Fig. 4

Melatonin and everolimus co-treatment enhanced autophagy activation in MCF-7 cells. (A) Melatonin, everolimus, and MEL/EVE were administered to MCF-7 cells in the absence or presence of the lysosomal inhibitor hydroxychloroquine (HCQ, 10 M, 40 min), and the levels of p62, LC3-I, and LC3-II protein expression were measured. As a loading control, β-actin was employed. Protein band quantitative studies were carried out and displayed as bar graphs. (Three separate tests were carried out). ImageJ software was used to calculate band intensity ratios for the p62/Actin and LC3-II/LC3-I ratios. (B) Representative immunofluorescence images showing autophagosomal dot formation in MCF-7 cells labeled with LC3 was seen and quantified in the absence or presence of the lysosomal inhibitor HCQ, (10 M, 40 min). (At least four separate tests were carried out). The inset in the lower left corner represents a magnified view of a selected region from the main image, highlighting the structural details of LC3 puncta. The bars represent mean ± SEM. *p < 0.05, **p < 0.005