Figure 1

Violacein selectively controlled AbAs viability and decreased their antioxidant defenses. (a) Violacein molecular structure. (b) Representative confocal images of p6 AbAs showing a fibrilar S100β staining (green) upon a 24 h treatment with 0.25% DMSO (VEH) or 100 nM violacein. Violacein reduced cellular density and modified S100β appearance showing more condensed cytoskeleton (green arrow) or nuclear envelope and altered substrate adhesion (white arrow). Hoechst 33342 stained cell nuclei (blue). Calibration: 50 µm. (c) Concentration-inhibition curves in confluent p6 AbAs (△), AA (○) or C6 (□) cells cultures treated with 0–1500 nM violacein (24 h, sulforhodamine B (SRB) assay). Solid lines represent the best fittings whereas dotted ones indicate 95% confidence intervals. (d) Violacein effects on AA and p6 AbAs migration assessed by percent of cell covered area upon 48 h of treatment with vehicle or 0–200 nM violacein. At 200 nM, violacein reduced AbAs migration when compared with vehicle (p = 0.0181). (e) Violacein inhibitory effects on AbAs mitochondrial NAD(P)H activity evaluated by MTT reduction related to Hoechst 33342 fluorescence. Reduction was found since 50 nM violacein compared with vehicle (p = 0.0334) or with AA submitted to the same concentration (p = 0.004). Hoechst 33342 was abbreviated as H. (f) Selective action of violacein on AbAs mitochondrial potential assessed with JC1. Decreases in AbAs were found at 30 nM (p = 0.0005) and higher concentrations. At 200 nM, violacein marginally decreased AA mitochondrial potential (p = 0.057). (g) Effects of violacein on levels of oxidative stress in AA and p6 AbAs assessed by carboxy-dichlorofluorescein (C-DCF) fluorescence and expressed as C-DCF/Hoechst 33342 fluorescence ratios related to vehicle. At 200 nM, violacein increased C-DCF relative emission in AbAs when compared with vehicle (p = 0.0047) or with AA (p = 0.001). (h) Effects of violacein on total glutathione levels were seen at 200 nM with reduced formation of glutathione-monochlorobimane (MCB) adducts in AbAs respect to the vehicle (p = 0.0007) but not in AA. Experiments and replicates appear in Table 1 for all of the figures. (d–g) symbols: □: AA; ■: AbAs.