Figure 6.

ATF4 promotes lung fibroblast activation. (A) qPCR analysis of ATF4-overexpressing normal fibroblasts (pcDNA3.1+HA-ATF4) shows enhanced profibrotic transcript levels compared with normal lung fibroblasts transfected with an empty vector plasmid. The results were analyzed by the unpaired Student’s t-test for comparisons between two groups with normal distribution. Data are presented as mean ± SD. ** p < 0.01, *** p < 0.001, and **** p < 0.0001. (B) HA-tagged ATF4, collagen I, α-SMA, fibronectin, and β-actin proteins were detected by western blot (N = 3 independent experiments). (C) The activation of fibroblasts was examined based on fibroblast contraction in 3D collagen matrices (N = 3 independent experiments). We used ImageJ software to measure the area of gels. The results were analyzed by the unpaired Student’s t-test for comparisons between two groups with normal distribution. Data are presented as mean ± SD. *** p < 0.001. (D) MRC5 cells were subjected to immunofluorescence with a vimentin antibody. Scale bars, 20 μm. ImageJ software was used to quantify the fluorescence intensity. The results were analyzed by the unpaired Student’s t-test for comparisons between two groups with normal distribution. Data are presented as mean ± SD. *** p < 0.001.