Fig. 3: Jmjd3 directly regulates anti-migratory and inflammatory genes in wound keratinocytes in response to IL-17A and direct inhibition of Jmjd3 in keratinocytes improves migration/wound closure rate. | Nature Communications

Fig. 3: Jmjd3 directly regulates anti-migratory and inflammatory genes in wound keratinocytes in response to IL-17A and direct inhibition of Jmjd3 in keratinocytes improves migration/wound closure rate.

From: IL-17A is increased in diabetic wounds and impairs keratinocyte function via histone demethylase JMJD3

Fig. 3: Jmjd3 directly regulates anti-migratory and inflammatory genes in wound keratinocytes in response to IL-17A and direct inhibition of Jmjd3 in keratinocytes improves migration/wound closure rate.

a ChIP analysis of H3K27me3 or IgG at indicated promoters in keratinocytes with (white) or without (blue) IL-17A stimulation. Itga3: n = 5 (unstimulated), n = 4 (IL-17A-stimulated) technical replicates, p = 0.0034, Timp1: n = 3 technical replicates, p = 0.0046, Ccl20: n = 3 technical replicates, p = 0.0059, Cxcl1: n = 3 technical replicates, p = 0.0020, Cxcl3: n = 3 technical replicates, p = 0.0258, Cxcl5: n = 3 technical replicates, p = 0.0174. n = 3 independent experiments. b qPCR analysis of keratinocytes treated with DMSO only (white), with IL-17A alone (blue), or with IL-17A and GSK-J4 (1 µM) (red). Itga3: n = 4 biological replicates, p < 0.0001 (DMSO vs. IL-17A), p = 0.0017 (IL-17A vs. IL-17A and inhibitor), Timp1: n = 6 biological replicates, p < 0.0312 (DMSO vs. IL-17A), p = 0.0029 (IL-17A vs. IL-17A and inhibitor), Ccl20: n = 3 biological replicates, p = 0.0008 (DMSO vs. IL-17A), p = 0.0428 (IL-17A vs. IL-17A and inhibitor), Cxcl1: n = 3 biological replicates, p = 0.0003 (DMSO vs. IL-17A), p = 0.0294 (IL-17A vs. IL-17A and inhibitor), Cxcl3: n = 3 biological replicates, p < 0.0001 (DMSO vs. IL-17A), p = 0.0003 (IL-17A vs. IL-17A and inhibitor), Cxcl5: n = 3 biological replicates, p < 0.0001 (DMSO vs. IL-17A), p = 0.0007 (IL-17A vs. IL-17A and inhibitor), n = 3 independent experiments. c Western blot of ITGA-3 expression in keratinocytes treated with DMSO only (white), with IL-17A alone (blue), or with IL-17A and GSK-J4 (red). Representative densitometry plot is shown. n = 3 independent experiments. d Protein quantification of lysates from keratinocytes treated with DMSO only (white), with IL-17A alone (blue), or with IL-17A and GSK-J4 (red). TIMP-1: n = 6 biological replicates, p = 0.0012 (DMSO vs. IL-17A), p = 0.0068 (IL-17A vs. IL-17A and inhibitor), CCL-20: n = 3 biological replicates, p < 0.0001 (DMSO vs. IL-17A), p = 0.0199 (IL-17A vs. IL-17A and inhibitor), CXCL-1: n = 3 biological replicates, p < 0.0001 (DMSO vs. IL-17A), p = 0.0158 (IL-17A vs. IL-17A and inhibitor), CXCL-3: n = 3 biological replicates, p < 0.0001 (DMSO vs. IL-17A), p = 0.0167 (IL-17A vs. IL-17A and inhibitor), CXCL-5: n = 3 biological replicates, p < 0.0001 (DMSO vs. IL-17A), p = 0.0005 (IL-17A vs. IL-17A and inhibitor), n = 3 independent experiments. e qPCR analysis of keratinocytes treated with a non-targeting control (siNTC) (white) or siJmjd3 (gray). Jmjd3: n = 6 (siNTC), n = 4 biological replicates (siJmjd3), p = 0.0192, Itga3: n = 6 (siNTC), n = 4 biological replicates (siJmjd3), p = 0.0019, Timp1: n = 3 (siNTC), n = 4 biological replicates (siJmjd3), p = 0.0205, Ccl20: n = 6 (siNTC), n = 4 biological replicates (siJmjd3), p = 0.0015, Cxcl1: n = 6 (siNTC), n = 4 biological replicates (siJmjd3), p = 0.0019, Cxcl3: n = 6 (siNTC), n = 4 biological replicates (siJmjd3), p = 0.0011, Cxcl5: n = 6 (siNTC), n = 4 biological replicates (siJmjd3), p = 0.0011, n = 3 independent experiments. f qPCR analysis of Jmjd3fl/fl K14cre+ (red) and Jmjd3fl/fl K14cre- (yellow) keratinocytes. n = 3 biological replicates, Itga3: p = 0.0184, Timp1: p = 0.0044, Ccl20: p = 0.0160, Cxcl1: p = 0.0371, Cxcl3: p = 0.0042. n = 3 independent experiments. g, h Scratch assays of primary murine (n = 3 biological replicates, p < 0.0001 (48 h)) and N/TERT (n = 3 biological replicates, p = 0.0340 (12 h)) keratinocytes treated with IL-17A alone (blue) or IL-17A and GSK-J4 (red). n = 3 independent experiments. i, j Scratch assays of primary murine (n = 3 biological replicates, p = 0.0016 (48 h)) and N/TERT (n = 6 biological replicates (IL-17A alone), n = 4 biological replicates (IL-17A and GSK-J1), p = 0.0223 (8 h), p < 0.0001 (12 h)) keratinocytes treated with IL-17A alone (blue) or IL-17A and GSK-J1 (red). n = 3 independent experiments. k Scratch assay of Jmjd3fl/fl K14cre+ (red) and Jmjd3fl/fl K14cre- (blue) keratinocytes. n = 3 biological replicates, p = 0.0198 (48 h). n = 3 independent experiments. Data were analyzed for variances, and 2-tailed Student’s t tests for (a), (e), (f) and 1-way ANOVA tests for (b), (d), (g–k) were performed. Data are presented as the mean ± SEM.

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