Fig. 3: NNMT knockdown elevates autophagy response via PP2A-mediated ULK1 activation.

a–b SK-Hep-1-N.C. and SK-Hep-1-shNNMT cells were transiently transfected with either empty vector (E.V.) or GFP-mCherry tandem fluorescence-tagged LC3 plasmid for 44 h, followed by treatment with or without 10 nM okadaic acid for 2 h and/or subjection to amino acid starvation for an additional 4 h in the presence or absence of 10 nM BafA1 treatment. a Confocal microscopy images for autophagosome/autolysosome analysis. Scale bar indicates 10 μm. b The histogram represents the % of LC3 puncta per cell, with yellow (autophagosome)/red dots (autolysosomes). The data are shown as the mean ± SD and are representative of three independent experiments (*p < 0.05). c Western blot analysis of autophagy markers in shNNMT cells. Left panel: SK-Hep-1 cells were treated with or without 10 nM okadaic acid for 6 h and subjected to amino acid starvation for an additional 6 h. Right panel: SNU-449 cells were exposed to glucose starvation for 6 h, followed by treatment with or without 10 nM okadaic acid for an additional 12 h. d Western blot analysis of autophagy markers in SK-Hep-1-N.C. and SK-Hep-1-shNNMT cells. The cells were transiently transfected with N.C. or PP2Ac siRNA for 36 h, followed by treatment with or without 10 nM okadaic acid for 6 h. Then, cells were challenged with amino acid starvation for an additional 6 h, followed by western blot. The indicated proteins were quantified using ImageJ software. Each band intensities were normalized to that of actin. The data are representative of three independent experiments