Fig 2: TRIP13 activates AKT/mTOR pathway by interacting with ACTN4. a ACTN4 was identified as a binding partner of TRIP13 by combining Co-IP and MS. b The AKT/mTOR pathway was affected by ACTN4 interference. c Co-IP was used to validate the interaction of TRIP13 and ACTN4. d Immunofluorescence used to verify the colocalization of TRIP13 and ACTN4. e-f qRT-PCR and western blot analysis of TRIP13 or ACTN4 mRNA expression in HCC cells transfected with shTRIP13 or siACTN4. g-h Cell migration and invasion were measured in the indicated cells. i EMT markers and reprehensive genes of AKT/mTOR pathway were measured in the indicated cells by western blot. *P < 0.05, **P < 0.01, ***P < 0.001

Fig 3: OTUD3 enhances the stability of ACTN4 through deubiquitylation. (A, B) co-IP experiments between endogenous OTUD3 and ACTN4 in HCCLM3 and Huh7 cells. ACTN4 was detected in the immunoprecipitation when the anti-OTUD3 antibody was used as bait. (C) Colocalization of OTUD3 and ACTN4 in HCCLM3 and Huh7 cells (Scale bar: 14μm). (D) HCCLM3 and Huh7 cells were treated with 15μM proteasomal inhibitor MG132 for the indicated time, and the levels of ACTN4 were then detected. (E) HCCLM3 cell transfected with OTUD3 shRNA or shNC together with stably OTUD3 overexpressing Huh7 cells and negative control were treated with 20μM cycloheximide (CHX). Cells were collected at different time points and were detected ACTN4 protein expression. (F) HCCLM3 cells with OTUD3 knockdown or OTUD3 overexpression were treated with MG132 (15μM). Cells were collected at 6 h and immunoblotted with the antibodies indicated. (G, H) the knockdown or upregulation of OTUD3 altered the ubiquitination of ACTN4 in both HCCLM3 and Huh7 cells. The cells in each group were treated with MG132 (15μM). The levels of ubiquitin-attached ACTN4 were detected by western blot analysis with ubiquitin (Ub) antibody.

Fig 4: ACTN4 is critical for OTUD3-mediated HCC cells progression in vitro and in vivo. (E) Western blot showing the overexpression of OTUD3 rescued the decreased ACTN4 protein level caused by ACTN4 knockdown. (F, G) EdU assay identifying the effect of OTUD3 upregulation on inhibited HCC cell growth caused by ACTN4 downregulation. (H) Quantification of HCC cell transwell migration and invasion results. (I, J) Tumor sizes and tumor weights of 24 nude mice (6 mice per group) were measured and corresponding tumor growth curves were obtained in the rescue experiments. (K, L) In vivo lung metastasis rescue experiment was examined in 24 nude mice (6 mice per group). Representative H&E staining of lungs are shown (Scale bar: 100μm; magnification: 200X), along with the number of lung metastases in the four groups of nude mice. The unpaired two-sided Student’s t test was used for comparing between two groups of equal variances. Error bars represent mean ± SD from three independent experiments. NS: not significant, *P< 0.05, **P< 0.01.

Fig 5: Characterization of RAW 264.7 macrophages-derived DEX-packaging MVs. (A) Size distribution analysis of MV-DEX by nanoparticle tracking analysis. (B) Transmission electron microscopy images of MV-DEX showed double-membrane vesicles. Boxed area is enlarged in the right. (C) Markers of macrophage (CD68, F4/80 and CD206) and microvesicles (actinin 4) were confirmed in MV-DEX by Western blot. (D) HPLC analysis revealed the concentrations of DEX in MVs were dependent on the applied drug doses. (E) RAW cells were incubated with 30 μmol/L fluorescein-DEX. MVs were isolated and observed under confocal microscopy. DEX was shown as the green fluorescent (E1) (scale bar, 1 μm). GECs were incubated with fluorescein-DEX packaging MVs for 12 h and then observed under confocal microscopy (E2) (scale bar, 10 μm). n=3. Data are presented as mean ± SD.