Fig 2: Curcumin suppresses NEDD4 expression at both mRNA and protein levels. (A) The mRNA expression of NEDD4 was measured by real-time RT-PCR in curcumin-treated glioma cells. (B) Alteration of NEDD4, Notch1 and pAkt expression levels was detected by western immunoblot analysis in curcumin-treated glioma cells. (C) Quantitative results are illustrated for the above immunoblotting images (B). *P<0.05 vs control.

Fig 3: Overexpression of NEDD4 enhances glioma cell proliferation, suppresses cell apoptosis and increases cell invasion. (A) Glioma cell growth was measured by MTT assay after NEDD4 overexpression in combination with 15 μM curcumin treatment for 72 h. *P<0.05, vs control; #P<0.05 vs curcumin treatment or NEDD4 cDNA transfection. (B and C) Apoptotic cells were accessed by flow cytometry after NEDD4 overexpression in combination with 15 μM curcumin treatment for 48 h. *P<0.05, vs control; #P<0.05 vs curcumin treatment or NEDD4 cDNA transfection. (D) Cell invasion were detected by Transwell chamber assay after NEDD4 overexpression in combination with 15 μM curcumin treatment for 20 h. *P<0.05, vs control; #P<0.05 vs curcumin treatment or NEDD4 cDNA transfection.

Fig 4: NEDD4 induced the degradation of HBx in a ubiquitin/proteasome-dependent manner via K48-linked ubiquitination. (A) Flag-HBx was transfected into 293T cells with Myc-NEDD4 or empty vectors. After transfection of plasmids for 48 h, protein lysates were prepared at the indicated time points after treatment with 10 μM cycloheximide, and the Flag-HBx protein levels were evaluated by western blotting. The results demonstrated that the Flag-HBx degradation rate was significantly higher when HBx was coexpressed with Myc-NEDD4. (B) Flag-HBx was transfected into 293T cells with Si-Vector or Si-NEDD4. After transfection for 48 h, protein lysates were prepared at the indicated time points after treatment with 10 μM cycloheximide, and the Flag-HBx protein levels were evaluated by western blotting. The results demonstrated that the Flag-HBx degradation rate was significantly lower with NEDD4 knockdown. (C) Flag-HBx and Myc-NEDD4 expression vectors were cotransfected into 293T cells for 48 h, after which 293T cells were treated with chloroquine (CQ, 50 µM) or MG132 (10 µM) for another 12 h. Western blotting demonstrated that MG132 reversed the degradation of Flag-HBx induced by Myc-NEDD4, while CQ had little effect. (D) Flag-HBx and HA-Ubiquitin was transfected into 293T cells with Myc-NEDD4 or empty vectors. After transfection of plasmids for 48 h, protein lysates were harvested and Flag-HBx was immunoprecipitated with anti-Flag antibody. Western blotting demonstrated that Myc-NEDD4 significantly induced the ubiquitination of Flag-HBx. (E) Flag-HBx and HA-K48-Ubiquitin was transfected into 293T cells with Myc-NEDD4 or empty vectors. After transfection of plasmids for 48 h, protein lysates were harvested and Flag-HBx was immunoprecipitated with anti-Flag antibody. Western blotting demonstrated that Myc-NEDD4 significantly induced the K48-linked ubiquitination of Flag-HBx. (F) Flag-HBx and HA-K63 -Ubiquitin was transfected into 293T cells with Myc-NEDD4 or empty vectors. After transfection of plasmids for 48 h, protein lysates were harvested and Flag-HBx was immunoprecipitated with anti-Flag antibody. Western blotting demonstrated that Myc-NEDD4 did not induce the K63-linked ubiquitination of Flag-HBx. (G) Flag-HBx and HA-Ubiquitin was transfected into 293T cells with Myc-NEDD4 or Myc-NEDD4 catalytically inactive mutant (cysteine 1197 to alanine). After transfection of plasmids for 48 h, protein lysates were harvested and Flag-HBx was immunoprecipitated with anti-Flag antibody. Western blotting demonstrated that Myc-NEDD4 catalytically inactive mutant did not induce the ubiquitination of Flag-HBx. Data in A and B are presented as the mean ± SD. *p < 0.05, NS, no significance (p > 0.05). (n = 3 independently replicated experiments). HA-UB, full-length pcDNA3.1(+)-HA tagged-Ubiquitin (ubiquitin B, UBB, Homo sapiens); HA-K48-UB, full-length pcDNA3.1(+)-HA tagged-Lys48 Ubiquitin (ubiquitin B, UBB, Homo sapiens); HA-K63-UB, pcDNA3.1(+)-HA tagged-Lys63-Ubiquitin (ubiquitin B, UBB, Homo sapiens).

Fig 5: HBx upregulation reverses the suppression of proliferation and mobility induced by NEDD4 overexpression in HBV-associated HCC cells in HepG2.215 cell line. (A) NEDD4 upregulation did not affect the mRNA level of HBx. (B) The protein level of HBx was downregulated after NEDD4 overexpressed. (C) HBx mRNA expression in HBx-overexpressing cells, NEDD4-overexpressing cells, cells overexpressing both HBx and NEDD4, and negative control cells. (D) Upon upregulation of HBx, NEDD4-induced proliferation suppression was recovered. (E) HBx upregulation compensated for NEDD4-induced migration inhibition. (F) NEDD4-induced invasion suppression was reversed after HBx upregulation. Data are presented as the mean ± SD. *p < 0.05, NS, no significance (p > 0.05). (OE-HBx+OE-NEDD4 represents upregulated HBx in NEDD4 overexpression HepG2.215 cells; OE-HBx represents HBx overexpression HepG2.215 cells; OE-NEDD4 represents NEDD4 overexpression HepG2.215 cells and OE-Vector represents the negative control) (n = 3 independently replicated experiments; scale bars: 50 μm).