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HSP70 processes Bay41-4109-induced aberrant polymers.
No full text(A) HepAD38 cells were transfected with two different siRNAs targeting Hsp70 or mock siRNA. At 2 d after transfection, the cell lysates were detected by western blot using the indicated antibodies. (B) HBc protein levels normalized to actin levels were quantified. The quantification results of HBc/actin ratio from two independent immunoblots are shown as relative percentages. (C) HepAD38 cells were treated with Bay41-4109 or DMSO or Bay41-4109 and MKT077 for 48 h. Cell extracts were then analyzed by western blotting using the indicated antibodies. (D) HBc protein levels normalized to actin levels were quantified. The quantification results of HBc/actin ratio from two independent immunoblots are shown as relative percentages. (TIF)</p
Particle gel assay of Bay41-4109-induced aberrant polymers.
No full textIndicated franctions of DMSO- or Bay41-4109-treated samples from the Fig 2 were subjected to particle gel assay. (TIF)</p
STUB1 mediates transport of Bay41-4109-induced aberrant non-capsid polymers to the perinuclear region.
No full text(A) HepAD38 cells were treated with 1 μM Bay41-4109 alone, Bay41-4109 and 10 mM 3-MA, or DMSO for 2 d. For STUB1 silencing, cells were transfected with si_STUB1 or si_control at 36 h before treatment with both Bay41-4109 and 10 mM 3-MA. Cells were immunostained for HBc (green) as indicated. Nuclei were stained with Hochest33342. The “enlargement” column shows enlarged images of the areas indicated by white boxes in the “merge” column. Scale bars are 30 μm in the “merge” column and 3 μm in the “enlargement” column. (B) Numbers of perinuclear HBc puncta (diameter exceeds 3 μM in any direction) per cell. 100 cells were counted. The error bars indicate ±SD. **p p value was calculated by unpaired two-tailed student’s t-test. (C) HepAD38 cells were treated with 1 μM Bay41-4109 and 10 mM 3-MA for 2 d. Cells were immunostained for HBc (green) and STUB1 (red) as indicated. Nuclei were stained with Hochest33342. Areas indicated by white boxes are enlarged. Arrows point to typical co-localized sites. Scale bar is 10 μm. (D) HepAD38 cells transduced with LV-sh_STUB1 or LV-sh_control were treated with 1 μM Bay41 or DMSO for 24 h, and all the cells were treated with 0.1 mM BafA1 for 12 h. Cell lysates were immunoprecipitated with HBc antibodies or mock antibodies. Proteins were detected by western blot using antibodies recognizing the respective proteins. (E) HepAD38 cells were transfected with two different siRNAs targeting BAG3 or mock siRNA. At 36 hours after transfection, cells were incubated in medium with 1 μM Bay41-4109 or DMSO for 2 d. Cell extracts were then analyzed by western blotting using the indicated antibodies (left panel). The quantification results of three independent immunoblots are shown as relative percentages (HBc/Actin) with mock transfection/transduction samples set to 100% (right panel). (F) HepAD38 cells were transfected with si_BAG3 or si_control. 36 h after transfection, HepAD38 cells were treated with 1 μM Bay41-4109 and 10 mM 3-MA, or DMSO for 2 d. Cells were immunostained for HBc (green) as indicated. Nuclei were stained with Hochest33342. The “enlargement” column shows enlarged images of the areas indicated by white boxes in the “merge” column. Scale bars are 30 μm in the “merge” column and 3 μm in the “enlargement” column.</p
The effect of STUB1 overexpression in Bay41-4109-treated HBV-infected HepG2-NTCP cells.
No full text(A) Schematic diagram of the experimental procedure for HepG2-NTCP cells in Fig 8B–8D. (B, C, D) HepG2-NTCP cells transduced with LV-STUB1 or LV-control were infected with HBV at MOI of 500 genome equivalents in the presence of 2% DMSO. Bay41-4109 (1 μM) or DMSO was added during HBV infection. The HBeAg level (B) and HBV DNA copies (C) in media were quantified at 2, 4, and 6 d after HBV infection. The error bars indicate ±SD.*p p p value was calculated by unpaired two-tailed student’s t-test. (D, E) Cells were immunostained for HBc (green) as indicated. Nuclei were stained with Hochest33342 (D) The fluorescence signal intensity was quantified by ImageJ (E). *p p value was calculated by unpaired two-tailed student’s t-test. (F) Schematic diagram of the experimental procedure of adding Bay41-4109 during (co-infection) or post HBV infection (post-infection) in LV-control- or LV-STUB1-transduced HepG2-NTCP cells. (G) Cells were treated with Bay41-4109 during (co-infection) or post HBV infection as descripted in Fig 8G. cccDNA were isolated by hirt extraction, and their levels were quantified by qPCR.</p
STUB1 promotes the Bay41-4109-induced degradation of HBc.
