Fig 2: NEDD4 family E3 ubiquitin ligase AIP4 promotes HBV naked capsid egress.(A) AIP4 ectopic expression dose-dependently promotes naked capsids secretion. HepG2 cells were co-transfected with 1.0 μg of HBc and the indicated amounts of Myc-tagged AIP4 (Myc-AIP4). The control vector was supplemented to normalize the total amount of transfected plasmids to 3.0 μg per transfection. Cells were harvested at day 3 post-transfection, and the expression of Myc-AIP4 and HBc was analyzed by Western blot, intracellular and extracellular capsid were detected by particle gel immunoblot. (B) Knockdown of endogenous AIP4 inhibits HBV naked capsid secretion. HepG2 cells were transfected with si-con or AIP4 siRNA (si-AIP4-1 and -2 with different sequence targets) for 24 h, followed by pHBc transfection for 3 more days. HBc expression and knockdown efficiency of endogenous AIP4 were analyzed by Western blot, intracellular and extracellular capsids were detected by particle gel. (C) Overexpression of AIP4 promotes the egress of HBV DNA-containing naked capsid. HepG2 cells were co-transfected with envelope-null pTREHBVDES and pTet-off together with a control vector or Myc-AIP4 for 5 days. The intracellular HBV total RNA, HBc, Myc-AIP4, cytoplasmic HBV capsid and DNA content, and core DNA intermediates were analyzed by Northern bot (upper panel), Western blot (upper middle panels), capsid gel (middle panels), and Southern blot (lower middle panel), respectively. Extracellular naked capsids and viral DNA content were detected by particle gel immunoblot with HBcAb and hybridization with (-) strand-specific HBV riboprobe, respectively. (D) AIP4 promotes the egress of HBV pgRNA-containing naked capsids. HepG2 cells were co-transfected with priming-defective and envelope-null pCMVHBVY63DΔS and control vector or Myc-AIP4 for 3 days. The intracellular HBV total RNA, HBc, Myc-AIP4, cytoplasmic HBV capsid and core DNA intermediates, and extracellular naked capsids were analyzed as above described. The intracellular and extracellular capsid-associated HBV RNA were detected by particle gel hybridization using a (+) strand-specific full-length HBV riboprobe.

Fig 3: Overexpression and knockdown of ITCH in HLECs altered the cell proliferation and intracellular superoxide content in high-glucose environment. (A) Change in cell proliferation in pEGFP-C1-ITCH-, MG132-treated and siRNA-ITCH group in high-glucose environment shown by the Cell Counting Kit-8 experiment. (B) Variation in intracellular peroxide content in the aforementioned groups. (C and D) Apoptotic rate of cells were detected by flow cytometry. Data are presented as the mean ± standard deviation and analyzed by ANOVA followed by Tukey-Kramer multiple comparison post hoc tests. *P<0.05 vs. HG3; #P<0.05 vs. pEGFP-C1-ITCH. HLECs, human lens epithelial cells; siRNA-ITCH, small interfering RNA-ITCH; NC, negative control.

Fig 4: Overexpression and knockdown of ITCH in HLECs and the changes in the downstream proteins. (A) Transfection of pEGFP-C1-ITCH plasmid and MG132 treatment showed changes in the expression of associated proteins: ITCH, Txnip, Trx and caspase-3 via western blotting in low- and high-glucose environment. (B) Transfection of siRNA-ITCH revealed changes in the expression of associated proteins in low- and high-glucose environment via western blotting. (C) Change in Trx activity in the pEGFP-C1-ITCH-, MG132-treated and siRNA-ITCH group in high-glucose environment via insulin disulfide reduction experiment. Data are presented as the mean ± standard deviation and analyzed by ANOVA followed by Tukey-Kramer multiple comparison post hoc tests. *P<0.05 vs. LG; #P<0.05 vs. HG3; ^P<0.05 vs. pEGFP-C1-ITCH. Txnip, thioredoxin interaction protein; Trx, thioredoxin; HLECs, human lens epithelial cells; siRNA-ITCH, small interfering RNA-ITCH; NC, negative control.

Fig 5: Merlin induces phosphorylation and degradation of AMOTL1 mediated by NEDD ubiquitin ligases. (A) AMOTL1 half-life is reduced upon Merlin expression in BC52 tet on AMOTL1. (Left) AMOTL1 levels following doxycycline withdrawal when cotransfected with Merlin vector (+) or control vector (−). (Right) Quantification of three independent experiments (Mann-Whitney test). (B) When Merlin expression is inhibited by shRNA, overnight MG132 treatment of BC52 does not modify AMOTL1 protein levels (sh Merlin#1 vs sh ctrl). p27 was used as a positive control of MG132 activity (representative Western blot at left). The average of four independent experiments is presented on the right graph (Kruskal-Wallis test). (C) AMOTL1 signal (in red) drops in BC52 tet on AMOTL1 cells expressing GFP-Merlin (left). Numbers refer to the intensity score (0: no signal, 1: intermediate, and 2: strong). Scale bar is 50 μm. The quantification of AMOTL1 intensity score upon expression of Merlin, a defective AMOTL1-binding Merlin mutant (1-532), or GFP alone is presented on the right graph. AMOTL1 staining was scored in 150 transfected cells (right, Kruskall Wallis test). (D) The same approach was used for AIP4, an ubiquitin ligase activity dead mutant of AIP4 (CS) or GFP-NEDD4 (n = 150 cells). GFP was used as a negative control (quantification on top with Kruskal-Wallis test, immunofluorescence at the bottom). Scale bar is 50 μm. (E) The level of endogenous AMOTL1 increases in BC52 cells when the expression of AIP4 and NEDD4 is inhibited with siRNA (right). [Left, quantification (n = 3) Kruskal-Wallis test]. (F) The specificity of the anti-phospho-AMOTL1 S262 was tested on immunoprecipitated GFP-AMOTL1 treated (+) or not (−) with alkaline phosphatase (top). (G) Merlin induces the phosphorylation of GFP-AMOTL1 in 293 HEK cells. The level of phosphorylation of AMOTL1 was quantified by Western blot on immunoprecipitated GFP-AMOTL1 and normalized from the amount of GFP-AMOTL1 (n = 4, Mann-Whitney test). (H) When tested using the same strategy as in panel A, Merlin expression showed no significant impact on the half-life of AmotL1S262A mutant (n = 4, Mann-Whitney test). For A, B, C, E, G, and H, values are provided as mean ± SEM.