Journal: Theranostics

Article Title: p-STAT3-elevated E3 ubiquitin ligase DTX4 confers the stability of HBV cccDNA by ubiquitinating APOBEC3B in liver

doi: 10.7150/thno.99407

Figure Lengend Snippet: Cucurbitacin I attenuates the HBV replication and enhances the sensitivity of PEG-IFN α against HBV in vitro . (A-G) HepG2-NTCP cells were subjected to HBV infection (MOI = 500) and treated with factors as indicated at 1 and 4 dpi (days post-infection). Analysis was performed 7 days post-infection. The levels of HBV DNA were measured by real-time PCR in the supernatant of HBV-infected HepG2-NTCP cells treated with cucurbitacin I (A). The viability of HBV-infected HepG2-NTCP cell treated with cucurbitacin I was evaluated by the MTS assays (A). The HBV DNA content was tested by real-time PCR (B). The levels of HBsAg and HBeAg were validated by ELISA as appropriate in the supernatants (C, D). The HBc expression levels were examined by immunofluorescence (E). ImageJ was used to quantitate the protein expression levels in the immunofluorescence analysis (F). The levels of HBV pgRNA were determined by RT-qPCR in the cells (G). (H) The Volcano plot of the differentially expressed genes (DEGs) that cucurbitacin I has a significant effect on HBV-infected hepatoma cells analyzed by RNA-Seq (the screening criteria were |FC| ≥1.5 & Padj<0.05). (I, J) The GO/KEGG enrichment analysis of DEGs between the control and cucurbitacin I groups ( n =3). The mean ± SD of at least three experiments is shown. Statistically significant differences are indicated as follows: * P < 0.05, ** P < 0.01, *** P < 0.001. Abbreviation: N.S., not significant, Cu I, Cucurbitacin I.

Article Snippet: Cucurbitacin I was purchased from MedChemExpress LLC (Shanghai, China).

Techniques: In Vitro, Infection, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Expressing, Immunofluorescence, Quantitative RT-PCR, RNA Sequencing, Control

Journal: Theranostics

Article Title: p-STAT3-elevated E3 ubiquitin ligase DTX4 confers the stability of HBV cccDNA by ubiquitinating APOBEC3B in liver

doi: 10.7150/thno.99407

Figure Lengend Snippet: Cucurbitacin I attenuates the HBV replication and enhances the sensitivity of PEG-IFN α against HBV in vivo . (A) Schematic representation of mouse experimental process. (B) The mice were administered by cucurbitacin I (0.5 mg/kg) once a day and PEG-IFN α (25 μg/kg) twice per week for 4 weeks. Serum samples were collected before treatment (day 0) and days 7, 14, 21, and 28 after treatment. A comparative evaluation of changes in the body weight of HDI mice treated with the indicated compounds. (C) Pathological characteristics were observed by H&E staining in the liver tissues from mice treated with the indicated compounds. Scale bars: 100 μm. (D) The levels of HBV DNA in the serum of HDI mice were tested by qPCR. (E) Immunohistochemical staining for HBsAg and HBcAg were conducted in liver tissues. Scale bars: 50 μm. (F) The HBcAg expression levels in liver tissue samples were quantified by using the software of ImageJ. (G) The levels of HBV pgRNA in liver tissues from HBV-infected mice were measured by RT-qPCR. The mean ± SD of at least three experiments is shown. Statistically significant differences are indicated as follows: * P < 0.05, ** P < 0.01, *** P < 0.001. Abbreviation: N.S., not significant, Cu I, Cucurbitacin I.

Article Snippet: Cucurbitacin I was purchased from MedChemExpress LLC (Shanghai, China).

Techniques: In Vivo, Staining, Immunohistochemical staining, Expressing, Software, Infection, Quantitative RT-PCR

Journal: Theranostics

Article Title: p-STAT3-elevated E3 ubiquitin ligase DTX4 confers the stability of HBV cccDNA by ubiquitinating APOBEC3B in liver

doi: 10.7150/thno.99407

Figure Lengend Snippet: Cucurbitacin I enhances the sensitivity of PEG-IFN α against HBV cccDNA by elevating the levels of APOBEC3B. (A, B) HepG2-NTCP cells were subjected to HBV infection (MOI = 500) and treated with factors as indicated at 1 and 4 dpi (days post-infection). Analysis was performed 7 days post-infection. The HBV cccDNA content was tested by Southern blot analysis (A) and qPCR (B). (C) The effect of cucurbitacin I on the expression levels of APOBEC3A and APOBEC3B was detected by Western blot analysis in HepG2 cells treated with IL-6 to induce STAT3 activation for 16 hours. (D) The effect of cucurbitacin I on the binding of APOBEC3B to HBV cccDNA was evaluated by ChIP-qPCR in HBV-infected HepG2-NTCP cells. (E) HepG2 cells were treated with DMSO or cucurbitacin I 48 h followed by cycloheximide (CHX, 100 μg/mL) treatment for the indicated time. Cells treated with IL-6 to induce STAT3 activation for 16 hours. The protein levels of APOBEC3B were examined by Western blot analysis. (F) HepG2 cells were transfected with pcDNA3.1 STAT3 (2 μg) for 48 h and then treated with MG132 (2 mM) for 24 h. Cells treated with IL-6 to induce STAT3 activation for 16 hours. The protein levels of APOBEC3B and STAT3 were examined by Western blot analysis in the cells. (G) Western blot analysis was performed to determine whether the effect of cucurbitacin I on APOBEC3B protein expression through STAT3 in HepG2 cells treated with IL-6 to induce STAT3 activation for 16 hours. (H) Schematic diagram of the mechanism of cucurbitacin I reducing the stability of HBV cccDNA by inhibiting the proteasome degradation of APOBEC3B. The mean ± SD of at least three experiments is shown. Statistically significant differences are indicated as follows: ** P < 0.01, *** P < 0.001. Abbreviation: N.S., not significant, CHX, Cycloheximide, Cu I, Cucurbitacin I, A3A, APOBEC3A, A3B, APOBEC3B.