No full text(A, B) HepAD38 cells were transfected with expression vectors coding for E3 ligases including CUL5, MDM2, STUB1, PRKN, or empty vector (A) or two different siRNAs targeting STUB1 or mock siRNA (B). At 36 hours after transfection, cells were incubated in medium with 1 μM Bay41-4109 or DMSO for 2 d. Cell extracts were then analyzed by western blotting using the indicated antibodies (left panel). The quantification results of three independent immunoblots are shown as relative percentages (HBc/Actin) with mock transfection/transduction samples set to 100% (right panel). The error bars indicate ±SD. Data were analyzed by one-way analysis of variance, followed by Tukey’s comparison test for all groups * indicates p p > 0.05. (C, D) HepAD38 cells were transduced with LV-STUB1 or LV-control (C) or with LV-sh_STUB1 or mock LV-sh_control (D). At 36 h after transduction, cells were treated with 1 μM Bay41-4109 and 50 μg/ml CHX for the indicated time. The proteins were detected by western blot using the indicated antibodies (left panel). The quantification results of HBc/actin ratio from two independent immunoblots are shown as relative percentages (right panel). The samples of CHX treatment at 0 h were set to 100%. The error bars indicate ±SD.* indicates p p p was calculated by unpaired two-tailed student’s t-test.</p
STUB1 protects cells from cytotoxity of Bay41-4109-induced aberrant non-capsid polymers.
No full textHepAD38 cells were cultured in the presence (right panel) or absence (left panel) of Dox. Cells were transduced with LV-sh_STUB1 or LV-sh_control. 36 h later, cells were treated with the indicated concentrations of Bay41-4109 for 6 d. A CCK-8 assay was used to quantify cell viability. The error bars indicate ±SD. **p < 0.01, p value was calculated by unpaired two-tailed Student’s t-test.</p
Co-sedimentation and co-localization of STUB1 with Bay41-4109-induced aberrant non-capsid polymers.
No full text(A, B) HepAD38 cells were treated with DMSO or increasing concentrations of Bay41-4109 (as indicated) for 36 h. Cytoplasmic lysates from each sample were condensed with a centrifugal filter (100-KDa cutoff) and subjected to iodixanol gradient ultracentrifugation. The levels of the HBc and STUB1 proteins in each fraction were then analyzed with dot blots (A), and were quantified based on two independent experiments by ImageJ (B). (C) HepAD38 cells were treated with 1 μM Bay41-4109 or DMSO as indicated for 48 h. The cells were then immunostained for HBc (green) and stub1 (red). Nuclei were stained with Hochest33342. Areas indicated by white boxes are enlarged. Arrows point to representive co-localized sites. The scale bar is 10 μm. (D) Quantification of the number of HBc puncta co-localized with STUB1 per cell. 100 cells were counted. The error bars indicate ±SD. **p < 0.01, calculated by unpaired two-tailed student’s t-test.</p
Proteasome inhibitors had no effect on HBc protein level in Bay41-4109-treated cells.
No full text(A, B) HepAD38 cells were treated with 1 μM Bay41-4109 or DMSO for 24 h, followed by treatment with the proteasome inhibitor lactacystin, lysosome inhibitor BafA1, or lysosome inhibitor Pep/E64 for another 24 h. Cell extracts were then analyzed by western blotting using the indicated antibodies (A). The quantification results of two independent immunoblots are shown as relative percentages (HBc/actin) with the DMSO-treated sample set to 100% (B). The error bars indicate ±SD. n. s. indicates p > 0.05, p value were calculated by unpaired two-tailed student’s t-test. (TIF)</p
STUB1 inhibits secretion of virions and HBeAg from Bay41-4109-treated HepAD38 cells, and reduces intracellular p17 level in p17-transfected cells.
No full text(A, B) HepAD38 cells transduced with LV-STUB1 or LV-control were treated with 1 μM Bay41-4109 or DMSO for 2 d. Capsids precipitated from cell lysates were subjected to agarose gel electrophoresis and western blotting using a rabbit polyclonal anti-HBc antibody (A). Capsid levels of two independent immunoblots were quantified by ImageJ (B). (C, D) HepAD38 cells transduced with LV-STUB1 or LV-control were treated with the indicated concentrations of Bay41-4109 for 6 d. Media were refreshed every 2 d. The number of HBV DNA copies (C) and HBeAg level (D) in media were quantified. The error bars indicate ±SD. **p < 0.01, calculated by unpaired two-tailed student’s t-test. (E, F) HepAD38 cells were transfected with pcDNA3.1-STUB1 or control plasmid, and pcDNA3.1-p25ΔCTD or pcDNA3.1-p25. At 1 d after transfection, cells were incubated in medium with 1 μM Bay41-4109 or DMSO for 2 d. Cell extracts were then analyzed by western blotting using antibodies recognizing myc tag or actin (E). The quantification results of three independent immunoblots are shown as relative percentages (p17/Actin or precore/Actin) with mock-treated samples set to 100% (F).</p
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