Fig. 1: NNMT negatively regulates autophagy in liver cancer cells.

a NNMT expression in normal tissue and various liver cancer cell lines was assessed by western blot analysis using whole-cell lysates. The NNMT protein level was analyzed by densitometry using ImageJ software. The relative ratio of NNMT levels was normalized to actin expression. b–e p62 and LC3-II levels were assessed by western blotting in SK-Hep-1-N.C., SK-Hep1-shNNMT, Hep3B-E.V., and Hep3B-NNMT OE cells. b–c Cells were cultured in growth medium for 120 h, followed by treatment with or without 10 nM BafA1 for an additional 4 h. d–e Cells were incubated under amino acid starvation (HBSS) for 4 h in the absence or presence of 10 nM BafA1. p62 and LC3-II levels were detected by western blot analysis. f SK-Hep-1-shNNMT cells were transiently transfected with either empty vector (E.V.) or green fluorescent protein (GFP)-mCherry tandem fluorescence-tagged LC3 plasmid for 44 h and exposed to amino acid starvation for an additional 4 h with or without 10 nM BafA1 treatment. Confocal microscopy images for autophagosome/autolysosome analysis. Scale bar indicates 10 μm. The histogram represents the % of LC3 puncta per cell, with yellow (autophagosome)/red dots (autolysosomes). Each bar represents the mean ± SD. g SK-Hep-1-shNNMT cells were transfected with plasmid expressing FLAG-tagged NNMT. After transfection for 44 h, cells were incubated under amino acid starvation for 4 h in the absence or presence of 10 nM BafA1. The expression level of LC3-II or p62 were quantified by ImageJ software. Bar graph represents the normalized band intensities of each protein to actin. The histogram bars represent the mean ± SD of three independent experiments (*p < 0.05, **p < 0.01, ***p < 0.001)