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. 2023 Dec 20;10(1):e23870. doi: 10.1016/j.heliyon.2023.e23870

Fig. 6.

Fig. 6

Activation of autophagy by melatonin in MCF-7 cells in vitro. (A) Detection of Autophagy by Lysotracker Green Dye in cultured MCF-7 cells. Representative immunofluorescence detection of lysotracker (Green staning) in MCF-7 cells after 24 h treatment with chloroquine and melatonin in different groups. Fluorescence Microscope was used to visualize the accumulation of lysosomes in terms of intensity of green signal around the nucleus of the cells. (i) Control (no melatonin & chloroquine; M‾C‾); (ii) Treated with Chloroquine (5 μm) but no melatonin (M‾C⁺) (iii) Treated with melatonin (1 mM) but no chloroquine (M⁺C‾) (iv) Both Melatonin (1 mM) & Chloroquine (5 μm) (M⁺C⁺).Higher accumulation of lysosome was observed in Melatonin treated group (iii) compare to control group (i) and Chloroquine treated group (ii) whereas both melatonin and chloroquine treated group (iv) shows reduced accumulation of lysosome compared to group (iii), as detected by lysotracker green dye intensity. Scale bar 75 μm. (B) (i) Representative immunoblots of p62, Beclin1 and LC3B protein are presented for MCF-7 cells exposed for 24 h treatment with Melatonin (1 mM) & Chloroquine (5 μm) in different experimental groups. (ii) Densitometric analysis of p62, Beclin1 and LC3B II/I ratio. Beta-actin was used as the loading control. Blots shown are representative of three different experiments with similar results. Densitometric data represented as Mean ± S.E.M of three separate immunoblots of each experimental group (n = 3). *p˂0.05; Control (M‾C‾) vs. M‾C⁺; #p <0.05; M‾C‾ vs. M⁺C‾ and @ p <0.05; M‾C‾ vs. M⁺C⁺. Control (no melatonin & chloroquine; M‾C‾); Treated with Chloroquine (5 μm) but no melatonin (M‾C⁺); Treated with melatonin (1 mM) but no chloroquine (M⁺C‾); Both Melatonin (1 mM) & Chloroquine (5 μm) (M⁺C⁺). (C) Immunohistochemical analysis of expression of LC3B protein performed in frozen breast tumor tissue sections. (i) Representative IHC images showing lower expression of LC3B protein (green fluorescence) in control tumor section and (ii) higher LC3B expression in melatonin-treated tumor tissue sections as revealed by standard fluorescent microscopy. (Red arrow indicates the expression of LC3B Protein). Refer to Supplementary Material for uncropped images as applicable.