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Fig. 4 | Respiratory Research

Fig. 4

From: Regulation of fibroblast Fas expression by soluble and mechanical pro-fibrotic stimuli

Fig. 4

a Normal lung fibroblasts were treated with/without TGF-β1 (2 ng/ml) for 6 or 24 h (or were untreated controls). Proteins in the cell-culture supernatants were qualitatively assessed with the RayBio Human Cytokine Antibody Array 7.1. Shown are the assay positive controls (upper left and bottom right, black-dashed box), Fas/TNFRSF6 (red box) and TIMP1/2 (blue box). b sFas levels were assessed in the cell culture supernatants of normal lung fibroblasts treated with/without TGF-β1 (2 or 10 ng/ml) for the indicated time points. Data shown have been pooled from at least 3 independent experiments included with each dose- and time-point, and data have been indexed to represent the “fold-change” compared to the average of the untreated fibroblasts. ** p < 0.01 and *** p < 0.001 vs. untreated controls. c sFas was measured in the conditioned media from fibroblasts were treated +/− Brefeldin A (100 ng/ml) for 15–30 min then +/− TGF-β1 (10 ng/ml) for 24 h. Cell culture supernatants assessed for Fas by ELISA. Levels below assay detection were censored to the lowest detected concentration. The data shown are from 3 independent experiments. d Normal fibroblasts were treated with/without TGF-β1 (10 ng/ml) for 24 h. After washing, intact cells were collected by scraping and subjected to flow cytometry for Fas. A representative histogram is shown (left) along with the mean fluorescence intensity of untreated and TGF-β1 treated cells (right). n = 3 independent experiments. The gating strategy used is shown in Supplemental Data Fig. 3. e Cell-surface Fas immunofluorescence staining in normal lung fibroblasts treated with or without TGF-β1 (10 ng/ml) for 24 h. f Fluorescence was quantified in 10 individual cells per condition (5 cells from each of two independent experimental replicates)

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