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. 2020 Jan 20;11(1):38. doi: 10.1038/s41419-020-2242-5

Fig. 4. NEDD4L depletion stabilizes the glutamine transporter ASCT2.

Fig. 4

a MIA PaCa-2 and Panc-1 cells stably expressing shCTL and shNEDD4L were cultured for 24 h after which the culture media was collected and cells enumerated. Glutamine consumption levels of the cells were analyzed by comparing glutamine levels from media alone without cells and the collected cell culture media using a metabolite analyzer. The glutamine consumption rate was normalized to the number of cells. Error bars indicate the mean ± SEM for three independent experiments. *P < 0.05. b shCTL and shNEDD4L MIA PaCa-2 cells were plated overnight and subsequently replaced with EBSS media for starvation. Cell lysates were prepared at the indicated time points and immunoblotted with antibodies against ULK1, pULK1, and ASCT2. β-actin was used as loading control. The WB band intensities were quantified by Image J and error bars indicate the mean ± SEM for three independent experiments. *P < 0.05; **P < 0.01. c HA-ubiquitin and FLAG-ASCT2 plasmids were co-transfected in HEK293T shCTL and shNEDD4L cells for 48 h before harvest. Cell lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted against FLAG and HA to observe ASCT2 ubiquitination. d shCTL and shNEDD4L MIA PaCa-2 cells stably expressing GFP-LC3 were reverse-transfected with control siRNA (siCTL), ULK1-specific siRNA (siULK1), or ASCT2-specific siRNA (siASCT2) for 48 h. Subsequently, cells were incubated in either nutrient-complete or amino acids-deprived medium for 4 h and imaged using a confocal fluorescence microscope. Scale bar: 20 μm (Magnification, ×400). Percent of GFP-LC3 puncta area was normalized to the DAPI-stained area per cell. The data are representative of at least three independent experiments. Error bars indicate the mean ± SEM for three independent experiments. *P < 0.05; **P < 0.01. e HeLa cells stably expressing mCherry-GFP-LC3 were reverse-transfected with siCTL, siULK1, or siASCT2 in addition to siCTL and siNEDD4L respectively. Cells were imaged under starvation as in (d). Percent of mCherry puncta area was normalized to the DAPI-stained area per cell. The data are representative of at least three independent experiments. Error bars indicate the mean ± SEM for three independent experiments. *P < 0.05; **P < 0.01.