Article Snippet: Cucurbitacin I was purchased from MedChemExpress LLC (Shanghai, China).

Techniques: Infection, Southern Blot, Expressing, Western Blot, Activation Assay, Binding Assay, ChIP-qPCR, Transfection

Journal: Theranostics

Article Title: p-STAT3-elevated E3 ubiquitin ligase DTX4 confers the stability of HBV cccDNA by ubiquitinating APOBEC3B in liver

doi: 10.7150/thno.99407

Figure Lengend Snippet: E3 ubiquitin ligase DTX4 contributes to HBV cccDNA and HBV replication. (A, B) HepG2 cells were treated with cucurbitacin I for 72 hours. Cells treated with IL-6 to induce STAT3 activation for 16 hours. The effects of cucurbitacin I on the mRNA and protein levels of HECW1 and DTX4 were detected by RT-qPCR and Western blot analysis in cells, respectively. (C-K) HepG2-NTCP cells were subjected to HBV infection (MOI = 500) and treated with factors as indicated at 1 and 4 dpi (days post-infection). Analysis was performed 7 days post-infection. The effects of HECW1 and DTX4 on HBV DNA levels were tested by real-time PCR in the supernatants (C). The levels of HBsAg and HBeAg were measured by ELISA as appropriate in the supernatants (D). The HBV DNA content was validated by real-time PCR (E). The levels of HBsAg and HBeAg were verified by ELISA as appropriate in the supernatants (F, G). The levels of HBV pgRNA were determined by RT-qPCR in the cells (H). The HBc expression levels were detected by immunofluorescence staining (I). The HBV cccDNA content was tested by qPCR (J) and Southern blot analysis (K). (L-O) The mRNA and protein levels of DTX4 were analysed by RT-qPCR and Western blot analysis in the liver tissues of HBV-infected ( n =5) and uninfected ( n =5) human liver chimeric mice and in liver biopsy specimens from clinical patients with a high HBV load ( n =6) and with a low HBV load ( n =6). The mean ± SD of at least three experiments is shown. Statistically significant differences are indicated as follows: * P < 0.05, ** P < 0.01, *** P < 0.001. Abbreviation: N.S., not significant, Cu I, Cucurbitacin I, A3B, APOBEC3B.

Article Snippet: Cucurbitacin I was purchased from MedChemExpress LLC (Shanghai, China).

Techniques: Ubiquitin Proteomics, Activation Assay, Quantitative RT-PCR, Western Blot, Infection, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Expressing, Immunofluorescence, Staining, Southern Blot

Journal: Theranostics

Article Title: p-STAT3-elevated E3 ubiquitin ligase DTX4 confers the stability of HBV cccDNA by ubiquitinating APOBEC3B in liver

doi: 10.7150/thno.99407

Figure Lengend Snippet: Cucurbitacin I limits the stability of HBV cccDNA and HBV replication by inhibiting p-STAT3/DTX4/MSL2/APOBEC3B signalling. (A-H) PHH and HepG2-NTCP cells were subjected to HBV infection (MOI = 500) and treated with factors as indicated at 1 and 4 dpi (days post-infection). Analysis was performed 7 days post-infection. The protein expression was verified by Western blot analysis in the cells (A). The HBV DNA content was tested by real-time PCR (B). The levels of HBsAg and HBeAg were measured by ELISA as appropriate in the supernatants (C, D). The levels of HBV pgRNA were determined by RT-qPCR in the cells (E). The HBV cccDNA content was examined by Southern blot analysis (F) and qPCR analysis (G). The binding levels of APOBEC3B to HBV cccDNA micro-chromosome were evaluated by ChIP-qPCR analysis in the cells (H). The mean ± SD of at least three experiments is shown. Statistically significant differences are indicated as follows: ** P < 0.01, *** P < 0.001. Abbreviation: Cu I, Cucurbitacin I, A3B, APOBEC3B.

Article Snippet: Cucurbitacin I was purchased from MedChemExpress LLC (Shanghai, China).

Techniques: Infection, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Southern Blot, Binding Assay, ChIP-qPCR