Fig. 6. Inhibitory effects of MnTBAP involve regulations between bleomycin-induced VEGF and Wnt signaling.

(A) CRL-1490 cells were left untreated or pretreated with MnTBAP (100 µM), CID 11210285 hydrochloride (500 nM), salinomycin (50 nM), combination of MnTBAP (100 µM) and CID 11210285 hydrochloride (500 nM), or a combination of MnTBAP (100 µM) and salinomycin (50 nM) for 1 h followed by bleomycin (10 mU/ml) treatment for 24 h. Cell supernatant were collected and analyzed for VEGF protein by ELISA. Plots are mean ± S.E.M (n = 4). *P <0.05 versus non-treated control. **P <0.05 versus bleomycin treatment (B) CRL-1490 cells were pretreated for 1 h with CBO-P11 (10 µM), and then treated with bleomycin (10 mU/ml or 25 mU/ml) for 24 h and probed for PLCγ1, PKCα, and CaMKII proteins. (C) Mice treated with bleomycin (1 U/kg) with or without CBO-P11 (0.3 mg/kg) or equal volume of saline as control were euthanized at day 28. Lung tissue homogenates obtained from control and treated mice were probed for non-canonical Wnt/Ca2+ signaling proteins PLCγ1, PKCα, and CaMKII. (D) CRL-1490 cells were pretreated for 1 h with CBO-P11 (10 µM), and then treated with bleomycin (10 mU/ml or 25 mU/ml) for 24 h and probed for LRP6, Wnt5AB, Dvl2, and β-catenin. (E) Mice treated with bleomycin (1 U/kg) with or without CBO-P11 (0.3 mg/kg) or equal volume of saline as control were euthanized at day 28. Lung tissue homogenates obtained from control and treated mice were probed for canonical Wnt signaling proteins LRP6, Wnt5AB, Dvl2, and β-catenin. Blots were reprobed with β-actin antibody to confirm equal loading of the samples. Representative blots from three independent experiments (in vitro) or three animals per sample group (lung tissue homogenates) are shown. The immunoblot signals were quantified by densitometry. Values are mean ± S.E.M. (n = 3). *P <0.05 versus non-treated control. **P <0.05 versus bleomycin treatment.