Figure 5.

STAT3 knock‐down prolongs OSM‐induced STAT1 phosphorylation and ISG expression. Human dermal fibroblasts were transfected with STAT3 siRNA (siSTAT3), control siRNA (siCtrl) or only treated with transfection reagent (NT). At day two post‐transfection, cells were stimulated with 20 ng/ml hOSM for indicated time period. Total RNA was prepared and subjected to reverse transcription. (A) STAT3, (C) IRF1, (D) DDX58, (E) IFIH1, (G) SOCS3 and (H) SOCS1 mRNA levels were analysed by qRT‐PCR. Relative mRNA levels were normalized to GAPDH, and fold changes were calculated relative to untreated, NT sample (set to 1). Shown are the means (n = 3) with S.E.M. Statistical significance was assessed by unpaired t‐test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, versus siCtrl. (B, F) Whole cellular extracts were prepared, and tyrosine phosphorylation of STAT3 and STAT1 (B) as well as expression of RIG‐I, MDA5 and IRF1 (F) were determined by Western blot analysis using specific antibodies. The blots were stripped and reprobed with antibodies recognizing the proteins irrespective of their activation status. Tubulin was included as loading control. Blots shown are representative for three experiments.