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. 2021 Jan 4;131(1):e139481. doi: 10.1172/JCI139481

Figure 8. Neutrophil elastase downregulates mRNA expression of fibulin-5 in saccular-stage mouse lung fibroblasts through EGFR/MEK/ERK signaling.

Figure 8

P5 mouse lung fibroblasts were grown to confluence and cultured with or without purified human neutrophil elastase (HNE) at 1 or 4 μg/mL or TGF-α (50 ng/mL) for up to 24 hours. In some experiments, fibroblasts were treated with AG1478 20 μM (EGFR inhibitor) or U0126 25 μM (MEK inhibitor) prior to HNE treatment. (A) TGF-α ELISA from conditioned media samples after 15 minutes of HNE treatment of saccular stage lung fibroblasts. Data are expressed as mean ± SEM, n = 4 per group. (B and C) Quantification of Fbln5 (B) and Eln (C) expression by qPCR in saccular-stage fibroblasts 24 hours after treatment with TGF-α. Data are expressed as mean ± SEM, n = 4 per group. (D) Expression of Eln and Fbln5 in fibroblasts treated with HNE ± AG1478 20 μM for 24 hours. Data are expressed as mean ± SEM, n = 3–4 per group. (E and F) Western blot analysis of phospho-Erk1/2 and total-Erk1/2 in cell lysates from fibroblasts treated with HNE for 15 minutes. Data are expressed as mean ± SEM, n = 3 per group. (G) Expression of Eln and Fbln5 in fibroblasts treated with HNE ± U0126 25 μM for 24 hours. Data are expressed as mean ± SEM, n = 3–4 per group. Data are representative of 3 independent experiments. *P < 0.05 compared with control by 2-tailed student’s t test (A, B, C, and F); *P < 0.05 compared with control by 1-way ANOVA and post hoc Tukey test (D and G).