Fig. 5: CCL20–integrin α5β1 drives fibroblast activation by enhancing TGF-β signaling. | Nature Communications

Fig. 5: CCL20–integrin α5β1 drives fibroblast activation by enhancing TGF-β signaling.

From: The CCL20–integrin α5β1 interaction enhances TGF-β/Smad signaling to promote fibroblast activation in pulmonary fibrosis

Fig. 5

a CoIP analysis of the interaction between LAP and integrin α5. b Ratio of active TGF-β to total TGF-β in the culture medium of CCL20-treated MRC-5 cells, as determined by ELISA (n = 3 technical replicates per group). c TGF-β-reporter luciferase activity in HEK293T cells in response to the cell supernatant of fibroblasts treated with or without CCL20 (n = 4 technical replicates per group). d Immunoblots showing the levels of phosphorylated and total Smad3 or Smad2 in primary fibroblasts following CCL20 stimulation (n = 3 technical replicates per group). e Immunoblots showing the levels of phosphorylated and total Smad3 or Smad2 in MRC-5 cells following CCL20 stimulation (n = 3 technical replicates per group). f IF staining for phosphorylated Smad3 in primary fibroblasts stimulated with CCL20. Scale bars, 20 μm. g IF staining for phosphorylated Smad3 in MRC-5 cells stimulated with CCL20. Scale bars, 20 μm. h Immunoblots showing the levels of phosphorylated and total Smad3 or Smad2 in primary fibroblasts treated with or without ATN-161 in the presence of CCL20 (n = 3 technical replicates per group). i IF staining for phosphorylated Smad3 in primary fibroblasts treated with or without ATN-161 in the presence of CCL20. Scale bars, 20 μm. j Immunoblots showing the levels of phosphorylated and total Smad3 or Smad2 in MRC-5 cells treated with or without ATN-161 in the presence of CCL20 (n = 3 technical replicates per group). k IF staining for phosphorylated Smad3 in MRC-5 cells treated with or without ATN-161 in the presence of CCL20. Scale bars, 20 μm. l IF staining for α-SMA in primary fibroblasts treated with or without SIS3 in the presence of CCL20. Scale bars, 20 μm. m IF staining for α-SMA in MRC-5 cells treated with or without SIS3 in the presence of CCL20. Scale bars, 20 μm. n Expression of Acta2 in primary fibroblasts treated with or without SIS3 in the presence of CCL20 (n = 3 technical replicates per group). o Expression of ACTA2 in MRC-5 cells treated with or without SIS3 in the presence of CCL20 (n = 3 technical replicates per group). p Schematic diagram of the method used to evaluate the therapeutic effects of ATN-161 on the PF model. q Hydroxyproline levels in lung tissues from the indicated mice after BLM exposure (n  =  7 samples for BLM + PBS, BLM + PBS + ATN161, BLM + CCL20 + ATN161, n  =  6 samples for BLM + CCL20 + PBS). r Masson’s trichrome staining of lung tissues from the indicated mice after BLM exposure. Scale bar, 100 μm. s The expression levels of Fibronectin and Acta2 in lung tissues from the indicated mice after BLM exposure (n = 6 mice per group). t Immunoblots showing the levels of phosphorylated and total Smad3 or Smad2 in primary lung fibroblasts isolated from the indicated mice (n = 3 samples per group). The data are presented as the means ± SEM. The data in b–e, s, t were analyzed via two-tailed Student’s t-test. The data in h–j, n–o, q were analyzed via one-way ANOVA. n.s., not significant; *p < 0.05; **p < 0.01; ***p < 0.001. Source data are provided as a Source data file.

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