Fig. 3

CSF3 induces pulmonary fibrosis through the CSF3R/STAT3 signaling axis. a qRT-PCR analysis of fibrosis markers (α-SMA, COL1A1, fibronectin) in human lung fibroblast cell line (HLF, left) and mouse primary lung fibroblast cells (MLF, right) treated with recombinant CSF3 (rCSF3) (200 ng/ml, 24 h). b and c Representative immunofluorescence images of α-SMA and COL1A1 (b), and Western blot analysis of fibrosis markers and STAT3 activation (p-STAT3) (c) in HLF and MLF cells treated with rCSF3. β-Actin was used as a loading control. Scale bars: 200 μm. d Hydroxyproline content assay in HLF and MLF cells treated with rCSF3. e Schematic illustration of the procedure used to induce lung fibrosis in C57BL/6 mice with recombinant mouse CSF3. BLM was administered IP on day 1, followed by IP injections of rCSF3 three times per week. f Quantification of the fibrosis area was performed using Orbit software based on Masson’s trichrome staining images. The green area represents the fibrotic region. Scale bar: 500 μm. g Hydroxyproline content in mouse lung tissues from groups treated with BLM and rmCSF3 as indicated. h–k qRT-PCR analysis of α-SMA (h), COL1A1 (i) and fibronectin (j) expression, and hydroxyproline content (k) assay in wild-type (CSF3^+/+) MLF cells and CSF3 knock-out (CSF3^−/−) MLF cells treated with BLM and rCSF3. l and m qRT-PCR analysis of fibrosis markers and CSF3 expression (l), and representative immunofluorescence images of α-SMA and COL1A1 (m) in HLF cells transfected with si-CSF3R and treated with rCSF3 (200 ng/ml, 24 h). Scale bars: 200 μm. n and o Western blot analysis of α-SMA and COL1A1 (n), and representative immunofluorescence images of α-SMA and COL1A1 (o) in CSF3^−/− MLF cells treated with BLM and rCSF3. β-Actin was used as a loading control. Scale bars: 200 μm. Statistical significance was determined using ANOVA with multiple comparison or t-test