Figure 7.

Compound 145 activates cAMP/PKA/CREB dependent pathway in TGF-β₁-induced MRC-5 cells. (A) Lung fibroblasts were cultured for 24 h and then serum-deprived for 2 h. The cAMP level was measured after 30 min of incubation with the studied compounds (10 µM) and TGF-β₁ (5 ng/mL). Each bar represents the mean (± S.E.M.) The cAMP concentration (fmol/mL/mg of protein; n = 4). (B) Protein kinase A activity in MRC-5 was determined after 30 min of incubation with the studied compounds (10 µM), and 30 min of incubation with TGF-β₁ (n = 4). (C) Representative immunoblot and densitometry analysis of p-CREB in MRC-5 (n = 4). The ROD signal was normalized to the GAPDH control level. Each bar represents the mean value (± S.E.M.). (D) Immunofluorescence staining for p-CREB protein: MRC-5 were fixed, permeabilized, blocked, and incubated with anti-p-CREB antibody followed by an Alexa Fluor 546 conjugated antibody, nuclei were stained with Hoechst 33342 dye. Microphotographs were taken using a Leica DMi8 microscope, 40× objective, bar = 50 µm. (E) Cells exhibiting co-localization of p-CREB and nuclear signal were counted in 20 randomly selected fields of view and expressed as a percentage fraction in the entire MRC-5 population. The results were considered statistically significant at the p level of 0.05, against control (*).