Figure 9: FNDC4 signals partly via STAT3.
From: FNDC4 acts as an anti-inflammatory factor on macrophages and improves colitis in mice
(a) Socs3 gene expression after 4 h incubation with 100 nM hFc-FNDC4 or hFc control. Mean±s.e.m., N=3. *P<0.05 (T-test). (b) Phosphorylation of STAT3. Bone marrow-derived macrophages were starved overnight followed by 30 minutes treatment with 200 nM or 300 nM hFc-FNDC4, 200 nM hFc served as control. The full-size, uncropped blot image is presented in Supplementary Fig. 11c. Similar effects were observed in two repeated experiments. (c) STAT3 DNA binding to the Socs3 promoter region. Macrophages were treated with hFc-FNDC4 or hFc for 6 h. Data presented as percentage of input. Mean±s.e.m., N=4. *P<0.05 (T-test). (d) STAT3 inhibition reverses FNDC4-mediated gene regulation. Bone marrow-derived macrophages were pretreated with 50 μM S3I-201 STAT3 inhibitor or DMSO as control for 45 min and subsequently treated with 100 nM hFc-FNDC4 or hFc control for 6 h in the presence or absence of S3I-201. Mean±s.e.m., *P<0.05 and q<0.10, One-way analysis of variance followed by Tukey’s post hoc tests and Benjamini-Hochberg false discovery rate-adjustment, N=4. (e) STAT3 inhibition reverses FNDC4-mediated improvements of macrophage survival. Bone marrow macrophages were pretreated with 50 μM S3I-201 STAT3 inhibitor or DMSO as control for 45 min and subsequently treated with 100 nM hFc-FNDC4 or hFc control for 48 h in starvation media in the presence or absence of S3I-201. NT, non-treated. Mean±s.e.m., *P<0.05, one-way analysis of variance followed by Tukey’s post hoc tests, N=8 per condition. (f) Proteins showing a >1.3-fold increase or decrease in phosphorylation status in primary bone marrow macrophages on 30-min treatment with hFc-FNDC4 or hFc control. The fold change was calculated as the signal ratio of the paired antibodies for each pair of site-specific antibody and phospho site-specific antibody. Average ratios of two duplicates per condition. * Indicate that the fold induction in phosphorylation status is due to a reduction in binding of the total protein (conformational change affecting the accessibility of the epitopes; the array is performed using non-denatured protein).
