Figure 4. Activity of IHCA gp130 mutants is selectively dependent upon JAK1. (A) Two different siRNAs silencing JAK1 (siJAK1#1 and #2) and two different siRNAs silencing JAK2 (siJAK2#1 and #2) or TYK2 (siTYK2#1 and #2) were tested for their ability to interfere with STAT3 activity in HepG2 cells co-transfected with a STAT3-luciferase reporter and an empty plasmid (EP)- or a plasmid coding for the Y186 gp30 mutant (n = 3). Where indicated, cells were treated for 3 h with 100 ng/mL interleukin-6 (IL-6). Shown are the mean luciferase activity ± SD relative to cells receiving the EP and a non-targeting control siRNA (siCTL). (B) Hep3B cells transfected with a plasmid encoding the Y186 gp130 mutant (filled circles) or the corresponding EP and then treated with 100 ng/mL IL-6 were exposed for 16 h to increasing concentrations of the JAK1 inhibitor ruxolitinib (n = 3). The data shown are the mean luciferase activities ± SD relative to Hep3B cells not treated with the inhibitor. (C) Hep3B cells transfected with constructs coding for nine different IHCA gp130 mutants (K173, V184, Y186, S187, V189, E195, D215, P216 and A418) or the corresponding EP were treated with 100 ng/mL IL-6 and exposed for 18 h to increasing concentrations of ruxolitinib. Shown are the mean luciferase activities ± SD from triplicate assessments relative to transfected cells not treated with the inhibitor. Statistical significance was determined by 2-tailed Student t test; **P < 0.01; ***P < 0.001.