Fig 2: NEDD4L depletion stimulates mitochondrial oxidative phosphorylation and cell survival via regulation of ULK1 or glutamine transporter ASCT2.a shCTL and shNEDD4L MIA PaCa-2 cells were reverse-transfected with siCTL, siULK1, or siASCT2 and incubated for 48 h. The levels of mitochondrial OCR in each knockdown were measured by an extracellular flux analyzer in basal media and in response to treatment with the indicated mitochondrial regulators. *P < 0.05; **P < 0.01. b HeLa cells were reverse-transfected with siCTL, siULK1, or siASCT2 in siCTL and siNEDD4L respectively. Cells were stained with MitoTracker Green and then treated with either vehicle (DMSO) or CCCP (5 μM) for 1 h. Subcellular mitochondrial morphology were imaged using a confocal fluorescence microscope. Scale bar: 5 μm (Magnification, ×1000). The average area of MitoTracker Green was quantified. 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. c shCTL and shNEDD4L MIA PaCa-2 cells were reverse-transfected with siCTL, siULK1, or siASCT2 for 24 h. Cells were then incubated in nutrient-complete media or glutamine-free (Starv) media for 48 h. Cell death was assessed using annexin V/propidium iodide (PI) staining by flow cytometry. The dead cell portion was measured based on the number of annexin V and PI single-stained cells. Error bars indicate the mean ± SEM for three independent experiments. **P < 0.01. d shCTL and shNEDD4L MIA PaCa-2 cells were treated with vehicle (0.5% DMSO), SBI 0206965 (ULK1 inhibitor), or GPNA (ASCT2 inhibitor) for 48 h. Cell death rate was analyzed as in (c) and normalized against death from vehicle, respectively. Error bars indicate the mean ± SEM for three independent experiments. **P < 0.01. e Cells as in (d) were treated with vehicle (0.5% DMSO), and the indicated mitochondrial OXPHOS inhibitors: rotenone, CCCP, oligomycin, and antimycin A. Cell death was analyzed as in (c). Error bars indicate the mean ± SEM for three independent experiments. **P < 0.01.

Fig 3: NEDD4L depletion stabilizes the glutamine transporter ASCT2.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.

Fig 4: Mitochondrial respiration increases in NEDD4L-deficient cells.a Mitochondrial oxygen consumption rate (OCR) of MIA PaCa-2 shCTL and shNEDD4L cells was measured by an extracellular flux analyzer in basal media and in response to treatment with the indicated regulators of mitochondrial activities, including 1 μM Oligomycin, 0.5 μM FCCP, and 0.45 μM Rotenone/Antimycin A, for the indicated time periods. The levels of OCR were normalized by cell numbers which were measured by image-based HCS system Operetta CLS immediately after the analysis. *P < 0.05; **P < 0.01. b Cells as in (a) were plated overnight and subsequently treated with vehicle (DMSO) or Oligomycin/Antimycin A (OA; 1 μM/3 μM) for 2 h to induce mitochondrial dysfunction. Subsequently, cells were stained with JC-1 mitochondrial dye and analyzed by flow cytometry. Error bars indicate the mean ± SEM for three independent experiments. *P < 0.05; **P < 0.01. c Cells as in (a) were plated overnight and subsequently treated with vehicle (DMSO), CCCP (10 μM), or Oligomycin/Antimycin A (OA; 1 μM/3 μM), respectively for 4 h and resolved in a SDS-PAGE gel. Samples were immunoblotted against NEDD4L, COXIV, TOM20, and a cocktail antibody of oxidative phosphorylation (OXPHOS) complex members. 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. d HeLa cells were reverse-transfected with either siCTL or siNEDD4L and were treated with vehicle (DMSO), CCCP (10 μM), or OA (1 μM/3 μM) for 1 h. Subsequently, the cells were stained with MitoTracker Green to visualize subcellular mitochondrial morphology using a confocal fluorescence microscope. Scale bar: 5 μm (Magnification, ×1000). The average area of MitoTracker Green signal was quantified and presented as an average from at least five different images in three independent experiments. **P < 0.01.

Fig 5: NEDD4L represses pancreatic cancer growth in xenograft mouse models by inactivation of autophagy.a Nude mice were injected subcutaneously with 5 × 106 shCTL or shNEDD4L MIA PaCa-2 cells. When the average tumor size reached 40 mm3, tumor size was measured at the indicated time points. After 4 weeks, the tumors were removed for morphological and histochemical examinations. Representative images of tumors from shCTL (top) and shNEDD4L cells (bottom) are shown. *P < 0.05. b Subcutaneous shCTL or shNEDD4L MIA PaCa-2 cell-derived tumors were established in 6-week old female mice. Then, chloroquine (CQ; 20 mg kg−1) was administered daily via intraperitoneal injection and tumor growth was assessed once the tumor volume reached 100 mm3 at the indicated time points. Data are shown as the mean of five mice in each group ± SEM. *P < 0.05. c Immunohistochemistry of NEDD4L and ULK1 proteins detected from tumor tissues in (a). Tumors derived from shCTL or shNEDD4L MIA PaCa-2 cells were dissected 6 weeks after cell inoculation and embedded in paraffin, and tumor sections were stained with antibodies against NEDD4L, ULK1, Ki67, or LC3B. Scale bar: 50 μm. d Representative images of H&E staining and immunohistochemistry of NEDD4L and ULK1 proteins in pancreatic tumor tissues from KPC mice. Pancreas from KPC mice were dissected at 20 weeks after birth and embedded in paraffin, and pancreas sections were stained with H&E and with antibodies against NEDD4L, ULK1, or Ki67. Scale bars: 50 μm.