Fig. 3

DNA-PKcs kinase activity is not required for innate DNA sensing by lung fibroblasts. a-d Lung fibroblasts were stimulated with 10 μM of hypomethylated CpG DNA (CpG) and/or treated with 500 nM Nu7441 or 300 nM BIBF-1120 for 24 h. After 24 h, RNA was extracted and transcriptomic analysis for myofibroblasts markers were performed. Shown is the average fold change of ACTA2 (a), COL1A1 (b), COL3A1 (c) & FN1 (d) transcript expression in CpG stimulated and/or Nu7441 or BIBF-1120 treated relative to untreated lung fibroblasts. n = 3 normal; n = 5 IPF. Data shown are mean ± s.e.m.; **p ≤ 0.01 as indicated by the bar. 2-way ANOVA followed by Tukey’s multiple comparisons test. e-h Lung fibroblasts were stimulated with 10 μM CpG or 20 ng/ml TGF-ß1 and/or treated with 500 nM Nu7441 for 72 h. Shown is the average concentration of secreted collagen 1 protein by two normal (e-f) and two IPF (g-h) lung fibroblasts treated in triplicate. Data shown are mean ± s.e.m. *p ≤ 0.05, **p ≤ 0.01, and ****p ≤ 0.0001 compared to vehicle group or as indicated by the bars. One-way ANOVA followed by Tukey’s multiple comparisons test. i-j Shown is the average fold change of IL-8 protein, secreted by normal (i) and IPF (j) lung fibroblasts 72 h after stimulation and/or Nu7441 treatment relative to vehicle treated cells. k-l Seventy-two hours after stimulation and/or treatment, cells were fixed, permeabilized and stained with anti-αSMA and ß-tubulin antibodies. Shown is the average ß-tubulin normalized expression of αSMA protein in stimulated and Nu7441 treated normal (k) or IPF (l) fibroblasts relative to their respective vehicle treated groups. i-l Data shown are mean ± s.e.m. n = 2 normal and n = 2 IPF treated in triplicate. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001 compared to vehicle group or as indicated by the bar. One-way ANOVA followed by Tukey’s multiple comparisons test