel) or with FLAG-PIAS3, STAT3-CFP and v-Src (lower panel). Stimulation was performed with 20 ng/ml IL-6 for 30 minutes. Fixed cells were stained for FLAG-tagged PIAS3 using a FLAG antibody and an antibody recognizing pY⁴¹⁶-Src, respectively. Cells were analyzed by confocal laser-scanning microscopy. Bars, 10 µm. (F) HepG2 cells were transiently transfected with v-Src, FLAG-PIAS3 and a 2M reporter gene construct as indicated. Reporter gene assays were performed as described in D.