Research Paper Advance Articles

Figure 2. Bleomycin treatment did not directly induce fibrosis in normal and iUIP PCLS. (A–G) Normal and iUIP PCLS samples were treated with the vehicle or 1 μM bleomycin for 120 h. The expression levels of Col1a1 (A), Acta2 (B), Il6 (C), Mmp3 (D), Mmp9 (E), Ifna2 (F), and Ifng (G) were determined using qPCR. Ifng expression was not detected in vehicle-treated normal and iUIP PCLS. The detection rates of samples with Ct values <40 are shown in graph (G). (H, I) PCLS samples were treated with the vehicle or 1 μM bleomycin for 6 (H) and 24 h (I). The expression of Col1a1 was determined using qPCR. (J) Masson’s trichrome staining of PCLS from normal and iUIP treated with the vehicle or 1 μM bleomycin for 48 h. (K–N) Percentage of fibrosis area ratio (K, L) were calculated based on the total area in each PCLS. (M, N) aSMA expression was determined using WB in PCLS from normal and iUIP treated with the vehicle or 1 μM bleomycin for 48 h. β-Actin was used as an internal control for WB. (O, P) PCLS samples were treated with vehicle or IL-6 (50 ng/mL) and IL-6R (50 ng/mL) for 120 (O) and 24 h (P). The expression of Col1a1 (O) and Ifng (P) was determined using qPCR. Hprt served as an internal control. The results are shown as mean ± SE of n = 5 mouse PCLSs at each stage (n = 3 mouse PCLS for iUIP fibrosis and n = 4 of normal in aSMA WB (M)). Asterisks indicate ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, and ^****P < 0.0001 compared with vehicle-treated normal PCLS. “ns”, not statistically significant. Scale bar indicates 150 μm.