Fig. 4: IL-31 exerted its effects through IL-31RA and STAT3.

a Representative western blot analysis of STAT1, STAT3, STAT5 and their phosphorylated forms in dcSSc DFs treated with rhIL-31 (50 ng/ml; left). Quantification of protein expression normalized to β-actin (right). Exact p values (10 min vs. 0 min, 20 min vs. 0 min) = 0.008, 0.008 (pSTAT1/STAT1); 0.008, 0.008 (pSTAT3/STAT3); 0.016, 0.040 (pSTAT5/STAT5). Relative fold differences (10 min, 20 min) = 3.63, 3.34 (pSTAT1/STAT1); 2.11, 7.23 (pSTAT3/STAT3); 1.36, 1.35 (pSTAT5/STAT5). b Representative ChIP assay on dcSSc DFs assessing the binding of phosphorylated STAT3 to the Col1a2 enhancer (left). Immunoprecipitated chromatin was analyzed by real-time PCR using specific primers for the HS4 region of the Col1a2 enhancer (right). Exact p value = 0.029. n = 4 biologically independent experiments. c Protein levels of type I collagen, IL-6, IL-33, and CCL2 were evaluated by ELISA in the supernatants of dcSSc DFs that were transfected with IL-31RA siRNA and treated with rhIL-31 (50 ng/ml). Exact p values (media vs. IL-31 + Ctrl siRNA, media vs. IL-31 + IL-31RA siRNA, IL-31 + Ctrl siRNA vs. IL-31 + IL-31RA siRNA) = 0.00000009, 0.111, 0.0000008 (Type I collagen); 0.0000009, 0.202, 0.000007 (IL-6); 0.002, 0.766, 0.005 (IL-33); 0.000003, 0.222, 0.00002 (CCL2). d Protein levels of Type I collagen were measured by ELISA in the supernatants of dcSSc DFs that were transfected with OSMR siRNA or treated with inhibitors for STAT1 (STAT1-I: Fludarabine; 50 µM), STAT3 (STAT-I: Stattic; 5 µM), or STAT5 (STAT5-I: CAS 285986-31-4; 50 µM), followed by the treatment with rhIL-31 (50 ng/ml). Exact p values = 0.238 (IL-31 + OSMR siRNA vs. IL-31), 0.175 (IL-31 + STAT1-I vs. IL-31), 0.008 (IL-31 + STAT3-I vs. IL-31), 0.548 (IL-31 + STAT5-I vs. IL-31). n = 5 biologically independent experiments, unless otherwise noted. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001. Significance was determined using two-tailed Mann-Whitney U test (a, b, and d) and one-way analysis of variance followed by Tukey’s post hoc comparison test (c). pSTAT1, phosphorylated STAT1; pSTAT3, phosphorylated STAT3; pSTAT5, phosphorylated STAT5. Source data are provided as a Source Data file.