Cell Physiol Biochem 2016;39: DOI: /

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NEDD4 Depletion Inhibits Hepatocellular Carcinoma Growth via Targeting PTEN Cell Physiol Biochem 2016;39:768-779 - DOI:10.1159/000445667 Fig. 1. NEDD4 overexpression negatively correlates with the cumulative survival of HCC patients. (A) Representative immunohistochemical staining images of NEDD4 in HCC tumor samples (×200). Fresh tissues from the same patient were determined by western blotting. The densitometry for the blots was shown as the ratio between NEDD4 and GAPDH separately. (B) Kaplan-Meier survival curves of low and high NEDD4 expression HCC patients. © 2016 The Author(s) Published by S. Karger AG, Basel - CC BY-NC-ND 4.0

NEDD4 Depletion Inhibits Hepatocellular Carcinoma Growth via Targeting PTEN Cell Physiol Biochem 2016;39:768-779 - DOI:10.1159/000445667 Fig. 2. NEDD4 silencing in Huh7 cells inhibits cell proliferation and induces cytoskeletal changes. (A) The transfection efficiency of NEDD4 siRNA was analyzed by Western blotting at 48 hours post transfection. The densitometry for the blots was shown as the ratio between NEDD4 and GAPDH separately. (B) Representative images of EdU-positive replicating cells captured using a fluorescent microscope, where red nuclei represent replicating cells. (C) Quantitative data of EdU proliferation assay showing a significant decrease in cell proliferation. (D) NEDD4 depletion results in reorganization of the actin cytoskeleton in Huh7 cells. Data represent the means ± SD of three separate samples (*, p < 0.05). © 2016 The Author(s) Published by S. Karger AG, Basel - CC BY-NC-ND 4.0

NEDD4 Depletion Inhibits Hepatocellular Carcinoma Growth via Targeting PTEN Cell Physiol Biochem 2016;39:768-779 - DOI:10.1159/000445667 Fig. 3. NEDD4 depletion in Huh7 cells inhibits cell migration and invasion. (A) Huh7 cells treated with NEDD4 siRNA were wounded using a pipette tip and then cultured for a further 72 hours. Representative images of wound-healing at different time points are shown. (B) Quantitative data of wound width in the wound-healing assay. (C) Huh7 cells treated with siNEDD4 were seeded in transwell dishes covered with Matrigel and were cultured for 24 hours. Representative images of invading cells were shown. (D) Quantitative data of invasive cells in cell invasion assay. Data represent the means ± SD of three separate samples (*, p < 0.05, **, p < 0.01). © 2016 The Author(s) Published by S. Karger AG, Basel - CC BY-NC-ND 4.0

NEDD4 Depletion Inhibits Hepatocellular Carcinoma Growth via Targeting PTEN Cell Physiol Biochem 2016;39:768-779 - DOI:10.1159/000445667 Fig. 4. NEDD4 depletion induces cell cycle arrest at S phase. (A) Cells were treated with NEDD4 siRNA for 48 hours. Representative images of cell cycle analysis show a significant increase in S phase. (B) The data of cell cycle assay are quantified and presented as a histogram (*, p < 0.05). (C) Representative images of cell apoptosis analysis. Q1, Q2, Q3, and Q4 indicate dead cells, apoptotic cells in late stage, apoptotic cells in early stage, and live cells, respectively. (D) The data of cell apoptosis assay are quantified and presented as a histogram. The data shows no change in apoptotic cells after treatment with siNEDD4. © 2016 The Author(s) Published by S. Karger AG, Basel - CC BY-NC-ND 4.0

NEDD4 Depletion Inhibits Hepatocellular Carcinoma Growth via Targeting PTEN Cell Physiol Biochem 2016;39:768-779 - DOI:10.1159/000445667 Fig. 5. NEDD4 regulates the protein levels of PTEN and the phosphorylation of AKT, ERK1/2, and STAT3 in Huh7 cells. (A) NEDD4 co-localizes with PTEN and knockdown of NEDD4 increases the protein level of PTEN. (B) Huh7 cells were treated with NEDD4 siRNA for 72 hours. The protein levels of PTEN and the phosphorylation of AKT, ERK1/2, and STAT3 were analyzed by immunoblotting with specific antibodies. The densitometry for the blots was shown as the ratio between p-ERK1/2 and ERK1/2, between p-AKT and AKT, between p-STAT3 and STAT3, between PTEN and GAPDH, between NEDD4 and GAPDH. © 2016 The Author(s) Published by S. Karger AG, Basel - CC BY-NC-ND 4.0