Fig 2: IL‐38 reduced the TNF‐α‐induced production of MMP‐9 and MMP‐2 in macrophages in vitro. (a) Representative western blot analysis of iNOS and GAPDH immunoreactivity in RAW 264.7 cells after TNF‐α stimulation (0, 1, 5, 10, 20, 50 ng/ml). (b) Bar graph showing relative densitometry of iNOS (n = 3, p = 0.0411 [Kruskal–Wallis test], p = 0.0233 [post hoc, 0 vs. 20 ng/ml]). (c) Representative western blot analysis of IL1RL2 and GAPDH immunoreactivity in RAW 264.7 cells after TNF‐α stimulation (20 ng/ml). (d) Bar graph showing relative densitometry of IL1RL2 (n = 6, p = 0.0450 [t test], p = 0.0043 [Mann–Whitney U test]). (e) Representative photomicrographs of morphological changes of RAW 264.7 cells after TNF‐α stimulation (20 ng/ml) with or without IL‐38 treatment (100 ng/ml). Scale bar, 500 μm. (f) Bar graph showing the ratio of spindle‐shaped cells among macrophages after TNF‐α stimulation (20 ng/ml) with or without IL‐38 treatment (100 ng/ml; n = 3, p < 0.0001 [ANOVA], p < 0.0001 [post hoc, Control vs. TNF‐α], p < 0.0001 [post hoc, Control vs. TNF‐α + IL‐38], p < 0.0001 [post hoc, TNF‐α vs. TNF‐α + IL‐38]). (g) Representative western blot analysis of iNOS, MMP‐9, MMP‐2, and GAPDH immunoreactivity in RAW 264.7 cells after TNF‐α stimulation (20 ng/ml) with or without IL‐38 treatment (100 ng/ml). (h) Bar graphs showing the relative densitometry of iNOS (n = 6, p = 0.0003 [ANOVA], p = 0.0016 [post hoc, Control vs. TNF‐α], p = 0.0019 [post hoc, Control vs. TNF‐α + IL‐38], p = 0.0112 [post hoc, TNF‐α vs. TNF‐α + IL‐38]), MMP‐9 (n = 6, p = 0.0002 [ANOVA], p = 0.0009 [post hoc, PBS vs. TNF‐α], p = 0.0470 [post hoc, PBS vs. TNF‐α + IL‐38], p = 0.0176 [post hoc, TNF‐α vs. TNF‐α + IL‐38]), and MMP‐2 (n = 6, p = 0.0097 [ANOVA], p = 0.1222 [post hoc, Control vs. TNF‐α], p = 0.3385 [post hoc, Control vs. TNF‐α + IL‐38], p = 0.0075 [post hoc, TNF‐α vs. TNF‐α + IL‐38]).