Fig 2: Effect of TL8-52 and PF-06651600 on cytokine signaling in macrophages. (A) BMDMs were isolated from wild type mice. Cells were incubated with the indicated inhibitors in μM for 2 h before stimulation with 10 ng/ml IL-4 for 30 min. The levels of phosphorylated STAT6 were then determined by immunoblotting. (B) As (A) except cells were stimulated 10 ng/ml GM-CSF and the levels of phosphorylated STAT5 determined. (C) As (A) except cells were stimulated 10 ng/ml IL-10 and the levels of phosphorylated STAT3 determined. (D) As (A) except cells were stimulated 10 ng/ml IFNβ and the levels of phosphorylated STAT1 determined. For A–D, levels of ERK1/2 were determined as a loading control.

Fig 3: Basal and phosphorylated expression of STAT3, STAT6, ERK1/2 and Akt, and activation of PI3K in C-WT, C-N and C-KD clones. WB was performed to determine the expression of relevant pathway components in C-WT, C-N-2 and C-4-1. The phosphorylation of kinases and transcription factors was detected in cells treated with recombinant IL-4 (0.4 nM for 30 min) and IL-13 (1 nM for 30 min) in comparison to untreated cells (-). β-actin was used as loading control. Abbreviations: Akt: protein kinase B; ERK: extracellular signal-regulated kinase; IRS: insulin receptor substrate; PI3K: phosphoinositide 3-kinase; STAT: signal transducer and activator of transcription.

Fig 4: IL-4-mediated induction of collagen 1 and collagen 3 expression is coupled to JAK/STAT signaling.N1 immortalized human prostate fibroblasts were serum-starved for 48 hr then treated with vehicle or IL-4 (20ng/ml) with or without 2 hr pre-treatment with 5um tofacitinib, a JAK2/JAK3 inhibitor, and maintained in serum-free media for an additional 24 or 48 hr. IL-4 significantly induced expression of collagen I (COL1) and collagen 3 (COL3) concurrent with induction of STAT6 phosphorylation, all of which was repressed upon pre-treatment with tofactinib (A). Western blots shown in A are representative of 3 replicate experiments. Densitometric data from the replicate experiments is graphed in B (COL1), C (COL3), and D (pSTAT6/STAT6) (S8 Fig). Significant differences are indicated as * p < .05; ** p < .01; *** p < .001; **** p < .0001.

Fig 5: IL-4 and growth factors amplify STAT6 activation. A, B) STAT6 activation measured in a luciferase reporter assay in BHK cells stimulated for 24 h with growth factors and a sub-maximal concentration of IL-4. A) IL-4 (3 IU/mL) and/or FGF-2 (20 ng/mL) and B) IL-4 (6 IU/mL) and/or IGF-1 (100 ng/mL). Shown is the mean and SEM of STAT6 activity determined by luminometry from a single experiment that was representative of 3 independent experiments. C, D) Immunoblots of cell lysates (not immunoprecipitation as shown in Fig. 8B) showing phospho-STAT6 in BMDMs stimulated for 20 min with L. donovani promastigotes and C) IL-4 (8 IU/mL) and/or FGF-2 (20 ng/mL) or D) IL-4 (20 IU/mL) and/or IGF-1 (100 ng/mL). Bars represent the mean and SEM of fold change with reference to the uninfected, unstimulated (Uns) controls calculated by densitometry analysis of immunoblot bands from 3 independent experiments. Also shown is a representative individual immunoblot. E) IL-4-mediated activation of STAT6 in infected BHK fibroblasts is reduced by inhibition of FGFR and IGF-1R but not ERK. The cells were exposed to the control (DMSO) or FGFR inhibitor (FGFRi; PD166866; 10 µM), IGFR inhibitor (IGFRi; PPP; 5 µM), or ERK inhibitor (ERKi; PD98059; 5 µM) for 1 hr and then stimulated for another 24 hrs with IL-4 (25 IU/mL) in the presence or absence of inhibitor. Shown is the mean and SEM of STAT6 activity determined by luminometry from a single experiment that was representative of 2 independent experiments.