Figure 3
From: ROS-Mediated Apoptotic Cell Death of Human Colon Cancer LoVo Cells by Milk δ-Valerobetaine
Autophagy induced by milk-δVB. (a) Representative images of green detection reagent imaged by confocal laser microscopy after 72 h in LoVo cells incubated with 40% v/v buffalo milk (milk), δVB (2 mM) and milk supplemented with δVB (milk + δVB). Scale bar, 50 μm. (b) As for flow cytometry analysis, at least 10.000 events were acquired in log mode. For the quantitative evaluation of green detection reagent, FlowJo V10 software was used to calculate median fluorescence intensities. (c) Bar graph of the fluorescence intensity values of green detection reagent-labeled vesicles and expressed as fold change of the control (Ctr). Analysis was carried out by determinations in triplicate of n = 3 experiments **P < 0.01 vs Ctr, †P < 0.05 vs milk, #P < 0.05 vs δVB. (d–g) Protein expression levels of LC3BII/ LC3BI, p62, Atg7 and Beclin 1, measured by Western blot in control LoVo cells (Ctr) or cells treated for 72 h with milk (40% v/v), δVB (2 mM) and milk+δVB. Lane 1 = Ctr, lane 2 = milk, lane 3 = δVB, lane 4 = milk + δVB. Protein content was calculated with Image J software 1.52n version and expressed as arbitrary units (AU). *P < 0.05 vs Ctr; **P < 0.01 vs Ctr; ††P < 0.01 vs milk. The full-length blots are showed in the supplementary information (Fig. S3).
