Figure 4.

Inhibitory effects of NK1 on the autophagic mechanisms

(A) Schematic representation of the key players in the autophagic processes.

(B) Luciferase assay on HeLa cells treated with NK1. The activation of several autophagic genes before and after NK1 treatment was measured by a luciferase assay. Notably, each vector codifies for the Firefly luciferase whose expression is controlled by the different autophagic gene promoters. The Relative LUciferase intensity (RLU) between mock and NK1 treated HeLa cells is represented by histograms. Means ± SEM were obtained from three independent experiments. ns = not significant. ∗∗∗ p value <0.001.

(C) Traffick-light HeLa cells stably expressing RFP-GFP-LC3 were grown on coverslip for 24 h before being treated or not with NK1 for 24 h at the concentration of 10⁻⁶ M. After treatment, cells were employed for immunofluorescence and confocal microscopy analysis. Single focal sections are shown. Images are representative of three independent experiments made in triplicates. Scale bar: 50 μm. The histograms on the right report the relative number of autophagosomes (RFP⁺-GFP⁺-LC3 structures, green bars) and of autolysosomes (RFP⁺-GFP^--LC3 structures, pink bars). Means ± SEM were obtained from three independent experiments. ns = not significant.